WO2003001892A2 - Heparin-binding factors in tissue repair, regeneration and bioengineering - Google Patents

Abstract

Methods for constructing stable bioactive mammalian embryonic epithelial tissues and organs are described herein as well as constructing "designer" kidneys, and treating renal failure in vivo with heparin-binding growth factors. A new active epithelial growth factor having the capability of effectuating induction of growth and morphogenesis has been described. As an example, construction of a kidney is described. A kidney so constructed requires no artificial support, nor porous man-made membranes or tubing to effectuate its biological function of filtering body fluids. A single donor embryonic kidney, or fragment thereof, can produce a great number of functional kidneys suitable for treating subjects with various kidney disorders. The in vivo produced kidney would be less, or not at all, antigenic when transplanted into a subject, because of its embryonic character and artificial propagation in culture. This method of producing a functional organ can be useful in cloning other organ structures containing inducible epithelial tissues.

Description

HEPARIN-BINDING FACTORS IN TISSUE REPAIR, REGENERATION AND BIOENGMEERING

BACKGROUND OF THE INVENTION

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK53507, RO1-DK51211 (to S.K.N) and an American Heart Association Scientist Development Award 9730096N (to K.T.B).

This application claims priority to U.S. Provisional application number

60/301,684, filed Jun. 28, 2000.

Field of the Invention

The present invention generally concerns methods of engineering epithelial tissues and organs in vitro.

The present invention particularly concerns new methods and procedures for propagating cloned kidney members from embryonic ureteric bud tips grown in vitro under specific culture conditions. More specifically, an epithelial growth and differentiation factors are described.

Description of Related Art

A portion of this work was described in Sakurai, H., Bush, K. T., and Nigam, S. K. (2001) Development 128(17), 3283-93, which is incorporated herein in toto.

Many epithelial organs such as kidney, lung and prostate undergo branching morphogenesis in the course of development. The kidney is formed by mutual induction between two precursor tissues derived from the intermediate mesoderm, the metanephric mesenchyme (MM) and the ureteric bud (UB) (Grobstein, 1953). The UB induces the MM to differentiate and form the proximal nephron, while the UB undergoes dichotomous branching and elongation as it invades the MM, ultimately forming the kidney collecting system (Saxen, 1987). Although it is believed that the MM directs UB branching morphogenesis, the exact nature of this directive signal(s) is unknown.

Soluble factors that have been hypothesized to function in such a morphogenetic capacity include hepatocyte growth factor (HGF) and epidermal growth factor (EGF) receptor ligands, which have been shown to induce branching tubular structures in epithelial cells cultured in collagen gels (Barros et al., 1995; Cantley et al., 1994; Montesano et al., 1991; Sakurai et al., 1997b). However, these studies have been largely carried out on adult cell lines. In a cell culture model that employs UB ells, an epithelial cell line derived from embryonic day 11.5 (El 1.5) mouse UB neither HGF, EGF receptor ligands, nor many other factors tested (alone or in combination), were able to induce UB cells to form branching tubular structures with lumens (Sakurai et al., 1997a). However, UB cells undergo branching tubulogenesis in the presence of a conditioned medium elaborated by a cell line derived from the MM also isolated from an El 1.5 mouse (BSN cells) (Sakurai et al., 1997a). This data suggests that other, yet to be identified, soluble factors present in BSN-CM are important for UB cell morphogenesis. These potentially novel factors that are presumably secreted by the MM may be as (or more) important for the development of the collecting system as those currently receiving attention.

This MM-derived cell conditioned medium (BSN-CM), when supplemented with GDNF, also induces the isolated rat UB (in the absence of MM) to undergo dichotomous branching reminiscent of that seen in the developing kidney (Qiao et al., 1999a). This indicates that the MM-derived cell line, presumably reflecting the MM itself, secretes soluble factors capable of inducing branching morphogenesis of the UB. This isolated UB culture system can serve as a powerful assay system since it directly assesses the effect of soluble factors on UB morphogenesis.

SUMMARY OF THE INVENTION

The primary object of this invention is to provide functioning replacement epithelial organs or functional fragments thereof that are suitable for transplanting into recipients suffering from a variety of life-threatening diseases or developmental anomalies.

Another object in accordance with the present invention is to generate functional mammalian epithelium-derived organs, or active fragments thereof from embryonic explants, tissues or cells utilizing in vitro culture techniques.

Another object of this invention is to define soluble inducing factors effective in transforming embryonic epithelial cells or tissues into regenerating functional organs, glands and the like.

A further, most preferred object is to provide a bank of embryonic organs and tissues capable of replacing diseased, or otherwise incapacitated vital organs and tissues, minimizing the need for matching donors and/or immunosuppressive drugs.

Yet another preferred object is to induce repair of epithelial organs and tissues severely damaged by trauma or ischemic disease.

A further object is to design functioning epithelial organs, such as kidney, with certain specific functions.

In accordance with these objects, this invention contemplates a method for constructing a functional mammalian tubulogenic organ or fragment thereof in vitro. The method involves culturing and propagating embryonic explants, tissues or cells by isolating said explants, tissues or cells and growing them in culture with specific soluble and insoluble inducers for sufficient periods of time to allow the cultured specimens to form multiple branches. The tips of these branches are then dissected out and recultured in the presence of serum, growth factor mix, mixture of conditioned and nutrient-rich medium for several generations to form 3-dimensional tubulogenic structures with multiple growing tips. This process can proceed ad infinitum under proper culture conditions having effective inducer substances.

The contemplated method further involves culturing and propagating embryonic mesenchymal tissues capable of inducing limited differentiation and directional growth to form functional organs or tissues. The mesenchymal or other inducing tissue fragments are dissected out at the time of induction, and cultured in the presence of serum, growth factor mix, and a mixture of appropriate conditioned medium and nutrient-rich medium. After several passages in primary culture, growing inductive tissue may be partitioned into multiple fragments. Each fragment can then grown separately in culture. Vasculogenesis within each fragment is induced by substrate deprivation and/or the addition of specific soluble factors.

Finally, a grown, vascularized mesenchymal tissue fragment is combined in coculture with a cultured tubulogenic fragment described hereinabove, in a matrix in which in vitro angiogenesis has begun. The two tissue fragments are grown in nutrient-rich medium conditions to enable continued vasculogenesis. Alternatively, the "cloned" kidney can be implanted for in vivo vascularization.

A more specific and preferred embodiment of this invention is a method for generating a functional mammalian kidney in vitro by culturing and propagating ureteric bud tissue. This method comprises isolating embryonic kidney rudiments by dissection, isolating ureteric bud tissue fragments from mesenchyme by incubating the kidney rudiments with a proteolytic enzyme in the presence of DNAase and/or by mechanical separation. The isolated ureteric bud fragments are suspended in a gel matrix and the gel/fragment composition is placed on porous polycarbonate membrane inserts in wells of tissue culture plates. Growth factors are added to the culture wells, and the gel composition comprising the bud fragments is maintained at the interface of air and medium until the fragments form multiple tubular branches inside the gel matrix. Individual distal branch tips formed during culture are dissected out and recultured in the presence of serum, growth factor mix, mixture of mesenchymal and ureteric bud cell conditioned medium and nutrient-rich medium for several generations.

The mechanical separation of tissue fragments can be accomplished by manual dissection or laser separation and capture. The growth factor mix includes a glial cell line-derived neurotrophic factor or functional equivalent thereof. The added conditioned medium contains a heparin-binding, growth promoting constituent and/or inducer of differentiation. For example, a potent inducer is pleiotrophin. The extracellular matrix gel comprises a mixture of type I collagen and Matrigel or a comparable support matrix.

An equally preferred embodiment in accordance with this invention is method for simultaneous in vitro culturing and propagation of metanephric mesenchyme. This method comprises dissecting out fetal kidney mesenchyme tissue at the time of induction, culturing fragments of the mesenchymal tissue in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium, and partitioning the cultured mesenchyme into multiple pieces. Each piece is grown separately in culture for several generations and grown mesenchyme is then subjected to substrate deprivation and/or additional growth factors in order to induce vasculogenesis.

A most preferred embodiment in accordance with this invention is a method for in vitro engineering and constructing a functioning mammalian kidney by culturing and propagating an isolated ureteric bud, permitting the cultured bud to form multiple branches, dissecting out the individual branch tips, and reculturing in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium for several generations. The method also comprises simultaneously culturing and propagating isolated embryonic or fetal metanephric mesenchyme by dissecting out fetal mesenchyme at the time of induction, culturing mesenchymal tissue in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium, potentially partitioning the mesenchyme into multiple pieces with the option of growing each piece separately, and inducing vasculogenesis by subjecting grown mesenchyme to substrate deprivation. The most preferred method then provides for recombining each vascularized mesenchyme piece with each cultured bud in a matrix in which in vitro angiogenesis has begun, and growing in richest medium conditions to ensure continued vasculogenesis.

Thus, in the most preferred embodiment, is a functional mammalian kidney constructed from isolated embryonic or fetal kidney tissue or cells cultured in rich medium that has present a mixture of growth factors and inducer substances, and comprises recombination of an isolated ureteric bud propagated in culture to produce a functioning nephron, and metanephric mesenchyme propagated from cultured embryonic mesenchymal tissue fragments or cells. Said mesenchyme has the capability of inducing differentiation and providing directional guidance to the branching tubulogenic bud.

Still further embodiments and advantages of the invention will become apparent to those skilled in the art upon reading the entire disclosure contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 indicates that BSN-CM is necessary for branching morphogenesis of isolated ureteric bud tissue.

Phase contrast photomicrographs of isolated ureteric buds cultured for

14 days in the presence (b) or absence (c) of BSN-CM in the presence of 10% FCS, 125 ng/ml GDNF, and 250 ng/ml FGF1. In the presence of BSN-CM, the T-shaped ureteric bud (a) underwent extensive branching morphogenesis (b). In the absence of BSN-CM, no significant growth/morphogenesis was observed (c). Bar = 500 μm.

Figure 2 describes the purification protocol for the isolated morphogenetic factor.

(A) Silver stained SDS-PAGE gel of active fractions from column chromatography of BSN-CM. (a) Lane 1, whole BSN-CM; (b) Lane 2, active fraction from heparin sepharose column; (c) Lane 3, active fraction from the Resource phenyl sepharose hydrophobic interaction column. (B) Elution profile from the Resource S cation exchange column of the active fraction from Resource phenyl sepharose column. A single, sharp protein peak was eluted at 0.4-0.6 MNaCl. Each of the individual 1 ml fractions eluted from the column are indicated by the red numbers along the x-axis. (C) Phase-contrast photomicrographs of isolated ureteric buds cultured for 7 days in the presence of each 1 ml fraction from the Resource S cation exchange column (1-8 in B) supplemented with 10% FCS, 125 ng ml GDNF, and 250 ng/ml FGF1. Fraction 4, which corresponded with the protein peak on the elution profile (B) exhibited potent morphogenetic activity. Bar = 500 μm. (D) Silver stained SDS-PAGE gel of each fraction (1-8) eluted from the Resource S cation exchange column (B). Fraction 4, which possessed potent morphogenetic activity (C) contained a single low molecular weight band, which was identified as pleiotrophin by mass spectrometry. (E) Immunoblot analysis of the individual fractions eluted from the Resource S cation exchange column (1- 8 in B). The blot was probed with anti-pleiotrophin antibodies. Left lane; 250 ng of human recombinant pleiotrophin as a positive control.

Figure 3 shows gel filtration chromatography profile of the eluate from the Resource S cation exchange column.

(A) Elution profile from a Superdex 200 gel filtration column of the peak fraction from the Resource S cation exchange column (Fig. 2B, fraction 4). A single protein peak was eluted at 15.93 ml, which corresponds to a relative molecular mass of 18 kDa. Each of the individual 1 ml fractions are indicated by the red numbers along the x-axis. (B) Immunoblot analysis of Fraction 3 (A) from the gel filtration column demonstrated the presence of pleiotrophin. rh-PTN, human recombinant pleiotrophin used as a positive control. (C) Phase contrast photomicrograph of isolated ureteric bud grown for 7 days in the presence of Fraction 3 supplemented with 10% FCS, 125 ng/ml GDNF, and 250 ng/ml of FGF1. Bar = 500 μm.

Figure 4 demonstrates that adsorption of pleiotrophin abolishes morphogenetic activity. (A) Silver stained SDS-PAGE gel of morphogenetically active fraction from Resource S cation exchange column. Lane 1, whole fraction; Lane 2, fraction incubated with polyA-sepharose beads. The protein band at 18 kDa was not detected following treatment with polyA-sepharose beads. (B) Immunoblot analysis of the morphogenetically active fraction from Resource S cation exchange column. Lane 1, recombinant human pleiotrophin (positive control); Lane 2, active fraction; Lane 3, active fraction treated with polyA- sepharose beads; Lane 4, protein bound to beads. The blot was probed with anti-pleiotrophin antibodies. PolyA-sepharose beads adsorb pleiotrophin present in the fraction eluted from the Resource S cation exchange column. (C) Phase contrast photomicrographs of isolated ureteric buds grown for 7 days in morphogenetically active fraction eluted from the Resource S cation exchange column with or without exposure to polyA-sepharose beads. In either case, the fraction was supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. Bar = 500 μm.

Figure 5 indicates that pleiotrophin-mediated UB branching morphogenesis is concentration-dependent.

(A) Phase contrast photomicrographs of isolated ureteric buds grown for seven days in DMEM/F12 supplemented with increasing concentration of purified pleiotrophin. In each case, the growth media was also supplemented with 10% FCS, 125 ng/ml GDNF and 250 ng/ml FGF1. The numbers in the upper-left-hand corner of each picture indicate the concentration of pleiotrophin in μg/ml. Clear differences in the phenotype depending on the concentration of pleiotrophin are exhibited. (B) Phase contrast photomicrographs of isolated ureteric buds grown for 11 days in the presence (1) or absence (2) of 250 ng/ml FGF1 diluted in DMEM/F12 supplemented with 2.5-5 μg/ml pleiotrophin, 10% FCS, and 125 ng/ml GDNF. Bar = 500 μm.

Figure 6 demonstrates pleiotrophin-induced UB cell tubulogenesis in vitro.

(A) Bar graph demonstrating the morphogenetic effects of pleiotrophin on UB cells grown in three-dimensional extracellular matrix gels. UB cells were suspended in 20% Matrigel, 80% collagen gel mixture and grown for 4 days in the absence (control) or presence of purified pleiotrophin (0.1-2.5 μg/ml). Whole BSN-CM served as a positive control. All conditions were supplemented with 1% FCS. The percentage of cells and colonies with processes was counted as an indicator for tubulogenic activity. 20 cells/colony were counted in three randomly selected fields for each condition. Data is presented as mean ± s.e.m., * p< 0.05 (by unpaired Student's t test). (B) Phase contrast photomicrographs of UB cells grown for 8 days in DMEM/F12 supplemented with 1% FCS (a) control and either BSN-CM (b) or purified pleiotrophin (c). BSN-CM and pleiotrophin induced the formation of branching tubules with lumens (compare b and c). Bar - 50 μm.

Figure 7 is an example of pleiotrophin expression in the embryonic kidney.

(A) Immunoblot detection of pleiotrophin. Lane 1, extract of whole embryonic day 13 rat kidney; Lane 2, conditioned medium collected from UB cells; Lane 3, conditioned medium from BSN cells. Whole kidney and BSN- CM were positive for pleiotrophin (arrow). (B) Embryonic day 13 mouse kidney frozen sections stained with anti-pleiotrophin antibody. Pleiotrophin localized at the basement membrane of developing UB (a). Normal goat IgG did not exhibit significant staining (b). Bar = 100 μm.

Figure 8 shows the effect of exogenous pleiotrophin on UB morphology in whole kidney organ culture. Fluorescent photomicrographs of embryonic day 13 rat kidneys cultured for 7 days in DMEM/F12 supplemented with 10% FCS in the absence (a, control) or presence of pleiotrophin (b, 2.5 μg/ml; c, 5 μg/ml). The UB was visualized with FITC-conjugated lectin from Dolichos biflorus. Bar = 500 μm.

Figure 9 shows Rat UBs that were isolated and suspended in extracellular matrix gels in the presence of: (A; control) GDNF+ FGF-1; (B) heparin column eluate + GDNF + FGF-1 ; (C) whole BSN-CM + GDNF + FGF-1 for 7 days. BSN-CM was fractionated on a heparin affinity column.

Figure 10 shows the chromatographic separation profile of active heparin eluate from a hydrophobic interaction (Resource phenyl sepharose) column. Fractions 5-11 eluted with decreasing ammonium sulfate gradient were subjected to isolated UB cultures, as well as SDS PAGE and silver staining (Figure 10 cont'd)

Figure lldepicts the chromatographic separation profile of active fractions eluted from eluted from an anion exchange (Resource Q) column with increased salt gradient (upper). Fractions 3-8 were subjected to isolated UB culture assay. Activity is shown in the lower photographs.

Figure 12 shows that a non-PTN fraction induced UB branching morphogenesis as well. (A) Fraction 4, obtained after three sequential column separations, contains several protein bands depicted by silver staining. (B) No PTN was detected by western blotting in this fraction. (C) Isolated UB cultured in the presence of fraction 4 with GDNF and FGF-1 for 8 days are shown.

Figure 13 shows growth an arborized structure from an isolated UB, which was subdivided into smaller fractions and induced into additional generations of UB cells that grow and branch in vitro. Days 0, 5 and 8 shown

Figure 14 shows UB generations that were recombined with freshly isolated metanephric mesenchyme, and they retained the ability to induce dramatic tubular epithelial differentiation of the mesenchyme. Days 0, 2 and 5 shown.

Figure 15 shows additional evidence of the ability to induce dramatic tubular epithelial differentiation of the mesenchyme. Days 3, 4 and 9 are shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

INTRODUCTION

Inventors have isolated a UB branching morphogenetic activity from the BSN-CM and identified it as an 18 kDa heparin binding protein, pleiotrophin. Pleiotrophin was originally discovered as a fibroblast prohferative factor (Milner et al, 1989) and a neurite outgrowth-promoting factor (Rauvala, 1989). Outside the nervous system, pleiotrophin is generally detected in those embryonic organs in which mesenchymal-epithelial interactions are thought to play an important role, such as salivary glands, lung, pancreas, and kidney (Mitsiadis et al., 1995; Vanderwinden et al., 1992). Although pleiotrophin has been shown to be mitogenic for certain epithelial cells (Li et al., 1990; Sato et al., 1999), there has been no compelling evidence for a key role for pleiotrophin during epithelial organogenesis. Here, Inventors have shown that purified pleiotrophin induces impressive branching morphogenesis of the isolated UB (in the presence of GDNF) as well as tubule formation in a UB cell line in vitro. Inventors propose that pleiotrophin is a key metanephric mesenchymally-derived factor that plays a critical role in branching morphogenesis of the UB during kidney development. MATERIALS AND METHODS

Unless otherwise stated, the incubations were performed at 37°C in an atmosphere of 5% C0₂ and 100% humidity. For the immunodetection of pleiotrophin either on western blots or frozen sections of El 3 mouse kidney, a goat anti-pleiotrophin antibody (R&D systems) was used.

Examplel

Cell culture and conditioned medium

BSN cells were grown to confluency in DMEM F12 supplemented with 10% fetal calf serum (FCS). The growth media was removed and the cells were then incubated in serum-free DMEM/F 12 for 3-4 days followed by collection of the conditioned medium (Qiao et al., 1999a). Swiss 3T3 cells (ATCC) were grown to confluency in DMEM with 10% FCS. Once the cells were confluent, the growth media was replaced with DMEM supplemented with 2% FCS and the cells were cultured for an additional 3-4 days. The conditioned medium was collected and used for the experiments. UB cells were cultured in DMEM supplemented with 10% FCS at 32°C in an atmosphere of 5% C0₂ and 100% humidity.

Example 2

Isolated ureteric bud and whole embryonic kidney culture Timed pregnant female Sprague-Dawley rats at day 13 of gestation (day

0 coincided with appearance of the vaginal plug) were sacrificed and the uteri were removed. The embryos were dissected free of surrounding tissues and the kidneys were isolated. For the culture of the whole kidney rudiment, 2-3 kidneys were applied directly to the top of a polyester Transwell filter (0.4 μm pore size; Corning-Costar). The Transwells were then placed within individual wells of a 24-well tissue culture dish containing 400 μl DMEM/F12 supplemented with 10% FCS with or without purified pleiotrophin. Following 7 days of culture, the kidneys were fixed in 2% paraformaldehyde and double- stained with fluorescein-conjugated Dolichos biflorus, a lectin which binds specifically to UB-derived structures (Laitinen et al., 1987), and rhodamine- conjugated peanut agglutinin, a lectin which binds to structures derived from the MM (Laitinen et al., 1987), as described previously (Qiao et al., 1999a). Fluorescent staining was detected using a laser-scanning confocal microscope (Zeiss).

In the case of culture of the isolated UB, the isolated kidneys were trypsinized for 15 min at 37°C in L-15 media containing 2 μg/ml trypsin (Sigma). Trypsin digestion was arrested by the addition of 10% FCS and the kidneys were removed to fresh L-15 where the UBs were isolated from surrounding MM by mechanical dissection. Isolated UBs were suspended within an extracellular matrix gel [ 1 : 1 mixture of growth factor reduced Matrigel (BD) and Type 1 collagen (BD)] applied to the top of a polyester Transwell filter (0.4 μm pore size; Corning-Costar). The Transwells were placed within individual wells of a 24-well tissue culture dish containing 400 μl of either whole BSN-CM, purified fractions of BSN-CM, or DMEM/F12 which were supplemented with human recombinant FGFl (250 ng/ml; R&D Systems), rat recombinant GDNF (125 ng/ml; R&D Systems) and 10% FCS and cultured as previously described (Qiao et al., 1999a). Phase-contrast photomicrographs of the developing UB were taken using a RT-Slider Spot Digital Camera (Diagnostic Instruments Inc.) attached to a Nikon Eclipse TE300 Inverted Microscope.

Example 3

Three-dimensional UB cell culture. Confluent monolayers of UB cells were removed from tissue culture dishes by light trypinization and the cells (20,000 cells/ml) were suspended in an extracellular matrix gel composed of 80% Type 1 collagen and 20% growth factor-reduced Matrigel (Sakurai et al., 1997a). 100 μl of the UB cell- containing gel was then aliquoted into individual wells of a 96-well tissue culture plate. After gelation, 100 μl of growth medium (DMEM/F12 with or without purified pleiotrophin) supplemented with 1% FCS was applied to each well and the cultures were incubated at 32°C in 5% C0₂ and 100% humidity. Following 4 days of culture, the percentage of cells/colonies with processes was counted as an indicator of the tubulogenic activity. Phase-contrast photomicrographs were taken as described above.

Example 4

Purification of morphogenetic factor

1.5-2 L of BSN-CM collected as described above was filtered to remove extraneous cellular debris using a 0.22 μm polyethersulphone membrane filter (Corning). The BSN-CM was then concentrated ~40-fold using a Vivaflow 200 concentrator with a 5 kDa molecular weight cutoff (Sartorius). After adjusting the salt concentration to 0.4 M NaCl, the concentrated BSN-CM was then subjected to sequential liquid column chromatography using an AKTA purifier (Amersham-Pharmacia). Initial fractionation was performed using a heparin sepharose chromatography column (HiTrap heparin, 5 ml; Amersham- Pharmacia). The flow-through fraction was collected and individual 5 ml fractions of the heparin-bound proteins were eluted via increasing concentrations of NaCl (0.4 M-2.0 M) buffered to pH 7.2 with 50 mM HEPES. Aliquots of each fraction were subjected to buffer exchange by dia-filtration using an Ultrafree 500 spin column (Millipore) according to the manufacturer's instructions and then tested for morphogenetic activity using the isolated UB culture system.

An active fraction corresponding to the 1.2~1.4 M NaCl eluate was identified based on its ability to induce branching morphogenesis of the isolated UB. After adjusting this fraction to 1.7 M ammonium sulfate (pH 7.2) it was subjected to further fractionation using a Resource phenyl sepharose hydrophobic interaction column (1 ml; Amersham-Pharmacia). The flow- through was collected and 1 ml fractions of bound proteins were eluted with decreasing concentrations of ammonium sulfate (1.7 M-0 M). After buffer exchange, the individual fractions were again tested for their ability to induce UB branching morphogenesis.

The morphogenetically active fractions from the hydrophobic interaction column were diluted 10-fold with 50 mM HEPES and applied to a Resource S cation exchange column (1 ml; Amersham-Pharmacia). The flow-through was collected and individual 1 ml fractions of bound proteins were eluted using increasing NaCl concentrations (0 M-2.0 M) and assayed for their ability to induce branching morphogenesis.

The active fractions from the Resource S cation exchange column were subjected to further fractionation using a Superdex 200 gel filtration column (Amersham-Pharmacia). Individual 1 ml fractions were collected and assayed for morphogenetic activity. In addition, the active fractions from the Resource S cation exchange column were subjected to SDS-PAGE and the proteins were visualized using coumassie blue (Colloidal Coumassie; Invitrogen) staining. Individual protein bands were cut out of the gels and submitted for microsequencing. Sequence analysis of the protein bands was performed at the Harvard Microchemistry Facility by microcapillary reverse-phase HPLC nano- electrospray tandem mass spectrometry (μLC/MS/MS) on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer.

RESULTS

Example 5

Conditioned medium secreted by metanephric mesenchyme-derived cells is required for isolated UB branching morphogenesis.

To identify mesenchymal factors that induce branching morphogenesis of the ureteric bud (UB), Inventors employed a metanephric mesenchyme (MM)-derived cell line (BSN cells) as a substitute for the embryonic MM (Sakurai et al., 1997a). These cells were derived from the embryonic day 11.5 (El 1.5) MM from a SV40 large T-expressing transgenic mouse and have been extensively characterized. BSN cells are positive for vimentin and negative for cytokeratin, E-cadherin and ZO-1 by immunostaining, as well as negative for Dolichos biflorus lectin-binding. By PCR the cells express WT1 and are negative for c-ret (Sakurai, unpublished observations), they also express mRNA for growth factors such as HGF and TGFβ by northern blot (Sakurai et al., 1997a). cDNA array analysis has confirmed their non-epithelial character (Pavlova et al., 1999). Most importantly, conditioned medium elaborated by BSN cells (BSN-CM) has been shown to act similar to the MM: it induces branching morphogenesis of cultured UB cells and the isolated UB (in the presence of GDNF).

UBs isolated from El 3 rat embryos, when suspended in an extracellular matrix gel and cultured in the presence of BSN-CM (with GDNF), grew to form impressive multiply branching tubular structures comparable to those seen in in vivo kidney development (though the growth was non-directional) (Fig. lb). As previously shown (Qiao et al., 1999a), in the absence of BSN- CM, however, the UBs failed to develop (Fig. Ic). Thus, BSN-CM apparently contains an additional soluble factor(s) necessary for epithelial cell branching morphogenesis. Using this isolated UB culture model as an assay, Inventors attempted to purify the key morphogenetic factor present in the BSN-CM.

Example 6

Pleiotrophin is a morphogenetic factor present in BSN-CM

SDS-PAGE and silver staining of BSN-CM revealed the presence of many protein bands (Fig. 2A). Liquid column chromatography was used to fractionate BSN-CM, and each fraction was tested for its ability to induce branching morphogenesis of the isolated UB. Of the multiple columns tested, a heparin sepharose column was found to adsorb most of the morphogenetic activity. Within this heparin-binding fraction, a fraction which eluted at a NaCl concentration of 1.2-1.4 M possessed particularly strong morphogenetic activity. Silver stain analysis of this fraction revealed the presence of prominent lower molecular weight (<20 kDa) protein bands (Fig. 2A). This active fraction was then applied to a Resource phenyl sepharose hydrophobic interaction column. A morphogenetic activity was eluted from this column at 1.4-1.2 M ammonium sulfate. Again, silver staining of this peak fraction revealed prominent low molecular weight protein bands (Fig. 2A). This active fraction was diluted 10-fold with 50 mM HEPES (pH 7.2) buffer and applied to a Resource S cation exchange column. The Resource S column chromatogram is shown in Fig. 2B. Each 1 ml fraction of the Resource S eluate was substituted for whole BSN-CM in the isolated UB culture and compared with BSN-CM itself. Of the 8 fractions eluted from the column, only Fraction 4, the peak protein fraction, induced significant UB morphogenesis (Fig. 2C, panel 4). SDS-PAGE analysis and silver staining of this peak fraction revealed the presence of a single protein band with an approximate molecular weight of 18 kDa (Fig. 2D, Lane 4). This protein band was subjected to in-gel digestion followed by tandem mass spectrometry and was identified as pleiotrophin. (This type of experiment was done 3 times during different purifications, and pleiotrophin was always detected by mass spectrometry). The presence of pleiotrophin in the active fraction (Fraction 4) was confirmed by immunoblot analysis using anti-pleiotrophin antibodies (Fig. 2E). The morphogenetic activity of individual fractions corresponded to the presence of pleiotrophin in that fraction, hi a similar fashion, further purification of the peak fraction from Resource S column was accomplished by applying the active fraction to a Superdex 200 gel filtration column. A single protein peak eluted at 15.93 ml (Fig. 3A), corresponding to a protein with a molecular weight of approximately 18 kDa, and was positive for pleiotrophin by immunoblot (Fig. 3B). This fraction induced isolated UB branching morphogenesis (Fig. 3C). Taken together, these results identify pleiotrophin as a morphogenetic factor present in BSN-CM.

Previous studies have found that pleiotrophin can be isolated to homogeneity from a conditioned medium elaborated by Swiss 3T3 cells (Sato et al, 1999). Thus, using this alternative purification procedure, a pure fraction of pleiotrophin was isolated from 3T3 conditioned medium (3T3-CM), as confirmed by silver stain, immunoblot analysis (Fig.s 4A and 4B) and mass spectrometry. Like the pleiotrophin that purified from BSN cells, this pure pleiotrophin was capable of inducing impressive branching moφhogenesis of the isolated UB (Fig. 4C, left panel). Thus, pleiotrophin purified from two different cell lines gave the same results. Interestingly, as Inventors have reported previously (Qiao et al, 1999a), ~10X concentrated whole 3T3-CM failed to induce branching morphogenesis of the isolated UB (data not shown), suggesting that 3T3-CM may contain an inhibitory factor. Nevertheless, to provide further confirmation that pleiotrophin is the factor inducing the morphogenetic changes observed in the isolated UB culture, Inventors took advantage of the documented ability of polyA-sepharose to adsorb pleiotrophin (Corbley, 1997). As seen in Figs 4A and 4B, treatment of purified pleiotrophin with polyA-sepharose beads results in the loss of detectable pleiotrophin, either by silver staining or immunoblot analysis. Importantly, this bead-depleted fraction was no longer capable of inducing UB branching moφhogenesis (Fig. 4C, right panel), providing further evidence that pleiotrophin is a moφhogenetic factor for UB branching moφhogenesis. It is worth adding here that insect cell-derived recombinant human pleiotrophin is incapable of inducing proliferation (Kurtz et al, 1995; Souttou et al, 1997; Zhang and Deuel, 1999), and in Inventors' experiments recombinant human pleiotrophin produced in the insect cell line (R&D systems) was also unable to induce UB branching moφhogenesis (data not shown). Example 7

The pattern of pleiotrophin induced UB morphogenesis depends upon its concentration.

During the course of purification, Inventors observed differences in the moφhology of the branching UB, depending upon the amount of pleiotrophin present in the fraction (detected by immunoblotting). This was examined more carefully using the purified protein in which the pleiotrophin concentration was determined by immunoblotting using recombinant human pleiotrophin as a standard. High concentration (>5 μg/ml) pleiotrophin resulted in robust proliferation with less elongation, while lower concentrations of pleiotrophin (156 ng/ml-2.5 μg/ml) induced dichotomous branching and elongation of the stalk (Fig. 5A), similar to that seen with whole BSN-CM. Example 8

Pleiotrophin and GDNF are required and sufficient to induce UB branching morphogenesis.

In the course of purification, variation in the inductive capacity of whole BSN-CM on UB branching was encountered. It was found that the addition of fibroblast growth factor 1 (FGFl) could potentiate the activity of the BSN-CM, although alone or in combination with GDNF it was not sufficient to induce isolated UB branching moφhogenesis (Fig. Ic). Based on this finding, the growth media (either BSN-CM or individual fractions) used in the culture of the isolated UB was supplemented with 250 ng/ml of FGFl . However, it was found that purified pleiotrophin supplemented with GDNF was capable of inducing UB branching moφhogenesis in the absence of FGFl, although the UB grew faster when FGFl was added to the culture (Fig. 5B). This result suggests that pleiotrophin and GDNF alone are necessary and sufficient for the observed branching moφhogenesis of the isolated UB, though a FGF-like activity could play a facilitory role in the process.

Example 9

Pleiotrophin also induces branching morphogenesis ofUB cells in three- dimensional culture. As discussed previously, it has been shown that El 1.5 mouse UB derived cells (UB cells) develop into branching tubular structures with lumens in the presence of BSN-CM. DNA array, PCR analysis and immunostaining have confirmed the epithelial and UB-like characteristics of these cells (Barasch et al, 1996; Pavlova et al, 1999; Sakurai et al, 1997a). Using this model for UB branching moφhogenesis, pleiotrophin was also capable of inducing the formation of branching structures of UB cells. As in the isolated UB culture model, the extent of UB branching moφhogenesis was found to be concentration-dependent, with higher concentrations resulting in more extensive growth and branching (Fig. 6A). Moφhologically, the structures were comparable to those induced by whole BSN-CM (Fig. 6B).

Example 10

Pleiotrophin is expressed in the embryonic kidney and secreted from MM- derived cells but not UB-derived cells.

By immunoblot, pleiotrophin was found in an extract of whole embryonic day 13 rat kidney (Fig. 7A, a). To determine whether epithelial cells or mesenchymal cells secrete pleiotrophin, conditioned medium derived from the UB cell line and the BSN cell line were compared. Only BSN-CM contained pleiotrophin (Fig. 7A, b). This is consistent with a previous in situ hybridization study (Vanderwinden et al., 1992), which showed that the developing rat kidney mesenchyme (as early as El 3 of development) expresses pleiotrophin mRNA, but the ureteric bud does not. Another study had suggested the presence of pleiotrophin in the basement membrane of epithelial tubules in the developing kidney of E13 mouse embryos (Mitsiadis et al, 1995). When Inventors examined frozen sections of mouse E13 kidneys stained with anti-pleiotrophin antibodies, a strong signal was observed in the basement membrane of the UB with weak staining in the surrounding MM (Fig. 7B). Since the MM expresses pleiotrophin mRNA at the earliest stages of kidney development (Vanderwinden et al, 1992), the data presented here suggest that pleiotrophin is secreted by the MM and binds to the basement membrane of the UB where it can exert its moφhogenetic function.

Example 11

Exogenous pleiotrophin affects UB morphology in embryonic kidney organ culture. While the spatiotemporal expression pattern and in vitro data from the isolated UB and the UB cell culture model strongly support a direct role for pleiotrophin in UB moφhogenesis, it was also important to determine its effect in a system that more closely approximates the intact developing kidney. Thus, Inventors applied pleiotrophin to whole embryonic kidney organ culture. Exogenously added pleiotrophin disproportionately stimulated growth of the UB (Fig. 8). Pleiotrophin-treated kidneys exhibited an expanded UB area in a concentration-dependent manner similar to that seen in the isolated UB culture (compare Figs 5A and 8). Furthermore, the central area of UB expansion became more prominent at higher concentrations of pleiotrophin. The whole kidney also appeared slightly larger following pleiotrophin treatment. Nephron induction visualized with PNA lectin appeared to be normal even in the presence of high concentrations of pleiotrophin (data not shown). Thus, not only isolated UB, but also the UB in the context of the whole embryonic kidney responded to pleiotrophin, supporting the notion that the UB is the target for pleiotrophin action in the developing kidney.

DISCUSSION

Early studies suggested an essential role for direct contact between the metanephric mesenchyme (MM) and the ureteric bud (UB) during metanephrogenesis. Induction of the isolated MM was reported to be inhibited by the placement of a filter with < 0.1 μm pore size between an inducer and the MM, suggesting an absolute requirement for cell contact between the MM and an inducer (Saxen, 1987). Recently, however, it has been demonstrated that a combination of soluble factors elaborated by an immortalized UB cell line supplemented with either fibroblast growth factor (FGF) 2, or a combination of FGF2 and transforming growth factor α are sufficient, in the absence of direct contact between the UB and MM, to induce the mesenchymal-epithelial transition and differentiation of the proximal nephron in cultures of isolated MM (Barasch et al, 1999; Karavanova et al, 1996). Likewise, it has recently been found that soluble factors produced by a MM cell line (BSN cells) supplemented with glial cell-derived neurotrophic factor (GDNF) are necessary and sufficient to induce extensive branching moφhogenesis of the UB (Qiao et al, 1999a). Thus, soluble factors play a key role in both aspects of the mesenchymal-epithelial interaction leading to the formation of a functionally mature kidney. This constitutes an important revision in thinking relating to kidney organogenesis.

The identification of specific MM-derived soluble factors mediating UB branching moφhogenesis remains a central question in this field. Hepatocyte growth factor (HGF) has been shown to induce the formation of branching tubular structures with lumens in three-dimensional cultures of epithelial cell lines derived from adult kidneys (i.e., MDCK and mlMCD cells) (Barros et al, 1995; Cantley et al, 1994; Montesano et al, 1991; Santos et al, 1993).

However, incubation of three-dimensional cultures of an embryonic cell line derived from the UB (UB cells) with HGF had only a slight moφhogenetic effect, and the formation of branching tubular structures with lumens was not observed (Sakurai et al, 1997a). Furthermore, HGF, alone or in the presence of GDNF, does not induce branching moφhogenesis of the isolated UB (as is seen with the MM cell conditioned medium) (Qiao et al, 1999a). These findings suggest that HGF is not an essential factor for early branching moφhogenesis of the embryonic UB, though it may play a facilitory role. This notion is supported by the fact that genetic deletion of hgf or its receptor (c- met) apparently has little if any effect on kidney development (Bladt et al, 1995; Schmidt et al, 1995).

Another group of soluble factors implicated in branching moφhogenesis of epithelial cells are the family of epidermal growth factor (EGF) receptor ligands. EGF receptor ligands are capable of inducing the formation of branching tubular structures with lumens in three-dimensional cultures of mlMCD cells, a kidney cell line derived from adult collecting duct cells (Barros et al, 1995; Sakurai et al, 1997b). However, as is the case with HGF, EGF receptor ligands are not capable of inducing the formation of branching tubular structures in three-dimensional cultures of the embryonically-derived UB cells (Sakurai et al, 1997a), nor are they capable of inducing branching moφhogenesis of the isolated UB (Qiao et al., 1999a). Deletion of the EGF receptor gene results in cystic dilation of collecting ducts in mice with certain genetic backgrounds, perhaps suggesting a role in final maturation of these structures (Threadgill et al, 1995). However, as with HGF, most experimental evidence indicates that the EGF receptor ligands are not essential for early steps in UB branching moφhogenesis.

In fact, among many growth factors hypothesized to play a role in kidney development, no single factor, or combination of factors, has been shown to induce UB cells to form branching tubular structures. Only the conditioned medium elaborated by the MM-derived cell line, BSN-CM, consistently induced UB cells in three-dimensional culture to form branching tubular structures with clearly distinguishable lumens (Sakurai et al, 1997a). Likewise, in the isolated UB culture system (in the presence of GDNF), no growth factor, alone or in combination, could induce the extensive branching moφhogenesis observed when the isolated UB was cultured with BSN-CM (Qiao et al, 1999a).

An essential role for GDNF in UB development is supported by a number of studies, including gene knockouts. For example, null mutations of gd f, its receptor, c-ret, or its co-receptor, gfr , result in abnormal kidney development, although variable phenotypes have been reported in the gdnf and c-ret knockout mice (Enomoto et al, 1998; Moore et al, 1996; Schuchardt et al, 1996). Moreover, although the prohferative effect of GDNF on UB cells has been debated (Pepicelli et al, 1997; Sainio et al, 1997), GDNF has been shown to initiate UB growth (Sainio et al, 1997), and it is required for branching moφhogenesis of the isolated UB (Qiao et al, 1999a). Nevertheless, GDNF is not sufficient to induce branching moφhogenesis of either the isolated UB (Qiao et al, 1999a) or cultured UB cells (Sakurai et al, 1997a), again consistent with the view that there are additional factors in BSN- CM which are critical to the branching moφhogenesis of the UB.

Studies in the developing mammalian lung and Drosophilα trachea indicate that members of the FGF family function in branching moφhogenesis of epithelial tissues (Hogan, 1999; Metzger and Krasnow, 1999; Zelzer and Shilo, 2000). Furthermore, null mutations of either fgfl oτfgflO have also been reported to affect kidney development (Ohuchi et al, 2000; Qiao et al, 1999b), although in both cases the kidneys appear to be modestly affected. For example, fgf7-mx\\ kidneys, there is a 30% reduction in the number of nephrons, and the kidneys appear to function normally (Qiao et al, 1999b). Moreover, since FGF7 is detected in the developing kidney only after several iterations of UB branching have already occurred, it is likely that other factors are necessary for the early steps of the branching program. In the case of FGF 10, the defect appears similar, although the phenotype has yet to be investigated in detail since the embryos die at birth due to severe lung defects (Ohuchi et al, 2000). Nevertheless, by potentiating the effect pf an essential branching moφhogen produced by the MM, certain FGFs may play a facilitory role in early UB branching moφhogenesis (see below).

In the present study, serial liquid column chromatographic fractionation of BSN-CM lead to the isolation of an active moφhogenetic fraction that contained an apparent single protein (capable of inducing branching moφhogenesis comparable to whole BSN-CM). This protein was identified as pleiotrophin (Fig. 2). Immunoblot analysis of BSN-CM (Fig. 7A) as well as in situ hybridization data of developing kidney (Vanderwinden et al , 1992), clearly demonstrated that the embryonic MM is a source of pleiotrophin. In addition to its ability to induce branching moφhogenesis in the isolated UB, pleiotrophin also induced a UB cell line to form branching tubular structures with lumens, and is thus the only soluble factor so far identified with this capability (Fig. 6). Based on these in vitro studies with the isolated UB as well as the UB cell line, Inventors propose that pleiotrophin could act as a UB moφhogenetic factor produced by the MM.

Pleiotrophin and another heparin binding growth factor, midkine, make up a distinct growth factor family. These proteins share about 50% sequence homology (Rauvala, 1989), both are expressed widely during organogenesis (Mitsiadis et al, 1995), and are highly conserved among species (Kurtz et al, 1995). Both have been implicated in neurite outgrowth (Li et al, 1990; Rauvala et al, 1994), a phenomenon that has some similarity to branching moφhogenesis (particularly as it occurs in cultured cells), and they exhibit a spatiotemporal expression pattern in other developing organ systems which suggest a role in mesenchymal-epithelial interactions (Mitsiadis et al, 1995). However, other than the finding that pleiotrophin enhances bone formation (Imai et al, 1998) and limb cartilage differentiation (Dreyfus et al., 1998), little is known about the role of pleiotrophin in organogenesis. It are important to confirm an in vitro role for pleiotrophin in branching moφhogenesis during epithelial organogenesis. To Inventors' knowledge, a pleiotrophin gene knockout has not been reported. However, an in vivo study, which utilized dominant-negative mutant chimera mice did suggest a role for pleiotrophin in spermatogenesis, although other organs including brain, kidney, and bone appear normal in these mice (Zhang et al, 1999). There has also been some question about the mitogenic activity of pleiotrophin (Hampton et al, 1992; Souttou et al, 1997; Szabat and Rauvala, 1996), which seems to be affected by the source and purification method (reviewed in (Zhang and Deuel, 1999)). Nevertheless, in Inventors' studies, pleiotrophin freshly purified to apparent homogeneity from either BSN cells or 3T3 cells induced impressive growth and branching moφhogenesis of the isolated UB.

A wide range of concentrations of pleiotrophin has been reported to exhibit biological activity (up to 50 μg/ml) in various systems (Imai et al, 1998; Li et al, 1990; Rauvala et al, 1994; Souttou et al, 1997). Pleiotrophin binds to the extracellular matrix, which may explain why concentrations of 200-600 ng/ml were required for moφhogenetic activity in the systems employed in Inventors' study (Figs 5A and 6). The UB cells and isolated UB were cultured within basement membrane Matrigel, which could conceivably bind a large fraction of the pleiotrophin. It seems improbable, though not inconceivable, that another protein could have co-purified with pleiotrophin through 4 very different chromatography steps and not been detected by silver staining and microsequencing. If such a protein were there, it would have to possess moφhogenetic activity in the subnanogram to nanogram range and have physical properties (i.e. size, charge, hydrophobicity) very similar to pleiotrophin.

To date, several glycoproteins, including brain-specific proteoglycans, the receptor type tyrosine phosphatase beta (Maeda and Noda, 1998; Meng et al., 2000) and syndecan-3 (Raulo et al, 1994) have been postulated to function as receptors for pleiotrophin. The UB has been shown to express syndecan-1 (Vainio et al, 1989), and while pleiotrophin is capable of binding to the syndecan-1 (Mitsiadis et al, 1995), it remains to be determined whether syndecan-1 mediates pleiotrophin binding and signal transduction during UB branching moφhogenesis. Whether proteoglycans serve as co-receptors for pleiotrophin, as is the case for FGF signaling (Schlessinger et al, 1995), or whether they directly transduce the pleiotrophin signal is presently unclear.

The possible involvement of proteoglycans in pleiotrophin-mediated branching moφhogenesis of the UB is particularly interesting in light of several studies demonstrating the importance of proteoglycans in UB development (Bullock et al, 1998; Davies et al, 1995; Kispert et al, 1996). In these studies, chemical or genetic depletion of sulfated proteoglycans inhibits UB branching moφhogenesis, and this is accompanied by decreased GDNF expression, and loss of c-ret at the UB tips (Bullock et al, 1998; Kispert et al, 1996). Even at early time points, when c-ret expression is still preserved, addition of exogenous GDNF alone does not completely restore UB branching moφhogenesis (Sainio et al, 1997), suggesting that other molecules are required in this process. One possibility is that depletion of sulfated proteoglycans also affects pleiotrophin-mediated signaling or binding.

Together, Inventors' results suggest that pleiotrophin functions as a MM-derived moφhogen acting upon the UB. Moreover, the results support the idea that UB branching moφhogenesis is likely to be regulated by more than a single factor. At least two soluble factors, GDNF and pleiotrophin are necessary for the moφhogenetic changes. GDNF may initiate the UB outgrowth (Sainio et al, 1997), and pleiotrophin may induce proliferation and/or facilitate branching (Figs 5 and 8). Whether pleiotrophin acts primarily through control of epithelial proliferation, survival, or elongation/branching requires further study. In vivo loss of function studies could potentially clarify the exact role of pleiotrophin in UB branching moφhogenesis. In addition, Inventors' data suggests that a FGF family member may play a facilitory role, since FGFl potentiates the effects of purified pleiotrophin on UB branching moφhogenesis, though by itself (with GDNF present) exerts little if any moφhogenetic activity. There may also be a similar set of inhibitory factors, which may include members of the transforming factor-beta family (Sakurai and Nigam, 1997). As previously argued (Nigam, 1995), gradients of positive and negative factors, most of which are matrix-bound, may exist in the mesenchyme and stroma. By regulating proliferation, apoptosis and the expression of moφhogenetic molecules at branch tips, branch points and stalks, the global and local balance of these stimulatory and inhibitory factors could be a crucial determinant of branching patterns during collecting system development. In addition, it is likely that sulfated proteoglycans must also be present either to maintain expression of these soluble factors or to secure their binding sites. At later stages, other soluble factors such as HGF and/or EGF receptor ligands may play supplementary roles, either during branching (particularly in the later stages) or shaping/maturation of tubular structures.

Lastly, it should be noted that the concentration-dependent moφhogenetic changes induced by pleiotrophin in the UB (Fig. 5A), raise the possibility that pleiotrophin represents a classical "moφhogen", in the sense of activin in early Xenopus development (Green and Smith, 1990). Such a molecule might be expected to produce different phenotypic changes in the responding tissue depending upon the concentration of the molecule to which it is exposed. In this regard, the basement membrane of the developing UB, to which pleiotrophin is localized, could potentially act as a "reservoir." Release of pleiotrophin from the basement membrane at the UB tips, perhaps through digestion by matrix degrading proteases, could produce a local concentration gradient, resulting in increased growth and proliferation of tips, while lower amounts of pleiotrophin along the length of the stalk would appear to induce elongation of the forming tubule. Such a concentration gradient of pleiotrophin could provide a basis for modulating the shape and directionality of the developing UB.

OTHER HEPARΓN-BINDΓNG GROWTH FACTORS

Example 12

Activities in BSN-CM are trypsin sensitive, heat sensitive, and larger than 8 kD.

When BSN-CM was treated with trypsin or exposure to prolonged heat (100 °C > 30 min), the moφhogenetic activity for the UB was completely abolished. Based on this result, it is likely that the moφhogenetic factor(s) in BSN-CM is protein in nature. Centricon filtration systems with different nominal molecular weight cutoffs were used to concentrate BSN-CM. Centricon filters with a 8 kD molecular mass cutoff membrane maintained biological activities in the retained fraction but not in the flow-through, suggesting the moφhogenetic activity is larger than 8 kD.

Example 13

Multiple morphogenetic factors are likely to be present in BSN-CM.

As we have already published, the moφhogenetic factor is heparin binding. Thus, a heparin affinity column (Hitrap Heparin; Amersham- Pharmacia) was first employed. Each fraction was assayed in isolated UB culture system in the presence of GDNF and FGF-1. Strong proliferative/moφhogenetic activity was observed in the fractions eluted with 0.9-1.25 M NaCl (Figure 9). These moφhogenetically active fractions were adjusted to 1.7 M ammonium sulfate and were applied to the Phenyl Sepharose column at pH 7.2. Isolated UB culture showed that several different activities were present in fractions eluted between 1.5-0.7 M ammonium sulfate. The 1.5-1.35 M eluate (fraction 6 in Figure 10) induced UB proliferation but had little effect on branching tubule formation or elongation. In contrast, the 0.9-0.7 M eluate (fraction 10 in Figure 10) exhibited branching moφhogenesis and elongation, but less robust proliferation. Interestingly, the activity found in fractions 7-9 suggested a combination of both fraction 6 and 10. This result suggests that although full-blown branching moφhogenesis (as seen in the UB culture in fraction 9) may require a combination of multiple factors (i.e., a mainly prohferative factor present in fraction 6 plus a mainly elongation/branching factor present in fraction 10), individual factors can be separated and purified. In fact, by SDS-PAGE and silver staining (Figure 10, lower left panel), fraction 6, which appears to be mainly prohferative, contains a few bands clustered between 18-31 kD, while fraction 10, which appears to promote elongation and branching, contains only one band visible at 31 kD.

Example 14

Sequential use of liquid chromatography columns has lead to the isolation of PTN and narrowed down non-PTN morphogenetic activity.

Sequential use of a hydrophobic interaction column, a cation exchange column, and a gel filtration column has lead to the purification of PTN from these heparin-bound active fractions. However, as discussed above, BSN-CM is likely to contain more than one moφhogenetic factors. In fact, while higher salt eluate fraction (fraction 6 in Figure 11) from phenyl sepharose column contained PTN by western blotting, lower salt eluate fraction from phenyl sepharose column (fraction 10 in Figure 12) did not. i addition, when moφhogenetically active fractions eluted from heparin column (adjusted to Tris HCl buffer pH 8.0) was applied to an anion exchange (Q) column, moφhogenetic activity was eluted at 0.15-0.5M NaCl fractions (4 and 5 in Figure 11). This moφhogenetic activity was preserved after applying these fractions to a gel filtration column. This Q column-bound activity is unlikely to be PTN because PTN (pl=9.3) should not bind to Q column at pH 8.0. By microsequencing analysis, a heparin binding growth factor heregulin was present in these fractions. This result was further confirmed by western blotting, which was positive for heregulin alpha in these fractions. Recombinant human heregulin alpha (250 μg/ml) induced isolated UB to grow to the similar moφhology as fractions 4 and 5 in Figure 11 in the presence of GDNF and FGFl . Thus, it is very likely that heregulin is one of the factors that induce UB growth.

Example 15

Existence of morphogenetic factors other than pleiotrophin

Heparin-bound fraction of BSN-CM is likely to contain many moφhogenetic/growth- promoting factors other than PTN. Existence of such factors is highly likely for the following reasons: 1) an active fraction eluted from anion exchange (Q) column is not likely to contain PTN (see Figure 11 ); 2) A fraction eluted from phenyl sepharose column at 0.7 M ammonium sulfate (fraction 10 in Figure 10), which induced elongation and branching of the UB tubules, should not contain PTN. Considering the relatively low resolution of hydrophobic interaction column, the existence of very low concentrations of PTN cannot be excluded, however, a dose dependent response suggests that it is unlikely that such a low concentration of PTN can induce UB moφhogenesis observed (see attached reprint); and 3) A moφhogenetically active fraction containing little, if any, PTN by western blotting was obtained by sequential chromatography over 3 columns including heparin sepharose column (Figure 12). A METHOD FOR 22V VITRO KIDNEY ENGINEERING AND LONG TERM PROPAGATION

In another embodiment, the goal is to create clonal subcolonies of specifically engineered, functional "designer" kidneys that are suitable for xenofransplantation. Inventors take advantage of their laboratory's expertise in mechanisms of normal renal development by utilizing techniques developed in their lab to isolate and nurture individual components involved in kidney development. Normal kidney development consists of the reciprocal interaction between the embryonic ureteric bud (UB) and the metanephric mesenchyme (MM), and the mechanisms involved in UB moφhogenesis have largely been worked out in Inventors' lab. This new proposed work is aimed at translating these discoveries to create numerous in vitro "designer" kidney from a single progenitor and consist of several clearly defined steps:

Background

Embryonic kidney development is initiated when the metanephric mesenchyme (MM) induces an epithelial outgrowth of Wolffian duct, termed the ureteric bud (UB). The MM induces the UB to elongate and branch, and through multiple iterations of this branching program, the UB subsequently develops into the renal collecting system. In turn, the branching UB initiates the reciprocal induction of the MM and stimulates it to epithehalize and to form the tubular nephron. By a poorly defined mechanism, these nephrons then connect with the UB-derived collecting system, allowing drainage of urine into the bladder. This process is repeated through successive iterations to achieve the approximately 1 million nephrons present in the adult human kidney.

For many years, this process of reciprocal induction was thought to depend on direct cell contact between the MM and UB. Although mesenchyme cleanly isolated from the UB could be induced to form tubules by nonspecific inducers such as spinal cord, the UB was not able to undergo branching morphogenesis in vitro when isolated from the surrounding mesenchyme. Several years ago, Inventors developed a breakthrough model system whereby the isolated UB undergoes impressive branching moφhogenesis in vitro when exposed to several growth factors ( Qiao, J., Sakurai, H., and Nigam, S. K. (1999)). These factors include glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor- 1 (FGFl), and proteins secreted by a mesenchymally- derived cell line, such as pleiofrophin. In addition, Inventors have since defined several of the key regulatory processes that govern UB branching moφhogenesis, such as the matrix-binding requirements vis a vis integrin expression, the dependence of branching moφhogenesis on heparan sulfate proteoglycans, and the roles of positive and negative modulators of branching ( Bush, K. T., et al (2001), Steer, D. L., et al (in prep)). Using high throughput gene analysis with Afrymetrix GeneChips, Inventors have also analyzed the genetic regulatory processes that underlie whole kidney development, isolated ureteric bud branching moφhogenesis, and isolated mesenchymal induction and epithelial differentiation ( Stuart, R. O., Bush, K. T., and Nigam, S. K. (2001)). In addition, other groups have recently defined growth factors present in media conditioned by ureteric bud cells that can induce differentiation of isolated mesenchyme cultured in vitro, such as leukemia-inhibitory factor (LIF) and FGF2 ( Barasch, J., et al (1999)). Whole embryonic kidneys transplanted into adult animals are tolerated and become functional ( Rogers, S. A., et al (1998)).

These advances have lead to the possibility of recombination of subcultures of each of the components of the kidney - the ureteric bud and the mesenchyme. As described below, recent work in Inventors' lab has shown that the isolated UB and mesenchyme can be recombined in vitro and grow in an autonomous fashion. The resultant kidney is moφhologically and architecturally indistinguishable from a "normal" kidney and potentially could be transplanted. Furthermore, Inventors can partition the kidney or the cultured isolated ureteric bud into smaller fragments and support the in vitro development of these subtractions through several "generations." Inventors then are able recombine these subtractions with fresh mesenchyme and witness induction of the mesenchyme and growth of the UB as in an otherwise normal kidney. Furthermore, these nascent nephrons formed contiguous connections with limbs of the branched UB. Consequently, Inventors may be able to develop a population of renal primordia suitable for transplantation and derived from a single progenitor.

Here Inventors utilize a novel, in vitro, approach to renal engineering that should lead to the ability to create colonies of "designer" kidneys suitable for xenofransplantation. In Inventors' approach, the embryonic ureteric bud is separated from the surrounding metanephric mesenchyme and each component is cultured in isolation. The UB and/or the MM is then modified in vitro in a tailored fashion to express a specific function and the components are recombined. The designer neo-kidney is then transplanted into host animals and functionality is assessed.

Significance

End-stage renal disease (ESRD) affects almost 350,000 people living in the United States with an incidence that has increased by over 50% in the past decade. Total Medicare expenditures on patients with ESRD exceed $11.3 billion ( USRDS. (2001 )). The two treatment modalities for ESRD, dialysis and transplantation, both have significant limitations. Patients on dialysis have an extremely high mortality rate, approaching 20% per year. Patient survival is markedly improved with renal transplantation; however, the number of renal transplants is severely limited by the short supply of available organs. Most patients with ESRD do not have a suitable living donor for transplantation. In addition, the number of cadaveric transplants performed in the United States has increased by less than 1000/year over the past decade ( USRDS). For this reason, the waiting list for renal transplants continues to grow and many patients die while awaiting a new kidney. Recently, several alternative modalities have been proposed. Other groups have demonstrated the functional viability of transplantation of embryonic metanephroi into adult animals. In one model, kidneys from embryonic rats are transplanted into an omental pouch created in the adult host animal. These prevascular donor kidneys are able to recruit a mostly host-derived vasculature and can form mature, functioning tubules. After several weeks of growth in the omental pouch, the transplanted kidneys are surgically connected to the host's ureter and can clear inulin and concentrate urine. Significantly, there appears to be a state of peripheral immune tolerance to transplantation of embryonic kidneys, and transplantation across the rat MHC does not appear to require adjuvant immunosuppression ( Hammerman, M. R. (2002); Rogers, S. A., and Hammerman, M. R. (2001)).

Inventors' lab has been focused on critically examining the interactions between epithelial tissues and surrounding mesenchymal tissues that underlie the molecular biology of kidney development. This has led to several fundamental advances in the field of developmental biology and Inventors feel that it is now time to translate these advances to clinical application. Therefore, Inventors propose to utilize several recent discoveries made in Inventors' lab that could markedly increase the therapeutic potential of a xenofransplantable kidney and provide significant competitive advantages over other proposed techniques. Preliminary Studies (see Figures 13 - 15)

In preliminary studies, Inventors have dissected the embryonic UB and separated it from the surrounding metanephric mesenchyme (MM) (Figure 13). They have been able to grow an arborized structure from the isolated UB, subdivide it into smaller fractions and induce additional generations of UB cells that grow and branch in vitro. Days 0, 5 and 8 shown. Preliminarily, Inventors were able to carry the subfractionation of the UB through three generations. The UB generations were then recombined with freshly isolated metanephric mesenchyme, and they retained the ability to induce dramatic tubular epithelial differentiation of the mesenchyme (Figs 14 and 15). Days 0, 2 and 5 shown. Furthermore, there appeared to be connections between induced tubules of the mesenchyme and terminal portions of the UB thereby providing a conduit between the tubule and urinary collecting system. The regenerated kidney thus generated opens up the possibility of uniquely tailoring specific components of either the nephron (derived from the mesenchyme) or the collecting system (derived from the UB) in vitro in a potentially functional and transplantable organ.

Although the concept of transplanting fetal organs is not novel, there are several advantages to the approach outlined here. First, by culturing the UB and MM in vitro, Inventors have the unique opportunity to modulate each of their functions in a site-specific manner. For example, transfection of the mesenchyme with constructs expressing organic ion transporters could lead to increased capability to handle drugs and toxins; insertion of genes coding for growth factors, such as insulin-like growth factor (IGF), could lead to markedly enhanced neo-kidney development and improved functionality; insertion of immunomodulatory elements, such as repressors of co-stimulatory molecules, could lead to improved immune tolerance; stimulation of branching in the UB could lead to an increased number of resultant nephrons and improved renal functionality. Thus, there are numerous of ways to design a neo-kidney with tailored function.

Second, by subcloning either kidneys or UBs, Inventors have the potential to develop a large number of kidneys derived from a single progenitor. Although the concerns surrounding limited supply of xenotransplantable tissue are less than that of allo-organs, the "clonality" of the designed neo-kidneys may lead to some distinct advantages.

Third, it may be possible to create a chimeric kidney using the UB as a scaffold and recombined with xeno-derived mesenchymal cells. These mesenchymal cells could be derived from embryonic stem cells that, when exposed to kidney-derived signals emanating from the UB, are induced to differentiate as renal mesenchymal cells and then undergo epithelialization. In normal adults, stem cells originating in the bone marrow repopulate portions of the kidney and differentiate into renal cells, and it is likely that embryonic stem cells also posses this ability. If it were possible to create such a chimeric kidney, it would greatly decrease the likelihood of immunologic problems that currently make xenofransplantation difficult.

Methods

Materials

Tissue culture media are obtained from Mediatech and bovine fetal calf serum obtained from Biowhittiker. Growth factor reduced Matrigel and Type I collagen are obtained from Becton Dickenson. FGFl and GDNF are obtained from R&D systems. FITC-conjugated DB are obtained from Vector Laboratories. Example 16

Generation of conditioned media

The Cellmax artificial capillary cell culture system is inoculated with BSN cells as previously described in Qiao, et al, 1999, and conditioned media harvested according to the manufacturers instructions.

Example 17

Culture and subculture of whole embryonic kidney

Uteri from timed pregnant Sprague-Dawley rats corresponding to gestational day 13 are harvested and embryos isolated. Embryonic kidneys are isolated and cultured on top of a Transwell filter in the presence of DMEM F12 media supplemented with 10% FCS. The kidneys are cultured at 37C in fully humidified 5% C02 atmosphere. At the specified time intervals, the embryonic kidneys were sectioned into thirds and subcultured on filters and with fresh media.

Example 18

Culture and subculture of isolated ureteric buds

Isolated ureteric buds are obtained from whole embryonic kidneys as previously described. Briefly, the embryonic kidney is digested with trypsin and the UB separated from the MM using fine-tipped needles. The UBs are suspended within a matrix containing growth factor reduced Matrigel and Type I collagen and buffered by HEPES, NaHC03, and DMEM to a pH of approximately 7.2. This mixture containing the suspended UB is applied to the top of the Transwell filter and BSN-conditioned media added to the well. The BSN conditioned media is supplemented with GDNF (125ng/ml) and FGFl (31 ng/ml) and 10% FCS, and the isolated UBs cultured at 37C and humidified 5% C0₂ atmosphere. At specified time intervals, the cultured UB is separated from the surrounding matrix by blunt microdissection, sectioned into thirds, resuspended in new matrix and cultured with fresh supplemented BSN conditioned media.

Example 19

Culture of isolated mesenchyme

Isolated metanephric mesenchyme is isolated as described above and cultured on top of the Transwell filter. DMEM/F12 media supplemented with FGF2 (lOOng/ml) and TGFα (10 ng/ml) is added to the well to prevent MM apoptosis as previously described.

Example 20

Recombination experiments

Using blunt microdissection with fine tipped needles, cultured or subcultured UBs are cleanly separated from surrounding matrix and placed on top of a Transwell filter in close proximity to MM that is either freshly isolated or cultured. BSN conditioned media supplemented with GDNF, FGFl and 10% FCS was added to the well.

Example 21

Immunofluorescence

Cultured or subcultured embryonic kidneys, isolated buds, and recombined kidneys are fixed in 4% paraformaldehyde and processed for immunofluorescent staining with either FITC-conjugated DB or antibodies. Immunofluorescence is detected with a Zeiss laser-scanning confocal microscope.

Summary

The care of end-stage renal disease in the United States is an expensive endeavor with limited treatment options. Novel therapies present the opportunity for significant clinical improvement as well as new commercial opportunities. Inventors' laboratory is particularly well-suited for translating the significant advances in the developmental biology of kidney development into new clinical approaches to treating renal disease. The approach detailed above, whereby neo-kidneys are designed to possess specific functions, such as improved immune tolerance or enhanced tubular secretion of substrate, offer original approaches to xenofransplantation. Furthermore, creating clonal populations of neo-kidneys creates the potential for development of ex vivo organ propagation from a single tissue. This approach is potentially applicable to other epithelial tissues such as lung and pancreas.

Commercial Potential and Competitive Advantage

Large numbers of patients are awaiting renal transplantation. The development of techniques that allow for safe xenotransplantation, especially without excessive immunosuppression, would have enormous commercial application. Xenotranplantation of embryonic tissue appears to be well tolerated. Inventors' methodology allows for the development of colonies of subcloned neo-kidneys that have been specifically tailored to express certain functions. TREATMENT OF ACUTE RENAL FAILURE WITH TUBULOGENIC GROWTH FACTORS

A novel growth factor-based therapy for acute renal failure (ARF).

ARF is primarily caused by acute tubular necrosis (ATN). ATN is manifested by renal cell death, and the recovery from ATN is characterized by a recapitulation of early developmental processes, particularly growth factor- induced tubulogenesis. Inventors have identified several key mediators of kidney development and are poised to explore their therapeutic potential in the treatment and prevention of ARF.

Background

Acute renal failure (ARF) is a common condition with a poor outcome. Nearly 200,000 patients in the United States develop ARF annually and the mortality rate remains high in spite of improved techniques of renal replacement therapy. At the present time, treatment of ARF is primarily supportive since no specific therapy is available. Recently improved understanding of the cellular and molecular mechanisms of ATN may be translated into new therapeutic approaches.

The most common etiologies of ARF are renal ischemia or the administration of a nephrotoxic drug. In many cases, severe ARF results from a combination of renal hypoperfusion and nephrotoxicity. Severe ARF is associated with injury to renal tubular cells leading to both cellular dysfunction and ultimately cell death. Thereafter, recovery from ARF is characterized by de-differentiation of the tubular epithelium, proliferation, and regeneration of the tubular cell. In many respects, these recovery processes recapitulate the processes fundamental to normal embryonic renal development, such as growth factor expression, matrix digestion, and intercellular tight junction modification(Nigam, S., and Lieberthal, W. (2000); Lieberthal, W., and Nigam, S. K. (1998); Lieberthal, W., and Nigam, S. K. (2000); Sakurai, H., et al (2001)).

The major aim herein is to determine if factors that play a role in the development of the kidneys in utero also are important in the recovery of the kidneys from injury. Two major foci of Inventors' laboratory are the developmental biology of the kidney and the cell biology of epithelial injury. Inventors have recently identified several growth factors, notably pleiotrophin, which have significant roles in normal renal development. Because of the magnitude of the problem of ARF and some tantalizing early studies, a great deal of effort has been expended in both academic and commercial arenas on the possibility that growth factors might be "magic bullets" in the setting of acute kidney injury. Nevertheless, even though multiple growth factors have been tried, consistent success in animal models and the clinic has not been achieved. Inventors have argued in editorial forums (Nigam, S., and

Lieberthal, W. (2000)) that the reason for these inconsistent results is that most of the growth factors used so far, though tubulogenic in vitro, are relatively weak stimulators of branching tubulogenesis. The most potent in vitro tubular moφhogens in this regard appear to be those that Inventors have purified and are continuing to purify from an embryonic kidney cell line ( Sakurai, H, et al (2001); Sakurai, et al (1997)). The conditioned medium from this cell line (BSN-CM), as well as purified subfractions, appear to contain among the most powerful tubulogenic and prohferative growth factors known and have not, to Inventors' knowledge, been studied in the context of acute renal failure. There is a very strong correlation between known branching tubular moφhogens and enhancement of recovery in experimental acute renal failure (see Table 1, at end)( Bush KT, S. H., Tsukamoto T. (1999)); but again, Inventors argue that only the less potent growth factors (in this context) have thus far been tried in ARF. This may be due to the only recent identification of the most potent in vitro tubulogenesis factors such as pleiotrophin (largely through the efforts of Inventors' group); in fact, using the assays described below, Inventors expect to fully purify the other potent activities over the next year or two. In fact, a provisional patent application for the use of pleiotrophin and other heparin binding tubulogenic growth factors in the context of acute renal failure has been filed by UCSD.

Inventors' laboratory also has a long history of studying the cell biology of epithelial injury using cell culture models of ATP depletion, thermal injury and oxidative stress. In previous work, Inventors have demonstrated that proteasome inhibitors and agents such as tunicamycin are cytoprotective and current efforts are underway to examine the cellular and molecular basis for this protective effect.

During normal kidney development, the MM secretes a variety of soluble factors that induce branching moφhogenesis of the epithelial ureteric bud (UB). Inventors' laboratory has recently identified potent tubulogenic factors in media conditioned by an immortilized cell line derived from the metanephric mesenchyme. When the UB is cleanly isolated from its surrounding mesenchyme, these factors have the remarkable ability to induce branching moφhogenesis independent of mesenchymal-cell contact (Qiao, J., et al (1999)). Furthermore, these factors can induce tubulogenesis when applied to a 3-dimensional culture of immortalized cells derived from the UB (Fig 13). Finally, Inventors have recently identified the heparin-binding protein pleiotrophin as a major tubulogenic component of the BSN-CM (Figure 14)( Sakurai, H, et al (2001); Sakurai, et al (1997)). Inventors are currently identifying other factors found in BSN-CM that may provide co-stimulatory or modulatory functions to pleiotrophin. It seems only logical to test these potent tubulogenic factors in in vivo models of ARF.

Methods

Example 22

Animals

Adult male rats (weighing 200-250 grams) are housed and fed on standard rat chow, ad libitum water ingestion and 12-hour cycles of light and dark. All animals are maintained and experiments conducted in accord with the National Institutes of Health (NTH) "Guide for the care and Use of Lab Animals."

Example 23

Induction of renal ischemia/reperfusion injury

Rats are anesthetized with an intraperitoneal injection of sodium pentobarbitol solution (50 mg/kg). The anesthetized animals are placed on a warming blanket and a midline abdominal incision made. Bilateral or unilateral occlusion of the renal pedicule are maintained for 40 minutes to induce ischemia and the incision temporarily closed until completion of vascular occlusion. If an arterial catheter is required for the experiment, one are placed in the femoral artery and exteriorized in the dorsal scapular region. If ureteral catheters are necessary, they are placed and exteriorized. Upon completion of ischemic period, the arterial occlusion are removed, the incision are sutured or stapled closed and the rats allowed to recover for designated reperfusion times. Example 24

Induction ofnephrotoxic injury

Injury are induced with either mercuric chloride or the antibiotic gentamicin. Mercuric chloride primarily induces injury and subsequent cell proliferation in proximal straight tubules (PST), whereas gentamicin predominantly injures proximal convoluted tubules (PCT). Gentamicin nephrotoxicity are induced by I.P. injections of 40mg/ml in 0.9 percent saline, divided with three daily injections over two days for a total of 400 mg kg. Mercuric chloride are administered at various doses (0.25,0.5,1.0 and 2.5mg/kg). These doses have been reported to induce renal injury ranging from minimal to marked.

Example 25

Combined injury

To mimic the usual clinical situation, some rats are exposed to either gentamicin or mercuric chloride after the ischemic injury. It is anticipated that the renal injury are especially severe in these animals.

Example 26

Purification of factors involved in embryonic nephrogenesis: BSN cell conditional media (BSN-CM) is collected after 2 to 4 days of BSN cell confluency (Sakurai, 1997), spun at low speed to remove cell debris and filtered (0.22 um filter). The media is then concentrated (Vivaflow 200, 5kDa cutoff), subjected to sequential liquid column chromatography and elution techniques, and final purification accomplished with HPLC and SDS-Page electrophoresis. The final purified ρrotein(s) is (are) submitted for microsequencing to an out side vender.

Example 26

Identification of factor activity via culture ofthe isolated ureteric bud

Isolated ureteric buds are obtained from whole embryonic kidneys as previously described. Briefly, the embryonic kidney is lightly digested with trypsin and the UB is separated from the MM using fine-tipped needles. The UBs are suspended within a matrix containing growth factor reduced Matrigel and Type I collagen and buffered by HEPES, NaHC03, and DMEM to a pH of approximately 7.2. This mixture containing the suspended UB is applied to the top ofthe Transwell filter and the purified factor is applied to the well. The factor is supplemented with GDNF (125ng/ml) and 10% FCS, and the isolated UBs are cultured at 37C and humidified 5% C0₂ atmosphere and branching moφhogenesis is assayed.

Example 27

Plasma and urine analysis

Plasma collections during the experiment are collected via the rat tail vein under isoflurane anaesthesia. A large blood volume are collected at the end ofthe experimental period by exangination under pentobarbitol (50mg/kg) anaesthesia. Plasma from these collections are to be analyzed for sodium, potassium, ionized calcium, ionized magnesium (Nova 8 Electrolyte Analyzer), BUN and creatinine by autoanalyzer (core facility). Urine collection during and at the end ofthe experiment are done in metabolic cages. The urine are analyzed colormetrically for creatinine, calcium, magnesium, phosphate and chloride and protein. Sodium and potassium are measured with a Nova 6 Electrolyte Analyzer. Example 28

Histology

Cross sections of kidney from each rat are fixed on a microscope slide and stained with hematoxylin and eosin. Slides are read for the presence or absence of tubular epithelial degeneration and/or necrosis.

Example 29

Immunohistochemistry

Tubular injury and cell proliferation are assessed on PCNA/PAS sections. Staining are done on 5-μm paraffin sections from ethacarn-fixed renal tissue. Proliferating cells are immunostained with a rabbit anti-mouse monoclonal antibody (PC 10 from Dako) directed to proliferating cell nuclear antigen (PCNA). After blocking (goat sera) and incubation with the primary antibody, the sections are incubated with biotinylated goat-anti rabbit antiserum in the presence of normal rat serum and stained by the avidin-biotinylated horseradish peroxidase complex (Vectastain, Vector Labs) using 3,3 '- diaminobenzidine as the chromogen. Sections will then be counterstained with methyl green and periodic acid-Schiff (PAS).

Example 30

Identification ofapoptosis

Identification and determination of apoptosis are done using the terminal deoxynucleotidyl transferse (TdT)-mediated UTP-biotin nick-end labeling (TUNEL) technique by using an Apoptag in situ apoptosis detection kit (Oncor, Gaitheburg, MD). Frozen sections (5um) are fixed in 10% neutral- buffered Formalin and postfixed in ethanol: acetic acid at -20 C° for comparison to control tissue after described technique.

Example 31

Immunodetection and localization ofpeptide(s)

Determination if the factor(s) is also present in adult rat kidney: After purification of unique factor(s) an antibody are generated by immunizing rabbits with purified protein (Multiple Peptide Systems, San Diego, Ca.). Kidney homogenates after ischemic and/or nephrotoxin injury are fractionated on 4-15% SDS polyacrylamide gels under reducing conditions and transferred to PVDF membranes. After blocking with phosphate buffered saline containing 5% nonfat milk, the blots were incubated primary antibody (rabbit anti-rat peptide) and visualized by enhanced chemiluminescence system (Pierce). If peptide is present by western blot analysis then the polyclonal are used for immunohistochemical detection and localization.

Competitive Advantage And Commercial Potential

Inventors have pioneered many ofthe in vitro techniques used to identify branching tubulogenic growth factors, and although a number of groups are now employing them, Inventors believe Inventors are the only group that consistently has them working in robust fashion. Inventors also believe Inventors are considerably ahead of other groups in the use of these assays to purify novel growth factors involved in branching tubulogenesis. Inventors believe that these factors would have significant roles in ameliorating the course of ARF. As noted earlier, ARF is a common, expensive, and serious clinical problem with profound implications on patient mortality. Therapeutic options, other than supportive measures, which have thus far been unable to improve the associated mortality, would have enormous commercial potential, especially in intensive care unit settings. The abovementioned examples define new heparin-binding proteins necessary in producing an active, functional embryonic kidney, other epithelial organ, or fragment thereof.

While the present invention has now been described in terms of certain preferred embodiments, and exemplified with respect thereto, one skilled in the art will readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the spirit thereof. It is intended, therefore, that the present invention be limited solely by the scope ofthe following claims.

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Claims

CLAIMSWhat is claimed is:

1. A method for inducing and regulating epithelial organogenesis, comprising: contacting developing epithelial tissue with an effective amount of a solution containing one or more mesenchymally-derived growth factor secreted by mesenchymal tissue in culture; and permitting the growth factor or an analogue thereof to react with the epithelial tissue for a sufficient period of time to bind to the tissue, wherein binding ofthe growth factor induces and regulates epithelial organogenesis.

2. A method for inducing and regulating branching moφhogenesis that is required in epithelial organogenesis, comprising: contacting developing organ epithelial tissue with an effective amount of a solution containing one or more mesenchymally-derived growth factor secreted by mesenchyme tissue in culture; and permitting the growth factor or analogue to react with the epithelial tissue for a sufficient period of time to induce branching moφhogenesis, wherein binding of the growth factor triggers tubular moφhogenesis, and consequently organogenesis.

3. The method according to claim 2, wherein the mesenchyme tissue is metanephric mesenchyme.

4. The method according to claim 2, wherein the mesenchymally-derived growth factor is secreted by metanephric mesenchyme tissue in culture.

5. The method according to claim 2, wherein the mesenchymally-derived growth factor is a heparin-binding protein.

6. The method according to claim 2, wherein the heparin-binding protein is pleiotrophin.

7. The method according to claim 2, wherein the mesenchymally-derived growth factor is pleiotrophin in conditioned medium of a metanephric mesenchymal cell culture.

8. The method according to claim 2, wherein the conditioned medium is elaborated by BSN cells (BSN-CM) in culture.

9. The method according to claim 2, wherein the growth factor is pleiotrophin isolated from conditioned medium of a metanephric mesenchymal (BSN-CM) culture.

10. The method according to claim 7, wherein the growth factor is a recombinant pleiotrophin.

11. The method according to claim 8, wherein the growth factor is an active peptide.

12. The method according to claim 9, wherein the growth factor is a peptidomimetic.

13. The method according to claim 2, wherein the growth factor is a pleiotrophin analogue.

14. The method according to claim 2, wherein the growth factor is a synthetic pleiotrophin analogue.

15. The method according to claim 2, wherein the tubulogenesis is further facilitated by other factors.

16. The method according to claim 2, wherein the tubulogenesis is further facilitated by other co-factors.

17. The method according to claim 2, wherein the epithelial tissue is kidney tissue.

18. The method according to claim 2, wherein the epithelial tissue is lung tissue.

19. The method according to claim 2, wherein the epithelial tissue is prostate tissue.

20. The method according to claim 2, wherein the epithelial tissue is neural tissue.

21. The method according to claim 2, wherein the epithelial tissue is glandular tissue.

22. The method according to claim 21, wherein the tissue is salivary gland tissue.

23. The method according to claim 2, wherein the contacting is in vitro.

24. The method according to claim 2, wherein the contacting is in vivo.

25. The method according to claim 2, wherein the contacting is ex vivo.

26. A method for in vitro constructing of a functional mammalian epithelial tissue, organ or a fragment thereof, comprising: first, culturing and propagating embryonic epithelial explants, tissues or cells by isolating the explant tissues or cells and growing them in a culture, permitting the culture to form multiple branches, dissecting out individual tips ofthe branches, reculturing the branch tips in the presence of a mixture of serum, growth factor mix, conditioned medium and nutrient-rich medium for several generations; second, culturing and propagating isolated embryonic or fetal mesenchymal tissue by dissecting out fetal mesenchyme tissue at the time of induction, culturing the mesenchyme tissue in the presence ofthe mixture of any of serum, growth factor mix, conditioned medium and nutrient-rich medium, partitioning the mesenchyme tissue into multiple pieces and growing each piece separately, and inducing vasculogenesis by subjecting grown mesenchyme tissue to substrate deprivation or addition of soluble factors; third, recombining each vascularized mesenchyme tissue with each recultured branch tip in a matrix in which in vitro angiogenesis has begun; and growing in medium conditions suitable to ensure continued vasculogenesis.

27. A method for in vitro culturing and propagating ureteric bud tissue, comprising: isolating embryonic kidney rudiments by dissection, isolating ureteric bud tissue fragments from mesenchyme, suspending the isolated ureteric bud fragments in a gel matrix; placing the gel matrix containing the fragments on porous membrane inserts in wells of tissue culture plates; adding a growth factor mix to the wells ofthe culture plates; maintaining the ureteric bud fragments in the gel matrix at an interface of air and medium until the bud fragments form multiple tubular branches inside the gel matrix; dissecting out outermost individual branch tips ofthe bud fragments formed during culture; and reculturing the dissected branch tips in presence of a mixture of any of serum, growth factor mix, cell-conditioned medium and nutrient-rich medium for several generations until many tissue cultures of ureteric bud tissue are grown.

28. The method according to claim 27, wherein the growth factor mix comprises: a glial cell line-derived neurotrophic factor or functional equivalent thereof.

29. The method according to claim 27, wherein the mixture of any ofthe serum, growth factor mix, and the added cell-conditioned medium includes the cell-conditioned medium, comprising: a growth promoting constituent or inducer of differentiation or moφhogenesis.

30. The method according to claim 29, wherein the growth promoting constituent or inducer of differentiation or moφhogenesis is pleiotrophin.

31. A method for in vitro culturing and propagating metanephric mesenchyme tissue, comprising: dissecting out fetal kidney mesenchyme tissue at the time of induction; culturing said mesenchymal tissue in the presence of serum, growth factor mix, mesenchymal and/or bud cell conditioned medium and nufrient-rich medium; partitioning the cultured mesenchyme into multiple pieces and growing each piece separately in culture; and subjecting grown mesenchyme to substrate deprivation or addition of vasculogenic growth factors in order to induce vasculogenesis.

32. The method according to claim , wherein the vasculogenic growth factor is pleiotrophin.

33. A method for in vitro engineering and constructing a mammalian kidney, comprising: isolating embryonic kidney rudiments by dissection, isolating ureteric bud tissue fragments from mesenchyme, suspending the isolated ureteric bud fragments in a gel matrix; placing the gel matrix containing the fragments on porous membrane inserts in wells of tissue culture plates; adding a growth factor mix to the wells ofthe culture plates; maintaining the ureteric bud fragments in the gel matrix at an interface of air and medium until the bud fragments form multiple tubular branches inside the gel matrix; dissecting out outermost individual branch tips ofthe bud fragments formed during culture; and reculturing the dissected branch tips in presence of a mixture of any of serum, growth factor mix, cell-conditioned medium, pleiotrophin and nutrient- rich medium for several generations until many tissue cultures of ureteric bud tissue are grown; contacting each cultured bud with pleiotrophin in a matrix in which in vitro angiogenesis has begun; and growing in medium conditions suitable to ensure continued vasculogenesis.

34. The method according to claim 33, wherein the propagated tissues are members of a group of tissues containing essentially epithelial cells.

35. The method according to claim 34, wherein the propagated tissues implanted into a recipient comprise lung or salivary gland.

36. The method according to claim 33, wherein the propagated tissues are implanted into a recipient for the puφose of regeneration.

37. The method according to claim 36, wherein the propagated tissues are implanted into a recipient for the puφose of repair of tissues or organs suffering ischemic or toxic insult.

38. The method according to claim 33, wherein the propagated tissues are implanted into a recipient for the puφose of protection and growth of tissue for transplantation.

39. The method according to claim 33, wherein the propagated tissues are implanted into a recipient for the puφose of treatment of chronic epithelial tissue disease.

40. The method according to claim 33, wherein the pleiotrophin is administered by gene therapy.

41. The method according to claim 33, wherein the propagated tissues implanted into a recipient are contacted with pleiotrophin in vivo.

42. A functional mammalian kidney engineered and constructed in vitro, comprising: an isolated ureteric bud propagated in culture to produce a functioning nephron; and heparin-binding factor metanephric mesenchyme tissue propagated from cultured embryonic mesenchymal tissue fragments or cells wherein the functional nephron and the heparin binding factor are combined and placed in culture to form a functional kidney or functioning fragment thereof.

43. The method according to claim 42, wherein the heparin-binding factor is pleiofrophin.

44. A method for treating acute renal failure, comprising: contacting a subject's injured kidney tissue in vivo with tubulogenic growth factors.

45. The method according to claim 44, wherein the growth factors are heparin- binding growth factors.

46. The method according to claim 44, wherein the growth factor is pleiotrophin.

47. The method according to claim 44, wherein the growth factor heregulin.

48. The method according to claim 44, wherein the growth factor is infused into a recipient for the puφose of repair of tissues or organs suffering ischemic or toxic insult.