AU1138899A - Glucose regulated gene - Google Patents

Description

WO 99/31134 PCT/CA98/01061 GLUCOSE REGULATED GENE CROSS REFERENCE TO RELATED APPLICATION This application claims priority from U.S. application no. 60/069,352, which is incorporated by reference herein in its entirety. 5 FIELD OF THE INVENTION The invention relates to an isolated glucose regulated gene and its protein expression product. The invention also relates to methods of modulating the gene for treatment of hyperglycemia, glomerulosclerosis and renal cell apoptosis. BACKGROUND OF THE INVENTION 10 (i) Renal Failure as a Complication of Diabetes Mellitus Renal failure caused by glomerulosclerosis is a major complication of insulin dependent ("IDDM") and non insulin dependent ("NIDDM") diabetes (1, 2). Renal failure is increasing in Europe and North America (3-5), due to a variety of factors, including an aging population, poor dietary habits, and longer survival of juvenile diabetics. About 15 25% of patients undergoing treatment of end stage renal disease (ESRD) in the US and Canada, have kidney failure (nephropathy) caused by diabetes (1). Although renal failure in diabetes is not well understood, significant advances have been made recently. There still remains a clear need to characterize the processes that cause diabetes related kidney failure. 20 Renal disease occurs more frequently in IDDM than in NIDDM, and there is a strong genetic component associated with the former (1). So far, the genes involved in this disease have not been identified. There has been some suggestion that certain Angiotensin Converting Enzyme (ACE) polymorphisms predispose to development of diabetic nephropathy (6). Only about 30-40% of IDDM patients eventually develop ESRD 25 (7). It would be helpful if genetic factors that protect the 60-70% majority of IDDM patients from progressive renal failure could be identified. (ii) Hyperglycemia as a Cause of Diabetic Nephropathy - the Role of the Mesangial Cell Diabetics have chronically elevated blood glucose levels (hyperglycemia). 30 Hyperglycemia contributes to development of microvascular and renal complications. There is no doubt that controlling blood sugar reduces these complications (8). Studies show that diabetic glomerulosclerosis is caused by expansion of the mesangial matrix (1). The main product of the mesangial matrix, collagen IV, is found throughout the expanded 1 WO 99/31134 PCT/CA98/01061 mesangium. This is characteristic of diabetic glomerulosclerosis (16, 17). The mesangial cell is now considered to be involved in initiation of diabetic glomerulosclerosis. Current investigation of renal failure centers around mesangial cell ("MC") responses to hyperglycemia. 5 Hyperglycemia either directly (9) or indirectly (10) leads to the increased production of growth factors, accumulation of excess extracellular matrix ("ECM")and creation of advanced glycosylation end products. These findings have been reproduced and corroborated in animal models of diabetes. There are factors in addition to hyperglycemia that contribute to 10 glomerulosclerosis and microvasular changes. As mentioned above, most IDDM patients do not develop diabetic renal disease, despite the presence of life long elevated blood sugars. Hyperglycemia is a necessary, but not sufficient condition for diabetic renal complications. Nevertheless, if hyperglycemia could be fully understood at the molecular 15 level, this would permit targeted therapeutic intervention to prevent the hyperglycemia induced component of diabetic complications. It would also help identify genes that afford cell protection or establish cell vulnerability to sustained, elevated glucose. Recently, there has been a recognition that diabetic renal disease in the presence of hyperglycemia is associated with apoptosis. 20 (iii) Hyperglycemia-induced Alterations in ECM-MC Signaling Although a number of different cellular metabolic pathways are known to be altered by exposure to elevated concentrations of glucose (17, 18), diacylglycerol ("DAG") induced protein kinase C ("PKC") activation (especially its P2 isoform) is probably the most important (13, 14, 17-19). PKC inhibition reverses many of the acute and chronic 25 effects of hyperglycemia on MC by blocking DAG binding to PKC (13). The sequence of events described below occurs in hyperglycemia. The model is derived from in vitro studies of MC response in primary culture to short term hyperglycemic conditions and in vivo investigations of early changes in renal functional parameters (increased glomerular filtration rate and urine protein excretion) in animal models such as streptozotocin treated 30 rats. High glucose enhances intracellular production of sorbitol via the aldose reductase pathway. This leads to an increase in intracellular osmolality (11). At the same time (ii) high glucose increases de novo synthesis of diacylglycerol (DAG) leading to activation and phosphorylation of protein kinase C (PKC). This is followed by a series 2 WO 99/31134 PCT/CA98/01061 of "downstream" events, including increased expression of various growth factors, most notably, transforming growth factor beta (TGFI3). TGFP3, in an autocrine manner, stimulates MC production of extracellular matrix (ECM) elements, fibronectin and collagen IV, while at the same time reducing ECM degradation by increasing levels of the 5 metalloproteinase inhibitor TIMP-2 (12). These effects are prevented by treatment with anti-TGFP3 antibodies. TGFP is critical in accumulation of ECM following short term exposure of MC to elevated glucose concentrations. DAG-induced activation of MC PKC32 is responsible for the acute and even certain chronic changes associated with diabetic microvascular and renal complications 10 (13). Administration of a specific PKC32 inhibitor-LY333531, appears to prevent the in vivo and in vitro sequelae of hyperglycemia, described above (14). PKC is a serine-threonine phosphorylation kinase. Many different PKC isoforms exist, and their specificity of action is attributable to their intracellular compartmentalization, which varies from cell to cell. All PKC isoforms contain 2 15 regulatory domains, C1 and C2, which bind DAG and Ca", respectively, in addition to binding a kinase domain. Under resting conditions, the kinase domain is inactive due to its interaction with the C1 domain. When DAG binds to C1, dissociation occurs, allowing ATP to bind to the kinase region. This activates PKC. A drug named LY333531 acts by competing with ATP for binding at the kinase domain. The effect of this drug is to block 20 PKC phosphorylation without affecting intracellular DAG levels (14,15). The PKC pathway is not well understood. Whether PKC activation is the dominant dysfunction in diabetic glomerulopathy is undetermined. Also unknown is whether other signaling pathways stimulated by hyperglycemia are capable of interacting with and modifying DAG induced PKC activation. 25 It would be helpful if DAG activation of PKC (via binding to the C1 domain) and its interaction with other metabolic changes in glomeruli and microvasculature during hyperglycemia were characterized. This would lead to new treatments to control and prevent damage to glomerular and microvascular function caused by hyperglycemia and diabetes. 30 (iv) Signaling Proteins that Belong to the Same Superfamily as PKC There has been also been growing interest in the characterization of a novel class of signaling proteins that belong to the same superfamily as protein kinase C, but lack its kinase activity. Unc-13, one of the members of this family, encodes a phorbol ester/ diacylglycerol-binding protein in C. elegans. Initial evaluation suggested it had a role in 3 WO 99/31134 PCT/CA98/01061 neurotransmitter release. (20-23). Mammalian homologues (muncl 3s), muncl3-1, -2, and -3, were originally cloned from rat brain and similar to Unc-13 in that both possess DAG and Ca 2 + binding domains (20). Syntaxin, synaptobrevin, SNAP 25 (24) and Doc2 (25) were found to coimmunoprecipitate with muncl 3s, consistent with the suggestion 5 that this new family of DAG binding proteins is involved in vesicle trafficking and neurotransmitter release. It would be helpful if the role of genes in this family was characterized so that its role in metabolism was understood. No characterization data to date has linked this gene to hyperglycemia or kidney failure. The function of these signaling proteins and related isoforms is largely unknown. 10 Nevertheless there is emerging evidence that DAG activated muncl 3 is involved in neurotransmission (24). In summary, there is recognition that non PKC DAG activated signaling pathways regulate important cellular functions. Since hypergelycemia results in increased intracellular DAG concentration, there is a need to identify and characterize the targets of 15 DAG that are involved in the microvascular and renal complications of diabetes. This would lead to new compounds and methods for treatment of these complications. SUMMARY OF THE INVENTION We cloned a gene from human MC, Hmuncl3, which is up-regulated by hyperglycemia. Hmuncl3 mediates some of the acute and chronic changes in MC 20 produced by exposure to hyperglycemia. These changes result in diabetic microvascular and renal damage, such as glomerulosclerosis and apoptosis. We have established the following: (a) Structure of Hmuncl3 and biologically functional equivalent nucleotide sequences: Hmuncl3 is a signaling molecule localized to the plasma membrane 25 of renal mesangial cells, cortical epithelial cells and other cells, The topological organization is illustrated schematically in figure 7. There are functional extracellular RGD domains, and intracellular C1 and C2 domains. There is also an intracellular regulatory domain on Hmuncl3 that targets and activates a serine threonine catalytic phosphatase subunit to the plasma membrane 30 (b) Function of Hmuncl 3 and biologically functional equivalent nucleotide sequences: The functional role for Hmuncl 3 involves intracellular signal transduction and regulation of cell attachment and migration. Hmuncl3 acts through modulation of phosphatase activity. In this way, Hmuncl13 phosphatase activation opposes 4 WO 99/31134 PCT/CA98/01061 downstream serine/threonine phosphorylation initiated in response to PKC and integrin activation. (c) Disease Model & Therapeutic Intervention: Hmuncl 3 is activated in response to hyperglycemia-induced increases in DAG, causing (i) stimulation of phosphatase 5 activity and, (ii) modulation of DAG-induced PKCP3 activation. We have identified a model which incorporates the two DAG activated pathways: (i) PKC dependent and (ii) Hmuncl3 dependent. These two pathways regulate two opposing cell phenotypes, PKC-proliferation and hmuncl 3-apoptosis. The over-expression of Hmuncl3 under hyperglycemic conditions and Hmuncl3 DAG-induced 10 apoptosisprove a role for Hmuncl 3 in diabetic renal cell injury. Modulation of Hmuncl3 and biologically functional equivalent nucleotide sequences is particularly useful for treatment and prevention of renal cell damage. The invention is an isolated nucleotide sequence encoding a glucose regulated munc polypeptide. The nucleotide is preferably from a kidney cell, human cortical 15 epithelial cell or a cell from testis, ovaries, prostate gland, colon, brain and heart, more preferably a mesangial cell or a kidney cortical epithelial cell. The nucleotide sequence preferably comprises a Hmuncl13 polypeptide and all or part of the amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO. 1]. The nucleotide sequence preferably comprises a Hmuncl 3 gene having all or 20 part of the nucleotide sequence in Figure 8 [SEQ ID NO. 2]. The molecule preferably comprises at least 40% sequence identity to all or part of the nucleotide sequence of Figure 8. The sequence is preferably selected from a group consisting of mRNA, cDNA, sense DNA, anti-sense DNA, single-stranded DNA and double-stranded DNA. The nucleotide encodes an amino acid sequence of the invention. The nucleotide sequence 25 that encodes all or part of a Hmuncl 3 polypeptide, preferably hybridizes to the nucleotide sequence of all or part of Figure 8 under high stringency conditions (e.g. a wash stringency of 0.2X SSC to 2X SSC, 0.1% SDS, at 65 0 C). The invention also includes an isolated munc polypeptide, with the provisio that the polypeptide is not found in a mammalian central nervous system. The polypeptide of 30 preferably has transmembrane ECM-cell signaling activity and DAG and Ca' activated phosphatase activity and more preferably includes all or part of the Hmuncl13 amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO: 1]. The invention also includes amimetic of the purified and isolated polypeptide. The polypeptide preferably has at least 40% sequence identity to all or part of the amino acid sequence (a) in Figure 1 [SEQ ID 5 WO 99/31134 PCT/CA98/01061 NO: 1] . The polypeptide is preferably from a mammalian kidney cell. It is useful for inducing apoptosis and vesicle trafficking. The invention also includes a recombinant DNA comprising a DNA molecule the invention and a promoter region, operatively linked so that the promoter enhances 5 transcription of said DNA molecule in a host cell. The invention also includes a system for the expression of Hmuncl3, comprising an expression vector and Hmuncl3 DNA inserted in the expression vector. The expression vector preferably comprises a plasmid or a virus. The invention also includes a cell transformed by the expression vector. The invention also includes a method for expressing Hmuncl 3 polypeptide comprising: 10 transforming an expression host with a Hmuncl 3 DNA expression vector and culturing the expression host. The method preferably also includes isolating Hmunc13 polypeptide. The expression host is preferably selected from the group consisting of a plant, plant cell, bacterium, yeast, fungus, protozoa, algae, animal and animal cell. The invention also includes a pharmaceutical composition, including at least all or 15 part of the polypeptide of the invention, and a pharmaceutically acceptable carrier, auxiliary or excipient. The invention also includes a pharmaceutical composition for use in gene therapy, comprising all or part of the nucleotide sequence of any of the invention and a pharmaceutically acceptable carrier, auxiliary or excipient. The pharmaceutical composition for use in gene therapy, preferably comprises all or part of an antisense 20 sequence to all or part of the nucleic acid sequence in Figure 8. Another embodiment of the invention is a kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the nucleotide sequence of the invention. A kit for the treatment or detection of a disease, disorder or abnormal physical state, preferably includes all or part of the polypeptide of the invention. 25 The kit may also comprise an antibody to the polypeptide. The disorder is preferably selected from a group consisting of insulin dependent and independent diabetes, glomerulopathy and renal failure. The invention also includes a NH2 SQRSNDEVREFVKL-COOH specific antibody, preferably a polyclonal antibody. The invention is also a method of medical treatment of a disease, disorder or 30 abnormal physical state, characterized by excessive Hmuncl 3 expression, concentration or activity, comprising administering a product that reduces or inhibits Hmuncl 3 polypeptide expression, concentration or activity. The product is preferably an antisense nucleotide sequence to all or part of the nucleotide sequence of Figure 8, the antisense nucleotide sequence being sufficient to reduce or inhibit Hmuncl 3 polypeptide 35 expression. The antisense DNA is administered in a pharmaceutical composition 6 WO 99/31134 PCT/CA98/01061 comprising a carrier and a vector operably linked to the antisense DNA.. The disease, disorder or abnormal physical state is preferably selected from a group consisting of insulin dependent diabetes and independent diabetes, glomerulonephritis and ischemic renal injuries. 5 The invention also includes a method of medical treatment of a disease, disorder or abnormal physical state, characterized by reduced Hmuncl13 expression, concentration or activity, comprising administering a product that increases Hmuncl 3 polypeptide expression, concentration or activity. The product is preferably a nucleotide sequence comprising all or part of the nucleotide sequence of Figure 8, the DNA being sufficient to 10 increase Hmuncl 3 polypeptide expression. The nucleotide sequence is preferably administered in a pharmaceutical composition comprising a carrier and a vector operably linked to the nucleotide sequence. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the invention will be described in relation to the 15 drawings in which: Figure 1. Protein sequence alignment of Hmuncl3 [SEQ ID NO: 1] (GenBank accession number AF020202) with rat muncl 3s. (a) Alignment of all four proteins. Only a partial (AA 251-2207) of rat muncl 3-3 is shown. (b) Alignment of the first 100 amino acid at the N-terminal of Hmuncl 3 and rat muncl 3-1. Identical residues are boxed. The dotted line 20 above the sequence indicates the C1 domain and the continuous line indicates the C2 domain as proposed by Brose et al. (7). Figure 2. Expression of Hmuncl3 in human MC culture in 5.5 mM D-glucose plus 9.5 mM L-glucose (L(15)) or 19.5 mM L-glucose (L) or 15 mM (D(15)) 25 mM mM D-glucose (D) and as described in Methods. Increased expression of Hmuncl3 after 25 mM D 25 glucose treatment is revealed by Relative RT-PCR (a) and Northern blot (b). All blots are representative of at least 3 different experiments using different total RNA preparations. Figure 3. Expression of Hmunc 13 (lane 7, 8) or muncl3-2 (lane 9) in human kidney MC (lane 7), cortical epithelial cells (lane 8) or rat kidney MC (lane 9). RT-PCR was performed using a pair of primers for both Hmuncl3 and rat muncl3-2 indicated in the 30 Methods which amplified a segment of 193 bp. A pair of primers for GAPDH generated a 453 fragment were used to PCR no RT RNA (lane 1-3) and RT products (lane 4-6) of human kidney MC (lane 1, 4), cortical epithelial cells (lane 2, 5) and rat MC (lane 3, 6). Figure 4. In vitro translation of Hmuncl 3. Note that a proportion of the highest MW band (170 kDa) in the absence of microsomal membranes (lane 1) is shifted to higher MW (180 7 WO 99/31134 PCT/CA98/01061 kDa) in the presence of microsomal membranes (lane 2). Lane 3 is the supernatant of derived from the in vitro translation reaction with microsomal membranes as detailed in Methods. Figure 5. Comparison of gene structure of Hmuncl3 to various isoforms of rat Muncl 3s. 5 Figure 6. Expression of rat muncl 3-2 in renal glomerulus of normal (A) or streptozotocin treated (B) rats detected by in situ hybridization. A PCR fragment of rat muncl 3-2 (residues 5487-5669) with a T7 promoter introduced in its sense primer was in vitro transcripted to anti-sense cRNA with DIG-labeled UTP. A section of normal and streptozotocin-treated rat kidneys on the same slide was hybridized with this probe and 10 the signal was detected by Rodamine-conjugated anti-DIG antibody and observed by confocal microscopy. A negative control with sense cRNA showed little staining in all sections (data not shown). Note the morphological changes in the glomerulus of streptozotocin-treated rat and the higher staining of mesangila cells. This study also confirms the expression of muncl 3-2 in renal tubular epithelial cells. 15 Figure 7. Structure model of Hmuncl13. Figure 8. DNA sequence of Hmuncl3 [SEQ ID NO: 2] (GenBank accession number AF020202) Figure 9. (i) Comparison of the structure of rat muncl3s and Hmuncl3. C1 represents the DAG binding (C1) domain; C2 represents the Ca2+ binding (C2) domain. (ii) 20 Comparison of the sequence of the C1 domain of rat muncl3-1 and hmuncl3. Continuous lines indicate identical amino acids and the dotted line indicates similar amino acids. Figure 10. (i) Immunoblot of Hmuncl3 and the C1 less mutant. Hmuncl 3-HA (Hmuncl13), C1 less mutant (C1 less) or empty plasmid, pCMV.SPORT (pCMV), were 25 transiently transfected into OK cells. Whole cell lysates were prepared and subjected to 6% SDS-PAGE. The blot was detected by anti-HA. Note the slightly decreased molecular weight of the C1 less mutant. (ii) Immunostaining of OK cells transiently transfected with hmuncl 3-HA (A-C, E-G) and C1 less mutant (D, H). Cells were stained with anti-HA then probed with anti-mouse IgG-rhodamine for detection of Hmuncl3 (A-C) and C1 less 30 mutant (D). The Golgi apparatus was detected by staining with WGA-FITC (E-H). Slides were observed by confocal microscopy using a laser scanning microscope with excitation wavelength at 568 nm for detecting rhodamine (A-D) and 488 nm for detecting FITC (E H). Cells were treated with vehicle (A, E), 0.1 pM PDBu for 3 h (B, D, F, H), 4 pM nocodazole + PDBu (C, G) as described in the Methods. Negative controls obtained by 8 WO 99/31134 PCT/CA98/01061 incubating with normal mouse IgG or immunostaining of cells transfected with empty plasmid (pCMVSPORT) yielded very little or no staining (data not shown). Arrowheads indicate co-localization of anti-HA and WGA staining. Note: Upper and lower panel pairs, i.e. A and E, B and F etc, represent anti-HA and WGA-FITC staining, respectively, of 5 identical fields. (iii) Immunoblots of whole cell lysates (panel A) and Golgi membrane preparations (panel B) from Hmuncl 3 transfected OK cells with (+) or without (-) PDBu treatment for 3 h. The whole cell lysates represent small aliquots of cells for Golgi membrane preparations. Equal amounts of protein were loaded onto each lane of panel A or B. The blots were 10 then detected by anti-HA antibody. Figure 11. (i) Double labeling of apoptotic cells and expression of Hmuncl3 or C1 less mutant. Hmuncl 3 (A-C, E-G) and Cl less mutant (D, H) transiently transfected cells were subjected to TUNEL labeled with fluorescein (E-H) and then subjected to anti-HA and anti-mouse IgG-rhodamine labeling for expression of Hmuncl3 and C1 less mutant 15 (A-D). Cells were treated with vehicle (A, E) or 0.1 l±M PDBu for 8 h (B, D, F, H) or 16 h (C, G). Cl less mutant transfected cells treated with vehicle exhibit a similar image as D and H (data not shown). Negative controls of TUNEL by incubating cells with labeling mix and no TdT yielded no staining of fluorescein (data not shown). Arrowheads indicate representative cells co-stained with anti-HA (upper panels) and TUNEL (lower panels) 20 from identical fields. (ii) Graphic representation of the percentage of transfected (immunostaining positive) and apoptotic (TUNEL positive) cells in Hmuncl3 or C1 less mutant (C1 less) transfected cells treated with or without PDBu for 8 or 16 h. Cell numbers were counted with an average of three low power views under the confocal microscope. Bars are representations of means ± SD of three experiments. 25 Figure 12. Genomic DNA breakdown in Hmuncl3 transfected cells by PDBu treatment. Genomic DNA obtained from empty plasmid (pCMV), Hmuncl3 or Cl less mutant transfected cells treated with vehicle (-) or 0.1 gM PDBu for 8 h or 16 h was subjected to 2 % agarose gel electrophoresis. Molecular size marker (M) is shown. Figure 13. Expression of rat muncl 3-1 in kidney of normal (A) or STZ-treated diabetic 30 (B-D) rat detected by in situ hybridization. Outer cortex (A, B), medulla (C) and a higher power view of outer cortex (D) from diabetic rat kidney are shown. Similar to diabetic rats, staining in the renal medulla for normal rat kidney is less than the cortex (data not shown). Note the increased expression of muncl 3-1 in the tubular epithelial cells as well 9 WO 99/31134 PCT/CA98/01061 as in certain glomerular cells. Negative controls with sense cRNA showed little staining in both normal and diabetic rat sections (data not shown). Figure 14. Expression of muncl3-1, muncl3-2 and muncl3-3 in the renal cortex of the normal rat and following 1 day (1d) and 11 day (1 d) of hyperglycemia in STZ-treated 5 rats. 18S ribosome RNA (18S) served as a housekeeping gene. Figure 15. Schematic representation of DAG activated branched signaling pathways involving PKC and Hmuncl3. DAG levels are increased by such factors as hyperglycemia, phospholipase C (PLC) P/y and phospholipase D (PLD) resulting in activation of both PKC and Hmuncl 3 and leading to two separate downstream signaling 10 pathways, respectively resulting in proliferation and differentiation (PKC) or apoptosis (Hmuncl 3). DETAILED DESCRIPTION OF THE INVENTION Isolation and Identification of Hmuncl3 We cloned a human muncl3 gene (Hmuncl3) and protein from kidney which 15 has an important role in cell signaling. This gene is regulated by glucose. Hmuncl3 contributes to the renal and microvascular complications associated with hyperglycemia in diabetes mellitus, through a variety of mechanisms including Hmuncl 3 linked apoptosis. We also have identified biologically functional equivalent nucleotide sequences and proteins. 20 We obtained the glucose regulated gene by differential display reverse transcription polymerase chain reaction (DDPT-PCR) of candidate genes differentially expressed in human MC exposed to hyperglycemic conditions, compared to controls. Using this screening procedure, we obtained a PCR product which was then used to clone the full length cDNA. This gene is similar to mammalian brain muncl 3s (it is a 25 differentially spliced isoform, munc 13-1 and munc 13-2). Hmuncl3 is detectable in both MC, epithelial and other cells. The presence of a Hmuncl 3 gene in MC which has similarity to rat muncl 3 was very unexpected because rat muncl 3 is believed to be localized only in the brain (20). We determined that Hmuncl 3 is a target for regulation by glucose in MC and 30 other cells. For example, the expression of Hmuncl 3 is up-regulated by hyperglycemia in cultured kidney MC and epithelial cells. Hmuncl3 protein is involved in the acute and chronic effects of hyperglycemia in MC and renal epithelial cells, and contributes to the development of diabetic glomerulopathy. Hmuncl 3 also interacts with the syntaxins. ) 10 WO 99/31134 PCT/CA98/01061 We then used a full length cDNA clone of rat muncl3-1 (a gene from rat brain with sequence similarity to Hmunc 13 and some similar functional domains) to show how the gene is regulated by glucose. In vitro experiments revealed that exposure of fibroblasts transfected with muncl 3-1 to phorbol esters caused translocation of munc 5 13-1 to the plasma membrane. We performed other in vitro experiments to show that, as a second messenger, DAG can activate either a PKC (proliferative) signaling pathway or alternatively, a Hmuncl 3 (apoptosis) signaling pathway. The combined action of these two pathways showed the functional responses of cells to stimuli such as hyperglycemia. Our results indicate that hyperglycemic activation of Hmuncl3 and 10 induction of apoptosis is a factor causing cell injury in diabetic nephropathy. Localization of Hmuncl3 We demonstrated the presence of Hmuncl3 in primary cultured human MC and in a human kidney cDNA library as well as muncl 3-2 in rat MC. A gene similar to muncl 3s has never previously been isolated outside the central nervous system. We also 15 confirmed that Hmuncl3 is expressed in the brain by PCR of a commercial human brain cDNA library (Gibco BRL) In vitro translation also indicates co-translational modification of Hmuncl 3. It is unlikely that this initiates N-glycosylation since addition of a competitive inhibitor of N-glycosylation, Ac-Asn-Tyr-Thr-NH2 (26), did not shift the band to lower molecular weight. 20 Hmuncl3 Protein Three Dimensional Structure Analysis of the hydropathy plot of Hmuncl 3 by Kyte-Doolittle analysis indicates that there are a few hydrophobic regions (residue 603-609, 817-825, 970-977, 1107 1111) with K-D values from 139 to 172. However, these are not typical transmembrane segments. It is possible that the full-length protein can fold in such a way that 25 hydrophobic loops can anchor to the membrane but that such folding is not possible for the partial length protein. Functional Domains of Hmunc13 Protein We reviewed the Hmuncl 3 sequence and compared different segments of Hmuncl3 with other amino acid sequences. 30 Hmuncl3 contains 1 C1 domain and 3 C2 domains. The N-terminal segment is more similar to rat muncl 3-1 and the C-terminal segment is more similar to rat muncl 3-2 which contains 1 C1 and 2 C2 domains. After further analysis of the Hmuncl 3 nucleotide sequence, we found that another AUG codon (residue 444-446) after the first C2 domain contains an optimal Kozak sequence (5'-CACCAUGG-3') (27). It is possible that 11 WO 99/31134 PCT/CA98/01061 Hmuncl3 mRNA serves as a bifunctional mRNA (27) that encodes two open reading frames, one for an isoform with 3 C2 domains (muncl3-1) and the other with only 2 C2 domains (muncl3-2). We discovered that, in addition to Cl and C2 domains (fig.5), a segment of 5 Hmuncl3 (aa 309-371) not present in rat muncl3s, has similarity to a segment of the delta isoform of the B' subunit of protein phosphatase 2Ao - a serine threonine phosphatase (28). This B' subunit has been shown to be a regulatory subunit of the multimeric PP2Ao. The catalytic subunit of PP2Ao associates with specific proteins (B') that serve a targeting and regulatory function. It is the regulatory subunits that determine 10 in vivo specificity of the phosphatase by targeting the enzyme to the subcellular location of their substrates, and also modulating phosphatase activity by reversible protein phosphorylation and binding of second messengers (29). We have also identified two RGD binding domains at aa39-41 and 769-771 in Hmuncl 3. The presence of these motifs indicates that Hmuncl 3 interacts with ECM 15 element receptors-integrins, such as vitronectin recetpor aVf and fibronectin receptor as3. Such interaction is important for cell survival. Over-expression of Hmuncl3, in response to DAG prevents engagement of integrins to ECM resulting in apoptosis. Taken together, the structural features of Hmuncl13 described above, show a multifunctional role that involves transmembrane ECM-cell signaling, as well as DAG and 20 Ca"+ activated phosphatase activity. Our finding that MC Hmuncl 3 is regulated by glucose also indicates that it modulates renal cell responses to hyperglycemia either directly or through interaction with PKC. We have also confirmed that Hmunc 13 is upregulated in the streptozotocin treated diabetic rat compared to normal rats (Fig. 6). Thus Hmuncl 3 is implicated in the 25 pathogenesis of diabetic nephropathy. Biologically Functional Equivalent Nucleotide Sequences The invention also includes nucleotide sequences that are biologically functional equivalents of all or part of the sequence in Figure 8. Biologically functional equivalent nucleotide sequences are DNA and RNA (such as genomic DNA, cDNA, synthetic DNA, 30 and mRNA nucleotide sequences), that encode peptides, polypeptides, and proteins having the same or similar Hmuncl3 activity as all or part of the Hmuncl3 protein shown in Figure 1. Biologically functional equivalent nucleotide sequences can encode peptides, polypeptides, and proteins that contain a region having sequence identity to a region of a Hmuncl3 protein or more preferably to the entire Hmunc 13 protein. 12 WO 99/31134 PCT/CA98/01061 Identity is calculated according to methods known in the art. The Gap program, described below, is most preferred. For example, if a nucleotide sequence (called "Sequence A") has 90% identity to a portion of the nucleotide sequence in Figure 8, then Sequence A will be identical to the referenced portion of the nucleotide sequence in 5 Figure 8, except that Sequence A may include up to 10 point mutations, such as deletions or substitutions with other nucleotides, per each 100 amino acids of the referenced portion of the nucleotide sequence in Figure 8. Nucleotide sequences biologically functional equivalent to the Hmuncl 3 sequences can occur in a variety of forms as described below. 10 A) Nucleotide sequences Encoding Conservative Amino Acid Changes in Hmuncl 3 Protein The invention includes biologically functional equivalent nucleotide sequences that encode conservative amino acid changes within a Hmuncl3 amino acid sequence and produce silent amino acid changes in Hmuncl 3. 15 B) Nucleotide Sequences Encoding Non-Conservative Amino Acid Substitutions, Additions or Deletions in Hmuncl3 Protein The invention includes biologically functional equivalent nucleotide sequence that made non conservative amino acid changes within the Hmunc 13 amino acid sequence to the sequences in Figure 8. Biologically functional equivalent nucleotide sequences are 20 DNA and RNA that encode peptides, polypeptides, and proteins having non-conservative amino acid substitutions (preferably substitution of a chemically similar amino acid), additions, or deletions but which also retain the same or similar Hmuncl 3 activity as all or part of the Hmuncl3 protein shown in Figure 1 or disclosed in the application. The DNA or RNA can encode fragments or variants of the Hmuncl 3 of the invention. The 25 Hmuncl 3 or Hmuncl 3 -like activity of such fragments and variants is identified by assays as described above. Fragments and variants of Hmuncl13 encompassed by the present invention should preferably have at least about 40%, 60%, 80% or 95% sequence identity or preferably at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to the naturally occurring nucleotide sequence, or corresponding 30 region. Most preferably, the fragments have at least 99.5% sequence identity to the naturally occurring nucleotide sequence, or corresponding region. Sequence identity (also known as homology) is preferably measured with the Gap program. Nucleotide sequences biologically functionally equivalent to the Hmuncl3 in Figure 8 include: 13 WO 99/31134 PCT/CA98/01061 (1) Altered DNA. For example, the sequence shown in Figure 8 may have its length altered by natural or artificial mutations such as partial nucleotide insertion or deletion, so that when the entire length of the coding sequence within Figure 8, is taken as 100%, the biologically functional equivalent nucleotide sequence preferably has a length of about 5 60-120% thereof, more preferably about 80-110% thereof. Fragments may be less than 60%.; or (2) Nucleotide sequences containing partial (usually 80% or less, preferably 60% or less, more preferably 40% or less of the entire length) natural or artificial mutations so that some codons in these sequences code for different amino acids, but wherein the resulting 10 protein retains the same or similar Hmuncl 3 activity as that of a naturally occurring Hmuncl 3 protein. The mutated DNAs created in this manner should preferably encode a protein having at least about 40%, preferably at least about 60%, at least about 80%, and more preferably at least about 90% or 95%, and most preferably 97%, 98% or 99% sequence identity (homology) to the amino acid sequence of the Hmuncl 3 protein in 15 Figure 1. Sequence identity can preferably be assessed by the Gap program. C) Genetically Degenerate Nucleotide Sequences Since the genetic code is degenerate, those skilled in the art will recognize that the nucleic acid sequence in Figure 8 is not the only sequences which may code for a protein having Hmuncl 3 activity. This invention includes nucleic acid sequences that 20 have the same essential genetic information as the nucleotide sequence described in Figure 8. Nucleotide sequences (including RNA) having one or more nucleic acid changes compared to the sequences described in this application and which result in production of a polypeptide shown in Sequence (a) in Figure 1 are within the scope of the invention. 25 D) Biologically Functional Equivalent Nucleic Acid Sequences Detected by Hybridization Other biologically functional equivalent forms of Hmuncl 3 -encoding nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA hybridization techniques. Thus, the present invention also includes nucleotide sequences that hybridize to one or more of the sequences in Figure 8 or its complementary sequence, and that encode 30 expression for peptides, polypeptides, and proteins exhibiting the same or similar activity as that of the Hmuncl 3 protein produced by the DNA in Figure 8 or its variants. Such nucleotide sequences preferably hybridize to one or more of the sequences in Figure 8 under moderate to high stringency conditions (see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 14 WO 99/31134 PCT/CA98/01061 Cold Spring Harbor, N.Y.). Preferable hybridization conditions are high stringency, such as 42 0 C for a 20- to 30-mer oligonucleotide, 65 0 C for a 200-500 bp DNA probe or 70 0 C for a 200-400 bp cRNA probe. The present invention also encompasses nucleotide sequences that hybridize to 5 genomic DNA, cDNA, or synthetic DNA molecules that encode the amino acid sequence of the Hmuncl 3 protein, or genetically degenerate forms thereof due to the degeneracy of the genetic code, under salt and temperature conditions equivalent to those described in this application, and that code on expression for a peptide, polypeptide, or protein that has the same or similar activity as that of the Hmuncl 3 protein. 10 A nucleotide sequence described above is considered to possess a biological function substantially equivalent to that of the Hmuncl 3 genes of the present invention if the protein produced by the nucleotide sequence displays the following characteristics (i) DAG activated transloaction of the protein in vivo from the cytosol to Golgi (as measured by immunocytochemistry, described in the Materials and Methods section), and (ii) the 15 protein activates apoptosis (if the protein is expressed in vivo, the protein's expression is preferably induced by DAG). Production of Hmuncl3 in Eukaryotic and Prokaryotic Cells The nucleotide sequences (also referred to as a DNA sequence or a nucleic acid molecule; these terms include either a full gene or a gene fragment.. It will be clear to a 20 person skilled in the art whether it is appropriate to use a nucleotide fragment that includes all or a fragment of a gene when practicing the invention) of the invention may be obtained from a cDNA library. The nucleotide molecules can also be obtained from other sources known in the art such as expressed sequence tag analysis or in vitro synthesis. The DNA described in this application (including variants that are biologically 25 functional equivalents) can be introduced into and expressed in a variety of eukaryotic and prokaryotic host cells. A recombinant nucleotide sequence for the Hmuncl 3 contains suitable operatively linked transcriptional or translational regulatory elements. Suitable regulatory elements are derived from a variety of sources, and they may be readily selected by one with ordinary skill in the art (Sambrook, J, Fritsch, E.E. & Maniatis, T. 30 (1989). Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press. New York; Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, if one were to upregulate the expression of the gene, one could insert the sense sequence and the appropriate promoter into the vector. Promoters can be inducible or constitutive, environmentally - or developmentally-regulated, or cell - or 15 WO 99/31134 PCT/CA98/01061 tissue-specific. Transcription is enhanced with promoters known in the art such as CMV, RSV and SV40. If one were to downregulate the expression of the gene, one could insert the antisense sequence and the appropriate promoter into the vehicle. The nucleotide 5 sequence may be either isolated from a native source (in sense or antisense orientations), synthesized, or it may be a mutated native or synthetic sequence or a combination of these. Examples of regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation 10 initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. Other regulatory regions that may be used include an enhancer domain and a termination region. The regulatory elements may be from animal, plant, yeast, bacterial, fungal, viral, avian, insect or other sources, including synthetically produced elements and mutated 15 elements. In addition to using the expression vectors described above, the polypeptide may be expressed by inserting a recombinant nucleotide sequence in a known expression system derived from bacteria, viruses, yeast, mammals, insects, fungi or birds. The recombinant molecule may be introduced into the cells by techniques such as Agrobacterium 20 tumefaciens-mediated transformation, particle-bombardment-mediated transformation, direct uptake, microinjection, coprecipitation, transfection and electroporation depending on the cell type. Retroviral vectors, adenoviral vectors, DNA virus vectors and liposomes may be used. Suitable constructs are inserted in an expression vector, which may also include markers for selection of transformed cells. The construct may be inserted at a site 25 created by restriction enzymes. In one embodiment of the invention, a cell is transfected with a nucleotide sequence of the invention inserted in an expression vector to produce cells expressing the nucleotide sequence. Another embodiment of the invention relates to a method of transfecting a cell with 30 a nucleotide sequence of the invention, inserted in an expression vector to produce a cell expressing the Hmuncl 3 protein. The invention also relates to a method of expressing the polypeptides of the invention in a cell. 16 WO 99/31134 PCT/CA98/01061 Probes The invention also includes oligonucleotide probes made from the cloned Hmuncl 3 nucleotide sequences described in this application or other nucleotide sequences of the invention. The probes may be 15 to 30 nucleotides in length and are 5 preferably at least 30 or more nucleotides. A preferred probe is 5' CCTCTCCATTGTGTTCATCACCAC-3' or at least 15 nucleotides of this sequence. The invention also includes at least 30 consecutive nucleotides of Hmuncl13 in Figure 8. The probes are useful to identify nucleic acids encoding Hmuncl 3 peptides, polypeptides and proteins other than those described in the application, as well as peptides, polypeptides, 10 and proteins biologically functionally equivalent to Hmuncl 3. The oligonucleotide probes are capable of hybridizing to one or more of the sequences shown in Figure 8 or the other sequences of the invention under stringent hybridization conditions. A nucleotide sequence encoding a polypeptide of the invention may be isolated from other organisms by screening a library under moderate to high stringency hybridisation conditions with a 15 labeled probe. The activity of the polypeptide encoded by the nucleotide sequence is assessed by cloning and expression of the DNA. After the expression product is isolated the polypeptide is assayed for Hmuncl 3 activity as described in this application. Biologically functional equivalent Hmuncl 3 nucleotide sequences from other cells, or equivalent Hmuncl 3 -encoding cDNAs or synthetic DNAs, can also be isolated by 20 amplification using Polymerase Chain Reaction (PCR) methods. Oligonucleotide primers, including degenerate primers, based on the amino acid sequence of the sequences in Figures 8 can be prepared and used in conjunction with PCR technology employing reverse transcriptase (E. S. Kawasaki (1990), In Innis et al., Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21) to amplify biologically functional equivalent 25 DNAs from genomic or cDNA libraries of other organisms. Alternatively, the oligonucleotides, including degenerate nucleotides, can be used as probes to screen cDNA libraries. Biologically Functionally Equivalent Peptides, Polypeptides, and Proteins The present invention includes not only the polypeptides encoded by sequences 30 presented in this application, but also "biologically functional equivalent peptides, polypeptides and proteins" that exhibit the same or similar Hmuncl 3 protein activity as proteins described in this application. The phrase "biologically functional equivalent peptides, polypeptides, and proteins" denotes peptides, polypeptides, and proteins that exhibit the same or similar Hmunc 13 protein activity when assayed. Where only one or 17 WO 99/31134 PCT/CA98/01061 two of the terms peptides, polypeptides and proteins is referred to below, it will be clear to one skilled in the art whether the other types of amino acid sequence also would be useful. By "the same or similar Hmuncl3 protein activity" is meant the ability to perform the same or similar function as the protein produced by Hmuncl 3. These peptides, 5 polypeptides, and proteins can contain a region or moiety exhibiting sequence identity (homology) to a corresponding region or moiety of the Hmuncl 3 protein described in the application, but this is not required as long as they exhibit the same or similar Hmuncl 3 activity. Identity refers to the similarity of two polypeptides or proteins (or nucleotide sequences) that are aligned so that the highest order match is obtained. Identity is 10 calculated according to methods known in the art, such as the Gap program, described below. For example, if a polypeptide (called "Sequence A") has 90% identity to a portion of the polypeptide in sequence (a) in Figure 1, then Sequence A will be identical to the referenced portion of the polypeptide in sequence (a) in Figure 1, except that Sequence A may include up to 10 point mutations, such as deletions or substitutions with other amino 15 acids, per each 100 amino acids of the referenced portion of the polypeptide in sequence (a) in Figure 1. Peptides, polypeptides, and proteins biologically functional equivalent to the Hmuncl 3 proteins can occur in a variety of forms as described below. A) Conservative Amino Acid Changes in Hmuncl3 Sequences Peptides, polypeptides, and proteins biologically functionally equivalent to 20 Hmuncl3 protein include amino acid sequences containing amino acid changes in the Hmuncl3 sequence. The biologically functional equivalent peptides, polypeptides, and proteins have at least about 40% sequence identity (homology), preferably at least about 60%, at least about 75%, at least about 80%, at least about 90% or at least about 95% sequence identity, to the naturally occurring polypeptide, or corresponding region. Most 25 preferably, the biologically functional equivalent peptides, polypeptides, and proteins have at least 97%, 98% or 99% sequence identity to the naturally occurring protein, or corresponding region or moiety. "Sequence identity" is preferably determined by the Gap program. The algorithm of Needleman and Wunsch (1970 J Mol. Biol. 48:443-453) is used in the Gap program. BestFit is also used to measure sequence identity. It aligns 30 the best segment of similarity between two sequences. Alignments are made using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. B) Fragments and Variants of Hmuncl3 Proteins The invention includes peptides, polypeptides or proteins which retain the same or similar activity as all or part of Hmuncl 3. Such peptides preferably consist of at least 5 35 amino acids. In preferred embodiments, they may consist of 6 to 10, 11 to 15, 16 to 25 or 18 WO 99/31134 PCT/CA98/01061 26 to 50, 50 to 150, 150 to 250, 250 to 500 or 500 to 750 amino acids of the Hmuncl 3. Fragments of the Hmuncl13 protein can be created by deleting one or more amino acids from the N-terminus, C-terminus or an internal region of the protein (or combinations of these), so long as such fragments retain the same or similar Hmuncl3 activity as all or 5 part of the Hmuncl 3 protein disclosed in the application. These fragments can be natural mutants of the Hmuncl 3, or can be produced by restriction nuclease treatment of an encoding nucleotide sequence. Fragments of the polypeptide may be used in an assay to identify compounds that bind the polypeptide. Methods known in the art may be used to identify agonists and antagonists of the fragments. 10 Variants of the Hmuncl3 protein may also be created by splicing. Variants can also be naturally occurring mutants of the Hmuncl13 disclosed in the application. A combination of techniques known in the art may be used to substitute, delete or add amino acids. For example, a hydrophobic residue such as methionine can be substituted for another hydrophobic residue such as alanine. An alanine residue may be substituted 15 with a more hydrophobic residue such as leucine, valine or isoleucine. An aromatic residue such as phenylalanine may be substituted for tyrosine. An acidic, negatively charged amino acid such as aspartic acid may be substituted for glutamic acid. A positively charged amino acid such as lysine may be substituted for another positively charged amino acid such as arginine. Modifications of the proteins of the invention may 20 also be made by treating a polypeptide of the invention with an agent that chemically alters a side group, for example, by converting a hydrogen group to another group such as a hydroxy or amino group. Peptides having one or more D-amino acids are contemplated within the invention. Also contemplated are peptides where one or more amino acids are acetylated 25 at the N-terminus. Those skilled in the art recognize that a variety of techniques are available for constructing peptide mimetics (i.e. a modified peptide or polypeptide or protein) with the same or similar desired biological activity as the corresponding protein of the invention but with more favorable activity than the protein with respect to characteristics such as solubility, stability, and/or susceptibility to hydrolysis and 30 proteolysis. See for example, Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989). The invention also includes hybrid genes and peptides, for example where a nucleotide sequence from the gene of the invention is combined with another nucleotide sequence to produce a fusion peptide. For example a nucleotide domain from a molecule 35 of interest may be ligated to all or part of a Hmuncl3 nucleotide sequence encoding 19 WO 99/31134 PCT/CA98/01061 Hmuncl3 protein described in this application. Fusion genes and peptides can also be chemically synthesized or produced using other known techniques. The variants preferably retain the same or similar Hmuncl 3 activity as the naturally occurring Hmuncl13 of the invention. The Hmuncl13 activity of such variants can 5 be assayed by techniques described in this application and known in the art of TUNEL and DNA fragmentation assay. Variants produced by combinations of the techniques described above but which retain the same or similar Hmuncl 3 activity as naturally occurring Hmuncl 3 are also included in the invention (for example, combinations of amino acid additions, deletions, 10 and substitutions). Fragments and variants of Hmuncl3 encompassed by the present invention preferably have at least about 40% sequence identity, preferably at least about 60%, at least about 75%, at least about 80%, at least about 90% or at least about 95% sequence identity, to the naturally occurring protein, or corresponding region or moiety. Most 15 preferably, the fragments have at least 97%, 98% or 99% sequence identity to the naturally occurring polypeptide, or corresponding region. Sequence identity is preferably measured with either the Gap or BestFit programs. The invention also includes fragments of the polypeptides of the invention which do not retain the same or similar activity as the polypeptides but which can be used as a 20 research tool to characterize the polypeptides of the invention. Enhancement of Hmuncl3 protein activity The activity of the Hmuncl 3 protein is increased by carrying out selective site directed mutagenesis. Using protein modelling and other prediction methods, we characterize the binding domain and other critical amino acid residues in the protein that 25 are candidates for mutation, insertion and/or deletion. A DNA plasmid or expression vector containing the Hmuncl 3 gene or a nucleotide sequence having sequence identity is preferably used for these studies using the U.S.E. (Unique site elimination) mutagenesis kit from Pharmacia Biotech or other similar mutagenesis kits that are commercially available. Once the mutation is carried out and confirmed by DNA 30 sequence analysis, the mutant protein is expressed using an expression system and its activity is monitored. This approach is useful not only to enhance activity, but also to engineer some functional domains for other properties useful in the purification or application of the proteins or the addition of other biological functions. It is also possible to synthesize a DNA fragment based on the sequence of the proteins that encodes 20 WO 99/31134 PCT/CA98/01061 smaller proteins that retain activity and are easier to express. It is also possible to modify the expression of the cDNA so that it is induced under environmental conditions other than hyperglycemia or in response to different chemical inducers or hormones. It is also possible to modify the DNA sequence so that the protein is targeted to a different 5 location. All these modifications of the DNA sequences presented in this application and the proteins produced by the modified sequences are encompassed by the present invention. Pharmaceutical Compositions Hmuncl3 or its protein and biologically functional equivalent nucleotide 10 sequences or proteins are also useful when combined with a carrier in a pharmaceutical composition. Suitable examples of vectors for Hmuncl 3 are described above. The compositions are useful when administered in methods of medical treatment of a disease, disorder or abnormal physical state characterized by insufficient Hmuncl 3 expression or inadequate levels or activity of Hmuncl3 protein. The invention also includes methods of 15 medical treatment of a disease, disorder or abnormal physical state characterized by excessive Hmuncl 3 expression or levels of activity of Hmuncl 3 protein, for example by administering a pharmaceutical composition comprising including a carrier and a vector that expresses Hmuncl 3 antisense DNA. The pharmaceutical compositions of this invention used to treat patients having 20 degenerative diseases, disorders or abnormal physical states of tissue such as renal and vascular tissue. There is evidence that apoptosis plays a role in renal diseases related to (1) glomerular inflammation (2) tubular ischemia, toxins and ureteric obstruction (E.G. Neilson and W.G. Couser, Immunologic Renal Disease, (1997, 309-329), 8), could include an acceptable carrier, auxiliary or excipient. In some diseases, apoptosis is protective. In 25 other cases, apoptosis may contribute to cell injury. Regulation of apoptosis plays a critical role in many different renal disease states including both glomerular and tubulointerstitial types of injury. The conditions which may be treated by the compositions include microvascular and renal complications of diabetes and disorders in which renal apoptosis plays a role. 30 The pharmaceutical compositions can be administered to humans or animals by methods such as aerosol administration, intratracheal instillation and intravenous injection. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. Nucleotide sequences and proteins may be introduced into cells using in vivo delivery vehicles such as liposomes. They may also be introduced into 21 WO 99/31134 PCT/CA98/01061 these cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to 5 patients, and such that an effective quantity of the nucleotide sequence or protein is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA). On this basis, the pharmaceutical compositions could include an active compound or 10 substance, such as a Hmuncl 3 gene or protein, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The methods of combining the active molecules with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the 15 active compound to specified sites within tissue. Heterologous overexpression of Hmuncl 3 as a Research Tool Expression vectors are useful to provide high levels of protein expression. Cell cultures transformed with the nucleotide sequences of the invention are useful as research tools. Cell cultures are used in overexpression and research according to 20 numerous techniques known in the art. A cell line (either an immortalized cell culture or a primary cell culture) may be transfected with a vector containing a Hmuncl 3 nucleotide sequence (or variants) to measure levels of expression of the nucleotide sequence and the activity of the nucleotide sequence. A polypeptide of the invention may be used in an assay to identify compounds that bind the polypeptide. Methods known in the art may be 25 used to identify agonists and antagonists of the polypeptides. One may obtain cells that do not express Hmuncl 3 and use them in experiments to assess Hmuncl 3 gene expression. Experimental groups of cells may be transfected with vectors containing different types of Hmuncl 3 genes (or genes similar to Hmuncl 3 or fragments of Hmuncl 3 gene) to assess the levels of protein produced, its functionality and the 30 phenotype of the cells produced. The polypeptides are also useful for in vitro analysis of Hmuncl3 activity. For example, the protein produced can be used for microscopy or X ray crystallography studies. Other expression systems can also be utilized to overexpress the Hmuncl3 in recombinant systems. 22 WO 99/31134 PCT/CA98/01061 Hmuncl 3 is a useful research tool. For example, in one embodiment, Hmuncl 3 cDNA is expressed after it is inserted in a mammalian cell expression plasmid (pCMVSPORT, Gibco BRL). In a variation, Hmuncl3 cDNA is inserted in an inducible mammalian cell expression plasmid (plND, Invitrogen). Hmuncl3 cDNA may also be 5 positioned in reverse orientation in plND as a negative control. One can also use N terminal c-myc tag and C-terminal HA tag Hmuncl3 in plND and pCMVSPORT. In a preferred embodiment, stable tansfected mouse mesangial, NIH 3T3, MDCK, HEK 293 and OK cell lines are created with an inducible Hmuncl3 plasmid. Gene Therapy 10 Since it is possible that certain diabetics may be protected from development of renal complications by either up or down regulation of Hmuncl3, gene therapy to replace or delete Hmuncl 3 expression could also be used to modify the development/progression of diabetic renal and vascular complications. In addition, the use of anti-sense DNA that inhibits the expression of hmuncl 3 will allow treatment of diabetic nephropathy in 15 humans. The invention also includes methods and compositions for providing gene therapy for treatment of diseases, disorders or abnormal physical states characterized by insufficient Hmuncl3 expression or inadequate levels or activity of Hmuncl3 protein (see the discussion of phamaceutical discussions, above). The invention also includes 20 methods and compositions for providing gene therapy for treatment of diseases, disorders or abnormal physical states characterized by excessive Hmuncl13 expression or levels of activity of Hmuncl 3 protein The invention includes methods and compositions for providing a nucleotide sequence encoding Hmuncl3 or biologically functional equivalent nucleotide sequence to 25 the cells of an individual such that expression of Hmuncl3 in the cells provides the biological activity or phenotype of Hmuncl3 protein to those cells. Sufficient amounts of the nucleotide sequence are administered and expressed at sufficient levels to provide the biological activity or phenotype of Hmuncl3 protein to the cells. For example, the method can preferably involve a method of delivering a gene encoding Hmuncl 3 to the 30 cells of an individual having a disease, disorder or abnormal physical state, comprising administering to the individual a vector comprising DNA encoding Hmuncl3. The method may also relate to a method for providing an individual with a disease, disorder or abnormal physical state with biologically active Hmuncl 3 protein by administering DNA encoding Hmuncl 3. The method may be performed ex vivo or in vivo. Gene therapy 35 methods and compositions are explained, for example, U.S. Patent Nos. 5,672,344, 23 WO 99/31134 PCT/CA98/01061 5,645,829, 5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346 and 5,670,488, 5,240,846. The method may also relate to a method for producing a stock of recombinant virus by producing virus suitable for gene therapy comprising DNA encoding Hmuncl3. 5 This method preferably involves transfecting cells permissive for virus replication (the virus containing Hmuncl2) and collecting the virus produced. The invention also includes methods and compositions for providing a nucleotide sequence encoding an antisense sequence to Hmuncl3 to the cells of an individual such that expression of the antisense sequence prevents Hmuncl 3 biological activity or 10 phenotype. The methods and compositions can be used in vivo or in vitro. Sufficient amounts of the nucleotide sequence are administered and expressed at sufficient levels to prevent the biological activity or phenotype of Hmuncl3 protein to the cells. Similar methods as described in the preceding paragraph may be used with appropriate modifications. 15 The methods and compositions can be used in vivo or in vitro. The evidence for in vitro usefulness is downregulation of Hmuncl 3 in hyperglycemia conditions can inhibit hyperglycemia induced renal cell injury. The invention also includes compositions (preferably pharmaceutical compositions for gene therapy). The compositions include a vector containing Hmuncl3 or a 20 biologically functional equivalent molecule or antisense DNA. The carrier may be a pharmaceutical carrier or a host cell transformant including the vector. Vectors known in the art are adenovirus and herpesvirus vectors. The invention also includes packaging cell lines that produce the vector. Methods of producing the vector and methods of gene therapy using the vector are also included with the invention. 25 The invention also includes a transformed cell, such as an MC cell or other cell described in this application, containing the vector and recombinant Hmuncl 3 nucleotide sequence or a biologically functional equivalent molecule. Preparation of Antibodies The Hmuncl 3 protein is also useful as an antigen for the preparation of antibodies 30 that can be used to purify or detect other muncl 3 or muncl 3-like proteins. Monoclonal and polyclonal antibodies are prepared according to other techniques known in the art. For examples of methods of the preparation and uses of monoclonal antibodies, see U.S. Patent Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705. Examples of the preparation 24 WO 99/31134 PCT/CA98/01061 and uses of polyclonal antibodies are disclosed in U.S. Patent Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147. Antibodies recognizing Hmuncl3 can be employed to screen organisms containing Hmuncl3 protein or Hmuncl 3-like proteins. The antibodies are also valuable for immuno-purification of Hmuncl3 or Hmuncl 3-like proteins from crude 5 extracts. We prepare two peptide specific polyclonal antibodies against a C-terminal segment (preferably all or part of NH2-SQRSNDEVREFVKL-COOH) and an N-terminal segment (preferably all or part of NH2-TIRQSDEEGPGEW-COOH) of Hmuncl3 which has ability to detect rat muncl13-1, 13-2 and 13-3. 10 Screening for Agonists and Antagonists of Hmuncl3 and Inhibitors of Hmuncl3 Protein As described above, munc13 is useful in a pharmaceutical preparation to treat diabetes or its complications. Hmuncl3 is also useful as a target. Chemical libraries are used to identify pharmacophores which can specifically interact with Hmuncl 3 either in 15 an inhibitory or stimulatory mode. The Hmuncl 3 targets that would be used in drug design include - e.g. the DAG binding site or some other functional domain specific to Hmuncl 3. Modulation of Hmuncl 3 expression is commercially useful for identification and development of drugs to inhibit and/or enhance Hmuncl 3 function directly. Such drugs 20 would be targeted to any of the following sites: the DAG, Ca*, phosphatase and RGD domains. The invention also includes methods of screening a test compound to determine whether it antagonizes or agonizes Hmuncl 3 protein expression. For example, one method involves testing whether a compound inhibits the translocation of Hmuncl 3 from 25 cytosol to Golgi as well as its apoptotic effect. The invention also includes methods of screening a test compound to determine whether it induces or inhibits Hmuncl 3 expression. For example, one method involves testing whether a compound inhibits the promoter activity of Hmuncl3. Expression of Hmuncl3 30 Hmuncl3 is expressed in MC, human cortical epithelial cells and cells from testis, ovaries, prostate gland, colon, brain and heart.. Experiments to determine where the gene is expressed were done with RT-PCR. The function of Hmuncl13 in other cells will be similar to that in renal epithelial cells such as in translocation and apoptosis 25 WO 99/31134 PCT/CA98/01061 Hmuncl3 has a C1 domain. A region of the Cl domain from C. elegans unc-13 binds to phorbol esters and DAG similar to PKC (21). We noted that the Cl domain is similar among C. elegans uncl3, rat muncl3s and Hmuncl3 (Fig. 1), so the Cl domain in the Hmuncl3 can also bind phorbol esters. Hmuncl3 is also involved in cell signaling 5 in response to DAG binding. Regulation of Hmuncl3 in the Kidney We found that expression of Hmuncl3 in cultured MC was up-regulated by high glucose treatment (25 mM D-glucose). Even 15 mM D-glucose is enough to stimulate the over expression of Hmuncl13 as revealed by Northern blot. There are reports indicated 10 that hyperglycemia increases PKC activity in MC (13, 14, 31). Furthermore, DAG levels are increased when cultured MC are exposed to hyperglycemia (17, 13). Since Hmuncl3 and PKC share similar binding capacities for phorbol esters and DAG and both PKC contain C2 domains, Hmuncl3 is part of an alternative cascade following DAG binding. Thus Hmuncl 3 is activated in response to hyperglycemic induced increases in DAG. 15 Even though Hmuncl 3 does not contain a kinase domain and cannot therefore serve as a downstream regulator by protein phosphorylation (20, 30), nevertheless it is possible that Hmuncl3 modulates intracellular events through competitive binding of PKC or by regulation of vesicle trafficking and exocytosis. Subcellular Localization of Hmuncl3 in vitro 20 Expression of epitope-tagged hmuncl 3 in OK cells show that Hmuncl 3 has a cytoplasmic distribution under basal conditions, but with PDBu stimulation, Hmuncl3 is translocated to the Golgi apparatus. This effect is unlikely to have taken place through activation of endogenous PKC, since the deletion mutant, C1 less mutant (without the DAG binding domain), showed no translocation. In a recent study reported by Betz et al. 25 (24), muncl 3-1 was localized to the presynaptic region in rat brain by immunocytochemistry. In transfected HEK 293 cells, green fluorescent protein tagged muncl 3-1, -2 and -3 are all translocated to plasma membrane following phorbol ester stimulation. The fact that hmuncl 3 is translocated to the Golgi apparatus in response to 30 phorbol ester activation compared to translocation of muncl 3-1, -2 and -3 to the plasma membrane is proves that Hmuncl 3 is a unique isoform of muncl 3s. This brings up the relationship of the DAG activated signaling pathways of muncl 3s and PKC. The multiplicity of PKC isoforms and the tissue specificity of PKC functional expression are well known (32). The muncl 3 pathway is also composed of tissue specific functionally 26 WO 99/31134 PCT/CA98/01061 different isoforms. However, unlike PKC, the muncl 3 proteins have no kinase domain (20, 33). The Golgi apparatus is involved in vesicular traffic. A number of SNARE proteins, such as yeast Sed5p (34) and mVps45 (35), mammalian syntaxin 6 (36), VAMP4, 5 Syntaxin 13 and mVtib (36), have all been reported to be localized to the Golgi. Rat muncl3-1 has been shown to interact with a number of proteins involved in vesicle docking and trafficking, such as syntaxin (24) and Doc2 (37). Interaction of muncl3-1 and Doc2 was stimulated by DAG and has been suggested to be involved in Ca 2 + dependent exocytosis (37). The finding in the present study that translocation of 10 Hmuncl3 to the Golgi after DAG stimulation is another indication that Hmuncl3 is a protein that participates in DAG regulated vesicle trafficking and exocytosis. Further studies are required to investigate if Hmuncl 3 interacts with other Golgi localized SNARE proteins or whether some SNARE proteins co-translocate to the Golgi with Hmuncl 3 after DAG stimulation. It has also been suggested that PKC plays a role in Golgi budding (for 15 review see 38). For example, a study in S. Cerevisiae implicated DAG as playing an important role in the formation of Golgi budding involving Secl4 (39). Since Hmuncl3 translocates to the Golgi after DAG stimulation, it would also be of interest to determine the role of Hmuncl3 is involved in Golgi budding and interaction with Secl4L, the partial mammalian homologue of yeast Secl4 (40). 20 Role of Hmuncl3 in Apoptosis We investigated the localization of Hmuncl 3 to determine whether exposure to phorbol esters had any effect on its intracellular translocation. In the course of carrying out these studies, we observed that cells transfected with Hmuncl 3 became rounded up and died following treatment with phorbol 12, 13-dibutyrate (PDBu), a phorbol ester 25 analogue. We examined the mechanism of phorbol ester induced cell death in the transfected cells. We showed that exposure to phorbol ester causes apoptosis through activation of Hmuncl 3. This shows the interaction between the diabetic state, activation of Hmuncl3 and cell damage. The induction of apoptosis in Hmuncl 3 transfected cells after PDBu stimulation 30 was unexpected. This effect is unlikely to have occurred through other DAG activated pathways since the C1 less mutant transfected cells were not apoptotic after PDBu treatment. PDBu is a reagent known to be a tumor promoter capable of stimulating cell proliferation through PKC activation (41). Although the role of PKC in apoptosis is not consistent in the literature (42, 43), the bulk of evidence shows that PKC, especially 35 PKCc, activated by phorbol esters such as PMA and PDBu, inhibits apoptosis (41-44). 27 WO 99/31134 PCT/CA98/01061 There is also a body of evidence suggesting that, in the case of PKC induced apoptosis, down-regulation rather than DAG activation of PKC is responsible for this effect (43, 45). Hmuncl3 Participates in a Signaling Pathway and Counterbalances DAG Activated PKC 5 Considering the functional characteristics of Hmuncl3 as and the known behavior of muncl 3-1, -2, and -3 in rat brain, we determined a model for the cellular activation of Hmuncl3 and PKC isoforms. Since both muncl3s and PKC have similar binding affinity to phorbol esters, our results showing that cells transfected with Hmuncl 3 become apoptotic after DAG treatment mean that Hmuncl3 participates in a signaling pathway 10 that serves to counterbalance DAG activated PKC. This concept is illustrated schematically in Figure 15. DAG acts as a secondary messenger to activate two alternate pathways - one pathway effected through PKC results in kinase activation and serine/threonine phospholylaton of downstream targets leading to cell proliferation while the other pathway effected through Hmuncl 3 induces apoptosis, preferably through 15 interaction involving vesicle trafficking. Pathogenesis of the Microvascular and Renal Complications of Diabetes. We have shown that in rat kidney, muncl3-1 and muncl3-2 are mainly localized to cortical tubular epithelial cells. Using both in situ hybridization and relative RT-PCR, we have also demonstrated that muncl 3-1 and muncl 3-2 are over-expressed in kidney 20 of STZ-treated diabetic rats. This result in rat kidney is consistent with our in vitro findings, showing that expression of Hmuncl 3 is up-regulated by high glucose treatment in cultured human mesangial cells. It has been reported that an increase in intracellular DAG levels is only detectable after 2 days of high glucose treatment (46). The fact that expression of both rat muncl3-1 and muncl3-2 is found to be increased after only 1 day 25 of hyperglycemia shows that over-expression of these genes is a consequence of hyperglycemia and not secondary to stimulation by DAG. Therefore, in diabetes, there are two mechanisms acting to increase activity of Hmuncl 3: (i) hyperglycemia itself, (ii) hyperglycemia-induced increase in cellular DAG (47-49). The over-expression of Hmuncl 3 is a major contributor to cell injury in diabetic nephropathy by inducing 30 apoptosis. In this regard, it is noteworthy that under hyperglycemic condition, renal tubular cells undergo apoptosis (50-51). Finally, since PKC inhibitors have been developed to treat diabetic nephropathy (49), a potential side effect of those inhibitors could result from overactivity of Hmuncl3. 28 WO 99/31134 PCT/CA98/01061 EXPERIMENTS Experiment 1 - DDRT-PCR DDRT-PCR carried out on RNA extracts from MC exposed to high vs. low glucose conditions yielded 10 bands which exhibited differences between high glucose treatment 5 and controls (both normal glucose and osmolarity controls) (data not shown). After the bands had been cut, reamplified, cloned and sequenced, the sequences were compared to the GenBank database. One of the cDNA sequences had identity to a segment (residues 3523- 3863) of rat muncl3-2 (20). Since rat muncl3-2 is viewed as having a potential signaling function particularly in neurotransmission and in addition has not 10 previously been reported in any tissue outside the brain, we elected to clone the full gene from human kidney and confirm the nature of its regulation by hyperglycemia. Experiment 2 -Cloning of Hmuncl3 As a first step we cloned a partial length cDNA from a commercial human kidney cDNA library using oligonucleotides derived from sequence information obtained from 15 DDRT-PCR comparing cells at 25 mM D-glucose vs. 5.5 mM D-glucose and osmolarity control (see Methods). Then, using the sequence of the partial length clone, we designed another oligonucleotide closer to the 5' end and proceeded to clone a full-length cDNA (6.3kb, pCMVSPORTHmuncl 3), which we have named Hmuncl3. This cDNA encodes a protein with a predicted molecular weight of 180.5 kDa. As shown in figure 1, kidney 20 Hmuncl3 contains 3 C2 domains and 1 C1 domain. The N-terminal segment of Hmuncl3 (residues 1-100) is similar to to rat muncl3-1 (Fig. lb). The next segment (residues 101-391) exhibits considerable variation in Hmuncl3 compared to rat muncl3s and unc-13 (7). The C-terminal segment of unc-13s is highly conserved among human, rat and C. elegans (Fig. 1, ref. 7). In particular, the protein segment from residue 392 to 25 1591 of Hmuncl3 is about 93% similar to rat muncl3-2 (residue 766-1985), 79% similar to muncl3-1 (residue 486-1735) and 74% similar to muncl3-3 (residue 1000-2207). In summary, the C terminus of renal Hmuncl 3 has strongest identity to rat muncl 3-2 whereas the N-terminal of Hmuncl 3 has strongest identity to rat muncl 3-1. Experiment 3 - Hyperglycemia Up-requlates Hmuncl3 mRNA Expression in Kidney MC 30 To confirm the differential expression of Hmuncl 3 under varying glucose concentrations two independent methods were employed. In a pilot study, by using ribonuclease protection assays, we have found that expression of Hmuncl 3 in human MC treated with 19.5 mM L-glucose + 5.5 mM D-glucose (osmolarity control) was not changed (data not shown). Therefore, in the following experiment, we only compared the 29 WO 99/31134 PCT/CA98/01061 difference of Hmunc-13 expression between high D-glucose and high L-glucose treated MC. We first used relative RT-PCR with 18S rRNA as a housekeeping gene. As shown in figure 2a, Hmuncl3 was up-regulated in the high-glucose (25mM) treated MC compared to osmolarity controls. In a more quantitative way, Northern blot analysis was 5 carried out on cells grown according to the same protocol. As revealed by relative RT PCR, Hmuncl3 expression was increased in MC after hyperglycemia (Fig. 2b). Quantitative desitometry analysis revealed 70% increase of Hmuncl 3 expression after exposure to 25 mM D-glucose treatment (p < 0.05, n = 5, student's t-test). As shown in figure 2b, Hmuncl3 expression in MC following exposure to 15 mM D-glucose was also 10 increased relative to osmolarity control but there was no statistically significant difference between 15 mM D-glucose and 25 mM D-glucose treated cells. Experiment 4 - Expression of Muncl 3 in Epithelial and Rat MC To show that muncl 3 is also expressed in other cell types in the kidney besides MC and that it is expressed in the rat MC as well as human, RT-PCR was performed 15 using a pair of primers specific for both Hmuncl 3 and rat muncl 3-2. As shown in figure 3, Hmuncl3 was detected in cultured human kidney cortical epithelial cells and muncl3-2 was also expressed in primary cultured rat MC. Genomic contamination is unlikely since no band was observed in the no RT control for the GAPDH housekeeping gene (Fig. 3). Experiment 5 - Hmuncl3 is Expressed as a 180 kDa Protein in vitro and is Membrane 20 Associated Using a cell free in vitro translation system, we have demonstrated that Hmuncl3 is expressed as a ~170 kDa protein (Fig. 4). This is close to the predicted MW (180.5 kDa) from the cDNA clone. A number of less prominent lower molecular weight bands is also present following in vitro translation because of either initiation of translation from 25 internal AUG codons rather than the first interaction site or a premature termination of translation. Also shown in figure 4 is that in the presence of canine pancreatic microsomal membranes, a proportion of Hmuncl3 protein is shifted to a higher molecular weight (~180 kDa) suggesting that it is membrane associated and undergoes co translational processing. Only the full-length protein is associated with the membrane 30 because the partial length in vitro translation products are not observed in the microsomal pellet (Fig. 4, lane 2). Experiment 6 - Translocation of Hmuncl 3 to Golqi apparatus after DAG treatment To study its cellular function, we elected to over-express Hmuncl 3 in opossum kidney (OK) cells, a cell line of renal epithelia origin and compare two constructs - an HA 30 WO 99/31134 PCT/CA98/01061 tagged Hmuncl3 and an HA tagged Hmuncl3 deletion mutant lacking the CI domain (Cl less mutant). Cells employed in the present study were grown on glass cover slips under growth arrested conditions with serum starvation. Transient transfection of OK cells was confirmed by Western blot analysis (Figure 10). As shown in Figure 10(i), an -180 kDa 5 protein was expressed in the Hmuncl 3-HA transfected cells and a -175 kDa protein was detected in the C1 less mutant transfected cells. No band was detected in cells transfected with empty plasmid, pCMVSPORT. Intracellular localization of Hmuncl13-HA in transfected OK cells was monitored by immunocytochemistry (ICC) using cells doubly labeled with anti-HA antibody (Fig. 10(ii), 10 upper panels) and wheat germ agglutinin (WGA) (Fig. 10(ii), lower panels). As indicated in Figure 10(ii), inspection of panel A reveals that Hmuncl3 exhibits a cytosolic distribution compared to the Golgi apparatus stained with WGA shown in Panel E. But after exposure to 0.1 gM PDBu, a DAG analogue, Hmuncl3 is translocated to the peri nuclear area (panel B) and co-localizes with WGA at the Golgi apparatus (compare 15 panels B and F). Translocation of Hmuncl 3 to the Golgi after PDBu treatment occurred in 15-30 min and became more obvious in 2-3 h. By contrast, when cells were transfected with the C1 less mutant, lacking a DAG binding domain, there was no translocation after PDBu treatment (refer to panels D and H) and Hmuncl 3 staining remained cytosolic. 20 When cells were treated with nocodazole, a drug that depolymerizes microtubules, (52), after PDBu treatment, the patterns of WGA and Hmuncl3 staining became identical and both revealed a dispersed Golgi pattern (compare panels C and E of Fig. 10 (ii)). Translocation of Hmuncl3 from cytosol to the Golgi apparatus after PDBu 25 treatment was also confirmed by immunoblot analysis of a Golgi membrane preparation, following subcellular fractionation. As shown in Figure 10 (iii), after PDBU treatment, Hmuncl3 is enriched in Golgi membranes compared to whole cell lysates.. Experiment 7 - Hmuncl 3 over-expressed cells are apoptotic after DAG treatment The PDBu induced translocation from cytosol to Golgi suggests that Hmuncl 3 has 30 functional implications. While attempting to study the effect of prolonged exposure to DAG activation on Hmuncl 3 transfected cells, we noticed that the cells rounded up and died. However, Hmuncl 3 transfected cells without PDBu treatment and cells transfected with the C1 less mutant, with or without PDBu treatment, were relatively healthy. This finding was somewhat unexpected since DAG has long been known as a carcinogen and 31 WO 99/31134 PCT/CA98/01061 a promoter of cell growth, and led us to investigate the possibility and conclude that treatment with phorbol ester is inducing apoptosis in cells transfected with Hmuncl 3. Using the TUNEL assay, we found that the number of apoptotic cells was significantly increased in hmuncl3 transfected OK cells after 8 h and 16 h of PDBu 5 treatment. These results are displayed in Figure 11 (i). The upper panels show the expression of Hmuncl3 in OK cells and the lower panels demonstrate the presence of fluorescein labeled TUNEL on the same cells. Inspection of panel F (8 h of PDBu treatment) and panel G (16 h of PDBU treatment) compared to panel E (treatment with vehicle control) reveals evidence of DAG induced increase in TUNEL staining cells. This 10 conclusion is further supported by the fact that cells transfected with the C1 less mutant, exhibit almost no labeling with TUNEL following exposure to PDBu for 16 h (compare panel H with panels F and G). The above results are also summarized in fiugure (ii). Finally, cells transfected with empty plasmid also showed almost no TUNEL labeling with or without PDBu treatment (data not shown). 15 To further confirm, a DNA fragmentation assay was employed. Further evidence of a breakdown in genomic DNA is revealed by the "laddering" pattern shown in Figure 12, obtained after 8 and 16 h of PDBu treatment in Hmuncl3 transfected cells. Experiment 8 - Expression of muncl3s in normal and STZ-treated diabetic rat kidney We have previously demonstrated that Hmuncl 3 is up-regulated by high glucose 20 treatment in cultured human mesangial (33). Since the main thrust of the present study was to investigate the functional role of Hmuncl 3, we documented its in vivo expression. Furthermore, confirmation of up-regulation of Hmuncl 3 by hyperglycemia in an in vivo state is necessary to show the role for this gene in diabetic nephropathy. We characterized Hmuncl3 expression in human kidney. We used an animal model of 25 diabetes- the STZ treated rat (the relevant isoforms being muncl 3-1, -2, and -3). As shown in Figure 13, muncl13-1 is expressed mainly in cortical tubular epithelial cells of both normal and STZ-treated diabetic rats. However, the expression level of muncl13-1 was higher in STZ-treated diabetic rat after 11 days of hyperglycemia. Expression of muncl 3-1 was significantly higher in certain glomerular cells of diabetic animals. But it is 30 impossible to identify these cells with any certainty at the resolution of confocal microscopy. However, because of our previous in vitro results (33), we determined that muncl3-1 is up-regulated in the mesangial cells. Increased expression level of muncl3-2 was also detected in diabetic rats with similar expression pattern as muncl 3-1. Possibly because of low basal expression, we could not obtain satisfactory in situ hybridization 35 data for muncl3-3 in rat kidney. 32 WO 99/31134 PCT/CA98/01061 To confirm the over-expression of muncl3-1 and muncl3-2 in diabetic rat kidney, we performed relative RT-PCR on renal cortical RNA preparation. Relative RT-PCR was chosen because low expression of muncl 3s in the rat kidney and a very low signal was detected in Northern blot analysis. As shown in Figure 14, compared to the 5 housekeeping gene, 18S ribosome RNA, expression of muncl13-1 is over-expressed in the renal cortex of the STZ-treated diabetic rat after only 1 day of hyperglycemia whereas expression of muncl13-2 is increased to a much lesser extent. Interestingly, muncl13-3 is down-regulated in the same animal model. We screen to detect a human homologue of rat muncl 3-3 in a commercial human kidney cDNA library (Gibco BRL) using PCR with 10 primers targeted to different regions of muncl 3-3. We determine the role of muncl 3-3 in diabetic nephropathy. MATERIALS AND METHODS MC basal culture medium (MsBM) and renal epithelial basal medium (REBM) were purchased from Clonetic, San Diego, CA. Fetal bovine serum (FBS), Dulbecco's 15 modified Eagle's medium (DMEM), penicillin, streptomycin, human kidney cDNA library, SuperScript II RNase H reverse transcriptase, dNTP, E.coli RNase H, Taq DNA polymerase, Genetrapper cDNA Positive Selection System, 100 bp DNA size markers, Klenow Fragment, m 7 G(5')ppp(5')G RNA capping analog, ElectroMAX DH10 OB cells and restriction enzymes were obtained from Gibco BRL, Burlington, ON, Canada. DNase I 20 and TSequence kit were purchased from Pharmacia Biotech, Uppsala, Sweden. TA cloning kit was from Invitrogen, San Diego, CA. RNeasy total RNA preparation kit, QIAshredder and QIAquick Gel Extraction kit were purchased from Qiagen, Chatsworth CA. SP6 RNA polymerase, human cyclophilin template, 18S rRNA primers and competimers were from Ambion, Austin, TX. Vent DNA polymerase was obtained from 25 New England Biolab, Inc, Beverly, MA. Rapid hybridization buffer and a -[ 32 P]-dATP (specific activity 800 Ci/mmol) were purchased from Amersham, Arlington Heights, IL.

[

35 S]-Methionine (specific activity, 1000 Ci/mmol) was from NEN Life Science Products, Boston, MA. Duralon-UV membranes was purchased from Stratagene, La Jolla, CA. Six percent denatured polyacrylamide solution was purchased from National Diagnostics, 30 Somerville, NJ. Oligonucleotides were synthesized by Gibco BRL. X-ray film was from Kodak, Rochester, NY. Flexi rabbit reticulocyte lysate system and canine pancreatic microsomal membranes were purchased from Promega, Madison, WI. Other chemicals with cell culture or molecular biology grade were obtained from local suppliers. 33 WO 99/31134 PCT/CA98/01061 Cell culture Primary cultures of human kidney MC and cortical epithelial cells were purchased from Clonetic. Human MC were plated onto 25 cm 2 culture flasks and incubated in MsBM containing 5.5 mM D-glucose with 100U/ml penicillin, 100 lag/ml streptomycin and 5% 5 FBS. Cells were subcultured at 80-90 % confluence. Cortical epithelial cells were grown in REBM supplement with 100U/ml penicillin and 100 g/ml streptomycin. Rat renal MC were prepared and cultured as previously described (53,54). Protocol for studying the effect of hyperglycemia on human kidney MC Human MC between passage 5-9 were used in this study. Three parallel 10 experimental conditions were employed: 25 mM D-glucose (hyperglycemia), 5.5 mM D glucose (low glucose control) and 25 mM L-glucose (osmolarity control). The details are as follows: for high glucose treatment, subconfluent MC were growth-arrested in MsBM + 0.5% FBS overnight and exposed to 5.5 mM or 25 mM D-glucose for 3 days with one change of medium on the second day. In parallel, L-glucose at the final concentration of 15 19.5 mM was added to the culture medium to serve as an osmolarity control. In order to investigate if any dose-dependency of Hmuncl 3 expression by D-glucose treatment, in Northern blot studies, we analyzed two more sets of human MC cultured in 15 mM D glucose or 5.5 mM D-glucose + 9.5 mM L-glucose for 3 days. We have found that changing the medium every two days at 25 mM D-glucose is enough to maintain 20 physiological pH in the medium (pH 7.4) (data not shown). At the end of the experimental treatment period, total RNA of the cells was prepared. Isolation of total RNA Total RNA from human MC and cortical epithelial cells as well as rat MC was prepared using an RNeasy total RNA preparation kit according to manufacturer's 25 instructions. Cell lysates were prepared following homogenization using a QlAshredder. DDRT-PCR DDRT-PCR was performed by modified methods published by Liang and Pardee (55) and Sokolov and Prockop (56). Total RNA from human kidney MC was incubated with DNase I to remove any contaminating genomic DNA prior to first strand DNA 30 synthesis. Reverse transcription (RT) was carried out by incubating a 20 .l reaction mixture containing 1 gg total RNA, 100 ng fully degenerate hexamer, 500 pM each of dATP, dGTP, dCTP and dTTP and 200 units of reverse transcriptase (SuperScript II RNase H) together with the buffer provided by the manufacturer. The reaction mixture 34 WO 99/31134 PCT/CA98/01061 was incubated at 42 0 C for 50 min. The reaction was terminated by heating at 70 0 C for 15 min. E. coli RNase H (2 units) was then added to the reaction mixture followed by incubation at 37 0 C for a further 20 min to remove RNA complementary to the cDNA. Demonstration that the RNA was free of genomic DNA was confirmed using a pair of 5 GAPDH specific primers (5'-ACCACAGTCCATGCCATCAC-3' and 5' GTCCACCACCCTGTTGCTGTA-3') to obtain PCR products before and after RT. We found that there was no amplification in the absence of RT but a strong band was present in the presence of RT (data not shown). PCR was carried out using two 10-mer oligonucleotides, 5'-CAAGCGAGGT-3' and 5'-GTGGAAGCGT-3'. In a total of 12.5 pl1, 10 the reaction mixture contained 1 p l of RNA with RT, 100 pM of each of dNTP, 4 pM of oligonucleotides, 1.5 mM of MgCI 2 , 0.1 mCi/ml of Ca-[ 32 P]-dATP and 1.25 unit of Taq DNA polymerase. PCR was carried out using a Perkin Elemer PCR System 2400 (Perkin Elemer, Foster City, CA) starting at 94 0 C for 1 min, 34 0 C for 1 min and 72 0 C for 1 min for 45 cycles. The resulting PCR products were subjected to 6% denatured polyacrylamide 15 gel electrophoresis (PAGE) using radiolabelled 100 bp ladder as size markers. The gels were then dried and exposed to x-ray film overnight. Bands which showed clear cut differences in high (25 mM) compared to low (5.5 mM) D-glucose or the osmolarity control (25 mM L-glucose) were excised by aligning the film with the gel followed by elution overnight in 10 mM Tris-EDTA buffer (pH 8.0). Eluted DNA was purified and 20 subjected to a second run of PCR by the same pair of 10-mer oligonucleotides under the same experimental conditions without radiolabelled dATP. Fresh PCR products from this last step were cloned into pCR2.1 using a TA cloning kit. Clones with inserts were sequenced by using a TSequencing kit with T7 promoter as a primer according to the manufacturer's instructions. The resulting DNA sequences were compared to the 25 GenBank database using BLAST search. Library Screening Screening of Superscript human kidney cDNA library was achieved using a Genetrapper cDNA Positive Selection System. Captured cDNAs were transformed to ElectroMAX DH 10OB competent cells by electroporation with an electroporation system 30 (BTX Inc., San Diego, Ca) setting at 16.6 kV/cm. We first used an oligonucleotide (5' GTGGTGATGAACACAATGGAGAGG-3') originally derived from sequence information following DDRT-PCR to capture a partial length of Hmuncl3. According to this sequence information, we then designed another oligonucleotide (5' TCCTGTTTGGGAGGAGAAGTTCC-3') closer to the 5' end of the sequence to capture a 35 full length clone. The resulting clone (pCMVSPORTHmuncl 3) was sequenced from both 35 WO 99/31134 PCT/CA98/01061 strands using standard techniques described above. The primers were SP6, T7 promoters or synthetic oligonucleotides derived from the sequence information. Alignment and analysis of sequences was performed with Genework 2.5.1 (Oxford Molecular Group, Campbell, CA) using a Macintosh computer. Comparisons of similarity 5 were performed using the Gapped BLAST search from GenBank. Relative RT-PCR and RT-PCR For relative RT-PCR, RT products previously described were subjected to PCR for 30 cycles using a pair of primers (5'-GGAGCAAATCAATGCCTTGG-3' and 5' TCGGATCCAATGTGCTCTGG-3') specific for Hmuncl 3, amplifying a 671 bp fragment. 10 18S rRNA was chosen as a housekeeping gene by using 18S rRNA primers and 18S rRNA competimers with a ratio of 1:2. These primers amplify a 488 bp fragment. Resulting PCR products were subjected to 1.2 % agarose gel electrophoresis. To determine muncl3 expression in epithelial and rat MC, we employed RT-PCR with a pair of primers (5'-GA(T)GTC(A)CTGAAGGAGCTCTGG-3' and 5' 15 AGGACA(T)GCACACTGCTTTGG-3') targeted to Hmuncl 3 and rat muncl 3-2 both of which yield a 193 bp fragment. RT were performed post DNase I treatment on total RNA extracted from these cells as described above. Northern Blot Analysis Total RNA (10 ptg) extracted from human kidney MC was subjected to 1 % 20 denatured formaldehyde agarose gel electrophoresis as described (36) then transferred to Duralon-UV membranes overnight and exposed to UV light for cross linking. An 32 p_ radiolabelled probe of Hmuncl3 were generated from a PCR fragment derived from pCMVSPORTHmuncl 3 (4095 - 4288) with ct-[ 32 P]-dATP using a Klenow Fragment and random hexamers. Membranes were pre-incubated with rapid hybridization buffer at 25 65 0 C for 15 min and then incubated with radiolabelled probes at 65 0 C for 2 hours. After removal of the radiolabelled probes, membranes were washed first in 2 x SSPE (1 x SSPE contains 150 mM NaCI, 20 mM NaH 2

PO

4 and 1 mM EDTA, pH, 7.4) with 0.1% SDS at room temperature for at least 20 min then twice with 0.1 x SSPE with 0.1% SDS at 65 0 C for 30 min each. After exposure to the Phosphor screen (Molecular Dynamics, 30 Sunnyvale, CA), the blots with Hmuncl3 probe were stripped with a boiling solution of 0.1 x SSPE with 0.1% SDS. The stripped membranes were reprobed with a 32 P-labelled human cyclophilin template. Radioactivity of each band in digital images was analyzed on a PC using ImageQuant 4.0 (Molecular Dynamic). 36 WO 99/31134 PCT/CA98/01061 In vitro Translation In vitro translation was performed according to previously published method (26). Plasmid with Hmuncl3 cDNA (pCMVSPORTHmunc13) was linearlized with Hind III. Linearlized DNA (1 p.g) was transcribed with SP6 RNA polymerase and m 7 G(5')ppp(5')G 5 RNA capping analog. Capped cRNA was extracted using an RNeasy total RNA preparation kit. Eluted cRNA was precipitated and resuspended in 5 41 diethylpyrocarbonated-treated water. In the presence of 1 gl of this cRNA product, in vitro translation was achieved using a Flexi rabbit reticulocyte lysate system according to the method provided by supplier. Translation products were detected by incorporating 1 10 gCilpl of [3 5 S] methionine in the reaction mixture. To determine co-translational processing, 1.5 equivalent of canine pancreatic microsomal membranes was added to 10 gl of in vitro translation reaction. The resulting reaction was centrifuged at 16,000 g for 15 min to pellet microsomes. In vitro translation products were subjected to 8% PAGE. The gel was stained with Commassie brilliant blue then destained. The stained gel was then 15 dried and exposed to x-ray film. Statistical Analysis Group differences in densitometry of the Northern blots were analyzed by Student's t-test using Systat 5.2.1. (Systat Inc., Evanston, IL) for the Macintosh. Significance level was set at p < 0.05. 20 Construction of HA-taqqed hmuncl 3 and truncated mutant without Cl domain We constructed an HA-tagged hmuncl 3, by taking advantage of an EcoN I restriction site (nucleotide 3949) close to the 3' end of the open reading frame of hmuncl3 constructed in pCMVSPORT (Gibco, BRL, pCMVSPORThmuncl 3), and used PCR to introduce the HA-tag at the C-terminal of hmuncl 3. A PCR fragment was 25 generated with Vent DNA polymerase, insert of pCMVSPORThmuncl 3 as a template and a pair of primers (5'-GAATACGGTTCTGGATGAGCT-3' and 5' gcggccgcTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCTCCCCTCCTCCGTGGAAC G -3') where the HA tag sequence is underlined and a Not I site is shown in lower case. A stop code (5'-TCA-3') was placed between the HA tag and the Not I site. The PCR 30 product was then incubated with 2 units of Taq DNA polymerase at 72 C for 15 min and extracted by phenol/chloroform and ethanol precipitation. The resulting pellet was resuspended and ligated to pCR2.1 by using a TA cloning kit. This plasmid was then digested with Not I and EcoN I, subjected to 1% agarose gel electrophoresis. The insert was purified and ligated to pCMVSPORThmuncl 3 previously cut with Not I and EcoN I. 37 WO 99/31134 PCT/CA98/01061 The resulting construct (hmuncl 3-HA) was sequenced to confirm the addition of the HA tag. To construct a deletion mutant lacking the C1 domain (C1 less mutant), we replaced the entire C1 domain (AA 478-528) with two residues Ala and Arg. Primers 5' 5 CGTTGGCGCGCCAGCGGGCTGCAGAAAAGAGC -3' (Asc I site is underline) and 5' CTGTCTCATCAAAGTACACC-3' were used to generate a PCR fragment with Vent DNA polymerase and pCMVSPORThmuncl 3 as a template. Another piece of PCR fragment was generated by primers of Sp6 promoter (5'-AGCTATTTAGGTGACACTATAG-3') and 51 10 GCTAGGCGCGCCGGAGTGGTGCACGAAATGG -3' (Asc / site is underline). The two PCR fragments were digested with Asc I, ligated with T4 DNA ligase, and the ligated product was subjected to 1% agarose gel electrophoresis to check the size and for purification. The gel purified ligated piece was further digested with Kpn I and BstZ17 I and ligated to Kpn I and BstZ17 / digested pCMV SPORThmuncl 3-HA. 15 Plasmids for cell transfection were prepared using a Midi plasmid preparation kit according to manufacturer's instructions. Cells and transfection OK cells were grown in MEM supplemented with 10% FBS and 100 U/ml penicillin and 100 p.g/ml streptomycin, and plated in 60 mm or 100 mm culture dishes or on glass 20 cover slips placed in 24 wells culture plates. Cells were transiently transfected (transfected rate 30-50%) with hmuncl 3-HA or C1 less mutant by using Lipofectamine Plus according to the manufacturers instruction, and maintained in serum free MEM overnight (3 h for apoptotic experiments) after 24 h of transfection. Cell monolayers were washed and fresh medium containing PDBu or the same amount of vehicle (DMSO at a 25 final concentration of 0.0001%) was added to the culture medium at a final concentration of 0.1 pM and cells were analyzed at different time points as indicated. For nocodazole treatment experiments, nocodazole in DMSO was added to the medium at a final concentration of 4 jtM for 1 h and followed by addition of PDBu at a final concentration of 0.1 pM. Cells were subjected to immunostraining after 3 h of PDBu treatment. An 30 identical quantity of DMSO was added to control cells. Immunocytochemistry Cells grown on cover slips were washed 3 times with iced cold Hank's solution, fixed and permeabilized with 100% methanol at -20 C for 5 min. The cover slips were then air dried, washed 3 times with PBS and incubated in blocking solution (PBS + 0.2% 38 WO 99/31134 PCT/CA98/01061 Tween-20 (PBST) containing 10% no-fat dry milk). Cells were then incubated with 0.02 mg/mi anti-HA for 30 min at room temperature followed by 0.02 mg/mi anti-mouse IgG rhodamine for 30 min. Cells were washed at least 8 times with PBST between incubation of anti-HA and anti-mouse IgG-rhodamine or after anti-mouse IgG-rhodamine. Cover slips 5 were then mounted on a glass slide and observed under a confocal scanning microscope. For labeling of the Golgi apparatus, 0.05 mg/ml WGA-FITC was added to the anti-mouse IgG-rhodamine. Immunoblot analysis and preparation of crude Golgi membrane Cells grown on culture plates were washed 3 times with ice cold Hank's solution 10 and scraped into 0.5 ml cell lysis buffer (50 mM Tris-HCI, 150 mM NaCI, 0.25% sodium deoxycholate, 1% NP-40, 1 mM EDTA and protease inhibitor cocktail, pH 7.5), and then rocked at 4 C for 45 min. The insoluble fraction was removed by centrifugation at 14,000 g for 5 min. Supernatants were subjected to 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. The membrane was 15 washed twice with TBS, blocked with TBS containing 0.1% Tween-20 (TBST) and 1% normal horse serum for 30 min and then incubated with 0.5 gg/ml anti-HA in TBST. After washing with TBST for at least 4 times, the membrane fraction was incubated with 0.2 pg/ml anti-mouse IgG-biotin, washed with TBST and then incubated with the A and B reagent mix in a Vector ABC staining kit according to manufacturer's instructions. The 20 blot was detected by ECL according to the manufacturer's instruction. Golgi membranes were prepared by a sucrose density method reported previously (57) with a protease inhibitor cocktail presented in all buffer solution. The band at the interface of 0.8M and 1.2M sucrose was collected and subjected to 6% SDS PAGE and immunobloting as described above. Protein concentration was determined by Lowry 25 assay with bovine serum albumin as standard using a DC Protein Assay kit following its instruction. Detection of apoptosis by DNA fragmentation Cleaved genomic DNA during apoptosis for cells grown on cover slips was detected by terminal deoxynucleotidyl transferase (TdT) - mediated dUTP nick end 30 labeling (TUNEL) using a in situ cell death detection kit following manufacturer's directions. Fluorescein labels were incorporated in nucleotide polymers. Negative controls were obtained by incubating label solution without TdT under the same conditions. After labeling for apoptosis, cells were further subjected to 39 WO 99/31134 PCT/CA98/01061 Immunocytochemistry as described above without fixation and permeabilization to detect expression of hmuncl 3 or its C less mutant. Genomic DNA fragmentation of cells grown on 60 mm culture dishes was analyzed by 2% agarose gel electrophoresis using the procedure described elsewhere 5 (58). Streptozotocin treated diabetic rat model Rats received a single injection of STZ (65 mg/kg body weight, i.p.) dissolved in 20 mM citric acid (pH 4.5). Blood glucose was monitored daily by tail blood sampling with a Medisense blood glucose sensor (Medisense Canada, Mississauga, ON, Canada). 10 Blood glucose was maintained at a concentration of 15-20 mM with 2 U NPH insulin daily (s.c.) after diabetes was confirmed by elevated blood and urinary glucose. Rats were sacrificed after 1 or 11 days of diabetes. Rat kidneys were collected as soon as possible, usually within 3-5 min, and processed for total RNA preparation or tissue preparation for in situ hybridization as described below. Control rats were injected (i.p.) with the same 15 amount of 20 mM citric acid and their blood glucose levels were also tested daily (< 5 mM). Relative reverse transcription polymerase chain reaction (RT-PCR) Total RNA from rat kidney cortex was prepared using a TRIzol reagent according to instructions provided by the manufacturer and then treated with DNase I. Confirmation 20 of no genomic DNA contamination in RNA preparations and relative RT-PCR were performed as described elsewhere (33). Primers for amplification of rat muncl3-1 are 5' CGTGACCAAGATGAGTACTCC-3' (sense) and 5'-CGAAGTCGTGTAGTAAGGCG-3' (anti-sense) yielded a fragment of 195 bp. Primers for rat muncl 3-2 are 5' GAGTCCTGAAGGAGCTCTGG-3' (sense) and 5'-AGGACAGCACACTGCTTTGG-3' 25 (anti-sense) yielded a fragment of 193 bp. Primers for rat muncl 3-2 are 5' AGATGACCTTGGCAAGTGC-3' (sense) and 5'-CGATACATCATGGATGGATGG-3' (anti sense) yielded a fragment of 198 bp. The sequence of PCR products was confirmed by cloning PCR fragments into pCR2.1 using a TA cloning kit and sequencing using a TSequencing kit with T17 promoter as a primer. 30 In situ hybridization Templates for in vitro transcription were generated by PCR with primers described above for three different isoforms, except that for anti-sense cRNA, addition of T7 promoter (5'-TAATACGACTCACTATAGGGA-3') was present in the sense strain and for 40 WO 99/31134 PCT/CA98/01061 sense cRNA, T17 promoter was present in the anti-sense strain. Anti-sense and sense cRNA for different isoforms were obtained by in vitro transcription. PCR templates (200 ng) were incubated with T7 RNA polymerase (40U), its reaction buffer provided by the manufacturer and DIG RNA labeling mix in a total volume of 40 RI at 37 C for 90min. 5 Twenty Rl recombinant RNA was purified by using a RNeasy total RNA preparation kit and its yield was estimated by A 260 . The remaining cRNA was subjected to ethanol precipitation and resuspended in nuclease-free water. All solutions used before the post-hybridization step were diethylpyrocarbonate (DEPC) treated or prepared in DEPC-treated water. Kidneys were quickly cut to 2 mm 10 thick blocks after dissection then put in phosphate-buffered saline (PBS, pH 7.4) containing 4% parafromaldehyde for 4 h at 4 C. The tissue was soaked in PBS containing 30% sucrose overnight at 4 C and then stored in liquid nitrogen. Frozen tissues were sectioned (10 pm) and placed on a poly-L-lysine coated glass slides. In order to ensure the same experimental conditions, kidney sections from control and 15 diabetic rats were placed on the same slide. Tissue slides were then dried at 40 C overnight and stored at -80 C for less then a week. On the day of hybridization, slides with tissue sections were dried at 40 C for 2 h then washed twice with PBS. Slides were then incubated with 0.3% Triton X-100 in PBS for 15 min at room temperature and washed twice with PBS afterward. Sections were incubated with 1 ptg/ml RNase-free 20 proteinase K in TE buffer (100 mM Tris-HCI, 50 mM EDTA, pH 8.0) for 30 min at 37 C and then fixed by incubating with PBS containing 4% parafromaldehyde for 5 min at 4 C. Sections were then washed twice with PBS and acetylated with freshly prepared 0.1 M triethanolamine buffer (pH 8.0) containing 0.25% acetic anhydride. Slides were then incubated first with 4x SSPE (lx SSPE containing 150 mM NaCI, 20 mM NaH2PO4 and 25 1mM EDTA, pH 7.4) containing 50% formamide at 37 C for 20 min and then overlaid with 75 l hybridization buffer (40% fromamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 10 mg/ml bovine serum albumin, 4x SSPE, 10 mM DTT, 0.4 mg/ml yeast t-RNA and 0.1 mg/ml poly(A) ) containing 50 ng of denatured DIG-labeled cRNA probe. Slides were incubated in a humid chamber at 42 C overnight. After hybridization, 30 slides were washed at least 4 times in lx SSPE at 37 C. Sections were incubated with 20 p.g/ml RNase A in NTE buffer (500 mM NaCI, 10 mM Tris-HCI, 1 mM EDTA, pH 8.0) at 37 C for 30 min and washed twice with 0.1x SSPE. Slides were washed and blocked in TBS (100 mM Tris-HCI and 150 mM NaCI, pH 7.5) containing 1% blocking reagent and then incubated with 0.02 mg/ml anti-DIG-rhodamine for 1 h. Slides were washed at least 35 5 x with TBS. Staining was assessed by a confocal scanning microscopy. 41 WO 99/31134 PCT/CA98/01061 The present invention has been described in detail and with particular reference to the preferred embodiments; however, it will be understood by one having ordinary skill in the art that changes can be made thereto without departing from the spirit and scope thereof. 5 All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. 42 WO 99/31134 PCT/CA98/01061 REFERENCES 1. S.M. Mauer. Structural-functional correlations of diabetic nephropathy. Kidney Int. 45: 612-622, 1994 2. A.E. Thomson. Non-insulin dependent diabetes mellitus in nephrology: where do 5 we go from here? Kidney Int. 52: S-83-S-84, 1997 3. T.F. Drury and A.L. Powell. 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Claims (42)

1. An isolated nucleotide sequence encoding a glucose regulated munc polypeptide.

2. The nucleotide sequence of claim 1, wherein the nucleotide sequence is isolated 5 from a kidney cell, human cortical epithelial cell or a cell from testis, ovaries, prostate gland, colon, brain and heart.

3. The nucleotide sequence of claim 2, wherein the kidney cell is a mesangial cell or a kidney cortical epithelial cell.

4. The nucleotide sequence of claim 1 or 2, wherein the glucose regulated munc 10 polypeptide comprises a Hmuncl3 polypeptide.

5. The nucleotide sequence of claim 4, wherein the Hmuncl3 polypeptide comprises all or part of the amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO. 1].

6. The nucleotide sequence of claim 1 or 2, wherein the nucleotide sequence comprises a Hmuncl3 gene. 15

7. The sequence of any of claims 1 to 6, comprising all or part of the nucleotide sequence in Figure 8 [SEQ ID NO. 2].

8. The sequence of any of claims 1 to 3, wherein the sequence comprises at least 40% sequence identity to all or part of the nucleotide sequence of Figure 8.

9. The sequence of any of claims 1 to 8 which is selected from a group consisting of 20 mRNA, cDNA, sense DNA, anti-sense DNA, single-stranded DNA and double stranded DNA.

10. A nucleotide sequence encoding the amino acid sequence of claim 4 or 5.

11. A nucleotide sequence that encodes all or part of a Hmuncl 3 polypeptide, wherein the sequence hybridizes to the nucleotide sequence of all or part of 25 Figure 8 under high stringency conditions.

12. The nucleotide sequence of claim 11, wherein the high stringency conditions comprise a wash stringency of 0.2X SSC to 2X SSC, 0.1% SDS, at 65 0 C.

13. An isolated munc polypeptide, with the provisio that the polypeptide is not found in a mammalian central nervous system. 30

14. The polypeptide of claim 13, wherein the polypeptide has transmembrane ECM cell signaling activity and DAG and Ca** activated phosphatase activity. 49 WO 99/31134 PCT/CA98/01061

15. A polypeptide comprising all or part of the Hmuncl 3 amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO: 1].

16. A mimetic of the purified and isolated polypeptide of any of claims 13 to 15.

17. The polypeptide of any of claims 13 to 15, which has at least 40% sequence 5 identity to all or part of the amino acid sequence (a) in Figure 1 [SEQ ID NO: 1].

18. The polypeptide of claim 13, wherein the polypeptide is from a mammalian kidney cell.

19. The polypeptide of claim 13 for a use selected from a group consisting of apoptosis and vesicle trafficking. 10

20. A recombinant DNA comprising a DNA sequence of any of claim 1 to claim 12 and a promoter region, operatively linked so that the promoter enhances transcription of said DNA sequence in a host cell.

21. A system for the expression of Hmuncl 3, comprising an expression vector and Hmuncl3 DNA inserted in the expression vector. 15

22. The system of claim 21, wherein the expression vector comprises a plasmid or a virus.

23. A cell transformed by the expression vector of claim 21 or 22.

24. A method for expressing Hmuncl 3 polypeptide comprising: transforming an expression host with a Hmuncl3 DNA expression vector and culturing the 20 expression host.

25. The method of claim 24, further comprising isolating Hmuncl 3 polypeptide.

26. The method of claim 24 or 25, wherein the expression host is selected from the group consisting of a plant, plant cell, bacterium, yeast, fungus, protozoa, algae, animal and animal cell. 25

27. A pharmaceutical composition, comprising at least all or part of the polypeptide of any of claims 13 to 19, and a pharmaceutically acceptable carrier, auxiliary or excipient

28. A pharmaceutical composition for use in gene therapy, comprising all or part of the nucleotide sequence of any of claims 1 to 12, and a pharmaceutically 30 acceptable carrier, auxiliary or excipient. 50 WO 99/31134 PCT/CA98/01061

29. A pharmaceutical composition for use in gene therapy, comprising all or part of an antisense sequence to all or part of the nucleic acid sequence in Figure 8.

30. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the nucleotide sequence of any of claims 1 to 12. 5

31. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the polypeptide of claim 13.

32. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising an antibody to the polypeptide of claim 13.

33. The kit of any of claim 30 to claim 32, wherein the disorder is selected from a 10 group consisting of insulin dependent and independent diabetes, glomerulopathy and renal failure.

34. A NH2-SQRSNDEVREFVKL-COOH specific antibody.

35. The peptide of claim 34, wherein the antibody is a polyclonal antibody.

36. A method of medical treatment of a disease, disorder or abnormal physical state, 15 characterized by excessive Hmuncl 3 expression, concentration or activity, comprising administering a product that reduces or inhibits Hmuncl 3 polypeptide expression, concentration or activity.

37. The method of claim 36, wherein the product is an antisense nucleotide sequence to all or part of the nucleotide sequence of Figure 8, the antisense nucleotide 20 sequence being sufficient to reduce or inhibit Hmuncl 3 polypeptide expression.

38. The method of claim 37, wherein the antisense DNA is administered in a pharmaceutical composition comprising a carrier and a vector operably linked to the antisense DNA.

39. The method of any of claims 36 to 38 wherein the disease, disorder or abnormal 25 physical state is selected from a group consisting of insulin dependent diabetes and independent diabetes, glomerulonephritis and ischemic renal injuries.

40. A method of medical treatment of a disease, disorder or abnormal physical state, characterized by reduced Hmuncl 3 expression, concentration or activity, comprising administering a product that increases Hmuncl 3 polypeptide 30 expression, concentration or activity. 51 WO 99/31134 PCT/CA98/01061

41. The method of claim 40, wherein the product is a nucleotide sequence comprising all or part of the nucleotide sequence of Figure 8, the DNA being sufficient to increase Hmuncl13 polypeptide expression.

42. The method of claim 41, wherein the nucleotide sequence is administered in a 5 pharmaceutical composition comprising a carrier and a vector operably linked to the nucleotide sequence. 52