WO2018075622A1 - Materials and methods for modulating insulin signaling and preserving podocyte function - Google Patents

Abstract

The present disclosure is directed to the modulation of impaired insulin signaling by contacting a cell with a ceramide and methods of preserving podocyte function.

Description

MATERIALS AND METHODS FOR MODULATING INSULIN SIGNALING AND

PRESERVING PODOCYTE FUNCTION

FIELD OF THE INVENTION

[0001] The present disclosure is directed to the modulation of impaired insulin signaling by contacting a cell with a ceramide and methods of preserving podocyte function.

BACKGROUND

[0002] Diabetes refers to a disease process characterized by elevated levels of plasma glucose or hyperglycemia in the fasting state or after administration of glucose during an oral glucose tolerance test. Persistent or uncontrolled hyperglycemia is associated with increased and premature morbidity and mortality. Often abnormal glucose homeostasis is associated both directly and indirectly with alterations of the lipid, lipoprotein and apolipoprotein metabolism and other metabolic and hemodynamic disease. Therefore patients with diabetes mellitus are at especially increased risk of macrovascular and microvascular complications, including coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy, and retinopathy.

[0003] There are two generally recognized forms of diabetes. In type 1 diabetes, or insulin- dependent diabetes mellitus (IDDM), patients produce little or no insulin, the hormone which regulates glucose utilization. In type 2 diabetes, or noninsulin dependent diabetes mellitus (NIDDM), patients often have plasma insulin levels that are the same or even elevated compared to nondiabetic subjects; however, these patients have developed a resistance to the insulin stimulating effect on glucose and lipid metabolism in the main insulin-sensitive tissues, which are muscle, liver and adipose tissues, and the plasma insulin levels, while elevated, are insufficient to overcome the pronounced insulin resistance. Insulin resistance is not primarily due to a diminished number of insulin receptors, but is due to a post-insulin receptor binding defect that is not yet fully understood. This resistance to insulin responsiveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle and inadequate insulin repression of lipolysis in adipose tissue and of glucose production and secretion in the liver.

[0004] The abnormally high blood glucose (hyperglycemia) that characterizes both type 1 and type 2 diabetes, if left untreated, results in a variety of pathological conditions, including premature blindness, nerve damage, cardiovascular disease, stroke, and kidney failure (Sheetz and King, JAMA 288:2579-2588 (2002)). For example, diabetic nephropathy is a major long- term complication of diabetes mellitus, and is the leading indication for dialysis and kidney transplantation in the United States (Marks and Raskin, Med. Clin. North Am. 82:877-907 (1998)). The development of diabetic nephropathy is seen in 25 to 50% of type 1 and type 2 diabetic patients. Accordingly, diabetic nephropathy is the most common cause of end-stage renal disease and kidney failure in the Western world. Diabetic nephropathy affects

approximately 20-40% of all individuals with diabetes (American Diabetes Association (2009) Diabetes Care 32:S 13-S61; herein incorporated by reference in its entirety), and is the single most common cause of end-stage renal disease (ESRD) in the United States, leading to the need for dialysis. Risk for the development of diabetic nephropathy is directly related to the cumulative duration and severity of hyperglycemia (Wingard et al. (1993) Diabetes Care 16: 1022-1025; herein incorporated by reference in its entirety), as well as the cumulative duration and severity of hypertension that commonly accompanies diabetes mellitus (Raile et al. (2007) Diabetes Care 30:2523-2528; herein incorporated by reference in its entirety), although other non-modifiable risk factors such as age and genetic background also influence that risk. This places individuals of African- American descent at particular risk, due to their

disproportionately higher prevalence and severity of hypertension (Lopes et al. (2004) J. Clin. Hypertens. 5:393-401; herein incorporated by reference in its entirety). The intensive control of both hyperglycemia and elevated blood pressure (BP) has clearly been shown to reduce the onset and severity of diabetic nephropathy. The pivotal Diabetes Complications and Control Trial (DCCT) in individuals with type 1 diabetes (N. Engl. J. Med. (19893) 329:977-986; herein incorporated by reference in its entirety) and the United Kingdom Prospective Diabetes Study (UKPDS) in individuals with type 2 diabetes (Lancet (1998) 352:837-853; herein incorporated by reference in its entirety) clearly established the benefits of intensive glycemic control for delaying the onset and worsening of diabetic nephropathy. Data from the UKPDS also clearly established the benefits of intensive BP control on delaying the progression of diabetes complications, including nephropathy (BMJ (1998) 317:703-713; herein incorporated by reference in its entirety).

[0005] Renal damage in diabetes, as with renal damage due to primary kidney disease, involves proteinuria of glomerular origin. Renal tubules of the kidneys retain plasma proteins by reabsorption of such proteins as they pass through the glomerular filtration barrier. Normal urine protein excretion is up to 150 mg/d. Therefore, the detection of abnormal quantities or types of protein in the urine is considered an early sign of significant renal or systemic disease. When proteinuria occurs, it can cause further renal damage through release of cytokines, inflammation of the renal tubulointerstitium, and progressive fibrosis. Although diabetic nephropathy is a major cause of proteinuria in the United States, proteinuria also occurs in many other disease states that affect protein reabsorption or affect the glomerular barrier, such as proliferative glomerulonephritis (e.g., immunoglobulin A nephropathy, membranoproliferative

glomerulonephritis, mesangial proliferative glomerulonephritis, anti-GBM disease, renal vasculitis, lupus nephritis, cryoglobulinemia-associated glomerulonephritis, bacterial

endocarditis, Henoch- Schonlein purpura, postinfectious glomerulonephritis, hepatitis C), and nonproliferative glomerulonephritis (e.g., membranous glomerulonephritis, minimal-change disease, primary focal segmental glomerulosclerosis (FSGS), fibrillary glomerulonephritis, immunotactoid glomerulonephritis, amyloidosis, hypertensive nephrosclerosis, light-chain disease from multiple myeloma, secondary focal glomerulosclerosis). Additionally, conditions such as obesity and hypertensive nephrosclerosis can cause glomerular hyperfiltration, leading to proteinuria.

[0006] The importance of insulin signaling for podocyte biology in diabetic kidney disease and proteinuria has been reported (Coward et al., Curr. Opin. Nephrol. Hypertens., 25_104-110, 2015 and Fornoni, N. Engl. J. Med., 363:2068-2069, 2010). Diabetic kidney disease (DKD) is the most common cause of end-stage renal disease (ESRD). Multifactorial intervention trials targeting glycemic control, blood pressure and lifestyle interventions have been demonstrated to slow but not halt the progression of DKD in both type 1 (Hovind et al., Diabetes Care, 26: 1258- 1264, 2003) and type 2 diabetes (Graede et al., N. Engl. J. Med., 358:580-591, 2008). Proteinuria is the first clinical manifestation of diabetic kidney disease (DKD) and inversely correlates with the number of podocytes in experimental animal models and in humans with DKD.

Characteristic histopathologic findings of DKD often precede the development of albuminuria, may occur in the setting of normoalbuminuric DKD and may predict the progressive nature of DKD (Meyer et al., Biabetologia, 42: 1341-1344, 1999; Pagatalunan et al., J. Clin. Invest., 99:342-348, 1997; Toyoda et al., Diabetes, 56:2155-2160, 2007; White et al., Diabetes, 51:3083- 3089, 2002), challenging the concept that albuminuria per se may cause progressive DKD. It has also been reported that insulin signaling to the glomerular podocyte is critical for normal kidney function (Welsh et al., Cell Metab. 12:329-340, 2010). [0007] There remains a need in the art to provide a means to restore insulin signaling in an impaired podocyte to restore kidney function in a subject in need thereof.

SUMMARY

[0008] In one aspect, described herein is a method of restoring insulin signaling in a cell that overexpresses sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) comprising contacting the cell with a ceramide in an amount effective to restore insulin signaling in the cell. In some embodiments, the cell is a podocyte. The contacting step can occur in vitro or in vivo.

[0009] In another aspect, described herein is a method of restoring insulin signaling in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to restore insulin signaling in the subject. In some embodiments, the ceramide is selected from the group consisting of ceramide 1-phosphate (C1P), N-acetylsphinhosine (C2- ceramide), N-hexanoylsphingosine (C6-ceramide) and N-octanoylspingosine (C8-ceramide). In some embodiments, the ceramide is C16:0 C1P.

[0010] The phrase "restoring insulin signaling" as used herein refers to the restoration of the ability of a podocyte overexpressing SMPDL3b to phosphorylate AKT in response to insulin stimulation and thus an improvement of albuminuira in subject received treatment (i.e., reduction in the amount of protein observed in the urine of the subject).

[0011] In some embodiments, the subject is suffering from a disorder associated with impaired insulin signaling. Exemplary disorder associated with impaired insulin signaling include, but are not limited to, diabetes, pre-diabetes, obesity, insulin resistance, polycystic ovary syndrome, diabetes related macrovascular complications (e.g., coronary heart disease, myocardial infarction, congestive heart failure, or stroke), and microvascular complications (e.g., neuropathy, nephropathy, or retinopathy). In some embodiments, the subject is suffering from diabetic nephropathy.

[0012] In yet another aspect, disclosed herein is a method of treating a disorder associated with impaired insulin signaling in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to restore insulin signaling in the subject. In some embodiments, the disorder is a proteinuric kidney disease. In other embodiments, the disorder is diabetic nephropathy. [0013] In yet another aspect, disclosed herein is a method of treating a disorder associated with albuminuria or proteinuria in a mammalian subject in need thereof comprising

administering a ceramide to the subject in an amount effective to improve proteinuria or albuminuria in the subject.

[0014] In any of the methods described herein, the ceramide is, in some embodiments, C1P. In some embodiments, the ceramide is C16:0 C1P.

[0015] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited within the body of this specification are expressly incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1A-1B shows that sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) overexpression suppresses insulin receptor B (IRB) signaling and facilitates insulin receptor A (IRA) signaling in human podocytes. (Figure 1A) Representative Western blot and bar graph analysis of fold change in phosphorylated AKT (pAKT, Ser473) over total AKT (tAKT) in wild type (WT) and SMPDL3b overexpression (SMP OE) human podocytes exposed to increasing concentration of insulin (Ins: 0, 0.1, 1 nM). While WT podocytes response to insulin stimulation (**p<0.01, ***p<0.005), SMP OE cells do not phosphorylate AKT to increasing concentration of insulin. Data are presented as mean values + SD (n=3). (Figure IB) Representative Western blot and bar graph analysis of fold change in phosphorylated p70S6 kinase (p-p70S6K, Thr389) over total p70S6 kinase (t-p70S6K) in WT and SMP OE human podocytes exposed to increasing concentration of insulin (Ins: 0, 0.1, 1 nM). Phosphorylation of p70S6K is significantly

(*p<0.05, **p<0.01) increased in SMP OE podocytes under insulin stimulation when compared to WT cells. Data are presented as mean values + SD (n=3).

[0017] Figure 2A-2D shows that SMPDL3b affects expression and localization of the insulin receptor (IR). (Figure 2A) Representative Western blot and bar graph analysis of total IR protein expression in WT and SMP OE podocytes. No changes in protein level expression were found among podocytes types. Data are presented as mean values + SD (n=5). (Figure 2B)

Representative Western blot and bar graph analysis of phosphorylated and total Cavl protein expression in WT and SMP OE podocytes. Phosphorylated Cavl (pCavl) and total Cavl (tCavl) protein levels expression are identical in both types of podocytes. Data are presented as mean values + SD (n=5). (Figure 2C) Representative Western blot and bar graph analysis of IR distribution at the plasma membrane (PM) and in the cytosolic (Cytosol) fractions in WT and SMP OE human podocytes. SMP OE podocytes show less IR at the plasma membrane compare to WT (***p<0.001). Na/K-ATPase - marker of the plasma membrane fraction, MEK - marker of the cytosolic fraction. Data are presented as mean values + SD (n=3). (Figure 2D)

Representative Western blot and bar graph analysis of phosphorylated and total Cavl at the plasma membrane (PM) and in the cytosolic (Cytosol) fractions in WT and SMP OE human podocytes. No difference was found in SMP OE podocytes when compared to WT. Data are presented as mean values + SD (n=3).

[0018] Figure 3A-3B shows that SMPDL3b interacts with caveolin-1 and two isoforms of insulin receptor (IR). (Figure 3A) Co-immunoprecipitation (Co-IP) experiments performed in HEK293 cells showing interaction between SMPDL3b and IR isoform A (IRA), IR isoform B (IRB), and caveolin-1 (Cavl). GFP-empty vector was used as a negative control (n=3). (Figure 3B) Co-IP experiments performed in HEK293 cells demonstrating that both isoforms of IR, IRA and IRB, interact with Cavl. SMPDL3b augments IRA/Cav-1 interaction, while suppressing IRB/Cav-1 interaction (n=3).

[0019] Figure 4A-4B shows the effect of SMPDL3b overexpression on Neutral Lipid Content in Human Podocytes. (Figure 4A) Oil red O staining of wild type (WT) and SMPDL3b overexpressing (SMP OE) podocytes and relative bar graph analysis (***p<0.001, t-test).

SMPDL3b overexpression results in accumulation of lipid droplets in SMP OE podocytes. Data are presented as mean values + SD (n=5). (Figure 4B) Total cholesterol content is significantly (**p<0.005, t-test) increased in SMP OE podocytes compared to WT, however no changes were found in content of total triglycerides or total phospholipids. Data are presented as mean values + SD (n=6).

[0020] Figure 5A-5E provides the results of electrospray ionization/tandem mas spectrometry analysis of sphingolipids in human podocytes. (Figure 5A) SMPDL3b overexpression podocytes (SMP OE) demonstrate less total ceramide compared to wild type cells (WT). (Figure 5B) SMP OE podocytes have decreased amount of total ceramide- 1 -phosphate. (Figure 5C) Ceramide- 1- phosphate (CIP) species in WT and SMP OE podocytes. (Figures 5D-F) No changes were found between WT and SMP OE podocytes in total sphingomyelin (Figure 5D), total sphingosine (Figure 5E) or total sphingo sine- 1 -phosphate amount (Figure 5F). *p<0.05, t-test. **p<0.05, two-way ANOVA. Data are presented as mean values + SD (n=3).

[0021] Figure 6 shows that pre-treatment with C1P16:0 restores ability of podocytes overexpressing SMPDL3b phosphorylate AKT in response to insulin stimulation. Pre-treatment with lOOuM of recombinant C1P C16:0 for lh rescue AKT phosphorylation in SMPDL3b overexpression (SMP OE) podocytes in response to InM insulin stimulation. *p<0.05,

***p<0.01, t-test; #p<0.05, two-way ANOVA. Data are presented as mean values + SD (n=3).

[0022] Figure 7A-7C shows that C IP replacement to diabetic mice for four weeks is not toxic and protects from albuminuria. (Figure 7A) Kidney cortex from 20 week old db/db mice demonstrate reduction in C16:0 ceramide when compared to db/+ controls. ** P<0.01 (Figure 7B) Daily intraperitoneal administration of 30 mg/kg C16:0 ceramide 1-phosphate to 12 week old db/db mice for 4 weeks resulted in improvement of albuminuria (urine Alb/Creat Ratio). ** P<0.01 (Figure 7C) Daily intraperitoneal administration of 30 mg/kg C16:0 ceramide 1- phosphate to 12 week old db/db mice for 4 weeks did not result in impairment of liver function tests (AST/ALT), kidney function (BUN) or circulating lipids (Total cholesterol and

triglycerides).

[0023] Figure 8A-8F shows the effect of SMPDL3b on related sphingolipids. Bar graph analysis of total sphingomyelin (Figure 8A), total Ceramide-1 -Phosphate (Figure 8B) and C1P species (Figure 8C) in wild type podocytes (WT) or podocytes overexpressing SMPDL3b (SMP OE). *p<0.05, **p<0.05, t-test. Data are presented as mean values + SD (n=3). (Figure 8D) Bar graph analysis of total Ceramide-1 -Phosphate content in HEK cells transfected with either wild type SMPDL3b (SMP) or the SMPH135A mutant (n=2). (Figure 8E) TLC lipid analysis in pCMV or SMPDL3b transfected cells demonstrating a concentration dependent generation of C6-ceramide from C6-NBD-ceramide- 1-phosphate in SMPDL3b transfected cells. (Figure 8F) Bar graph analysis of Ceramide-1 -Phosphate production in HEK cells transfected with either WT or the H135A phosphodiesterase mutants of SMPDL3 (n=2).

[0024] Figure 9A-9E describes the phenotypes of non-diabetic and diabetic pSMPDL3bfl/fl mice. (Figure 9A) Bar graph analysis of kidney weight to body weight ratios in pSmpdl3b-fl/fl mice (fl/fl) and pSmpdl3b-+/+ mice (+/+) (Figure 9B) Representative PAS staining and (Figure 9C) Picrousirius staining of kidney sections from 36 old week pSmpdl3b-+/+ and pSmpdl3b-fl/fl mice demonstrating absence of a glomerulo sclerotic phenotype. (Figure 9D) Dot plot analysis of Alb/Creat Ratios (ACRs) in 20 weeks old pSmpdl3b-+/+ and pSmpdl3b-fl/fl mice, (figure 9E) Dot plot analysis of ACRs in pSMPDL3bfl/fl;db/db and pSmpdl3b-+/+;db/db mice (n=6) ***p<0.001, **p<0.01

DETAILED DESCRIPTION

[0025] The present disclosure is based, at least in part, on the discovery that

sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) overexpression in a cell (i.e., podocyte) results in decreased production of Ceramide- 1 -Phosphate (CIP) and impaired insulin signaling. As demonstrated herein, CIP replacement in SMPDL3b over-expressing cells restores inulin signaling, both in vitro and in vivo.

[0026] In one aspect, described herein is a method of restoring insulin signaling in a cell that overexpresses SMPDL3b comprising contacting the cell with a ceramide in an amount effective to restore insulin signaling in the cell.

[0027] Also provided is a method of restoring insulin signaling in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to restore insulin signaling in the subject. Also provided is a method of restoring or preserving podocyte function in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount to restore or preserve podocyte function in the mammalian subject.

[0028] In some embodiments, the subject is suffering from a disorder associated with impaired insulin signaling. Exemplary disorders include, but are not limited to, diabetes, pre-diabetes, obesity, insulin resistance, polycystic ovary syndrome, diabetes related macrovascular complications (e.g., coronary heart disease, myocardial infarction, congestive heart failure, or stroke), and microvascular complications (e.g., neuropathy, nephropathy, or retinopathy).

[0029] In one aspect, the subjects suitable for treatment with a ceramide as described herein include subjects with diabetes or a diabetes-related condition involving, e.g., impaired glucose tolerance, impaired insulin sensitivity, impaired insulin production. Such conditions and disease states include diabetes mellitus, type I diabetes, type II diabetes, gestational diabetes, metabolic syndrome, metabolic syndrome X, syndrome X, insulin resistance syndrome, Reaven's syndrome, CHAOS, and malnutrition-related diabetes mellitus. Such patients are at risk for and/or experience an increased incidence and severity of renal dysfunction and renal disease, as described below. [0030] In some embodiments, the subject has a renal disease. The term "renal disorder", "renal disease" or "kidney disease" means any alteration in normal physiology and function of the kidney. This can result from a wide range of acute and chronic conditions and events, including physical, chemical or biological injury, insult, trauma or disease, such as, for example, hypertension, diabetes, congestive heart failure, lupus, amyloidosis, multiple myeloma, vasculitis, sickle cell anemia and various inflammatory, infectious and autoimmune diseases, HIV-associated nephropathies etc. This term includes but is not limited to diseases and conditions such as kidney transplant; nephropathy; primary glomerulopathies (focal segmental glomerulosclerosis), Minimal Change disease, Membranous GN, IgA Nephropathy, chronic kidney disease (CKD); Glomerulonephritis; inherited diseases such as polycystic kidney disease; Acute and chronic interstitial nephritis, Mesoamerican Nephropathy, nephromegaly (extreme hypertrophy of one or both kidneys); nephrotic syndrome; Nephritic syndrome, end stage renal disease (ESRD); acute and chronic renal failure; interstitial disease; nephritis; sclerosis, an induration or hardening of tissues and/or vessels resulting from causes that include, for example, inflammation due to disease or injury; renal fibrosis and scarring; renal-associated proliferative disorders; and other primary or secondary nephrogenic conditions. Fibrosis associated with dialysis following kidney failure and catheter placement, e.g., peritoneal and vascular access fibrosis, is also included.

[0031] In some embodiments, the subject is suffering from diabetic nephropathy or diabetic kidney disease. The terms "diabetic nephropathy" and "diabetic kidney disease" are used interchangeably herein. Renal disorders or kidney diseases may also be generally defined as a "nephropathy" or "nephropathies". The terms "nephropathy" or "nephropathies" encompass all clinical-pathological changes in the kidney which may result in kidney fibrosis and/or glomerular diseases (e.g. glomerulosclerosis, glomerulonephritis) and/or chronic renal insufficiency, and can cause end stage renal disease and/or renal failure. Some aspects of the present disclosure relate to compositions and their uses for the prevention and/or treatment of hypertensive nephropathy, diabetic nephropathy, and other types of nephropathy such as analgesic nephropathy, immune- mediated glomerulopathies (e.g., IgA nephropathy or Berger's disease, lupus nephritis), ischemic nephropathy, HIV-associated nephropathy, membranous nephropathy, glomerulonephritis, glomerulosclerosis, radiocontrast media-induced nephropathy, toxic nephropathy, analgesic- induced nephrotoxicity, cisplatin nephropathy, transplant nephropathy, and other forms of glomerular abnormality or injury; or glomerular capillary injury (tubular fibrosis). In some embodiments, the terms "nephropathy" or "nephropathies" refer specifically to a disorder or disease where there is either the presence of proteins (i.e., proteinuria) in the urine of a subject and/or the presence of renal insufficiency.

[0032] In some embodiments, the subject is suffering from a disorder associated with albuminuria or proteinuria. Exemplary disorders associated with albuminuria include, but are not limited to, chronic kidney disease, proliferative glomerulonephritis (e.g., immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, mesangial proliferative

glomerulonephritis, anti-GBM disease, renal vasculitis, lupus nephritis, cryoglobulinemia- associated glomerulonephritis, bacterial endocarditis, Henoch- Schonlein purpura, postinfectious glomerulonephritis, or hepatitis C), and nonproliferative glomerulonephritis (e.g., membranous glomerulonephritis, minimal-change disease, primary focal segmental glomerulosclerosis (FSGS), fibrillary glomerulonephritis, immunotactoid glomerulonephritis, amyloidosis, hypertensive nephrosclerosis, light-chain disease from multiple myeloma and secondary focal glomerulosclerosis).

[0033] Ceramides

[0034] Ceramides are composed of spingosine and a fatty acid. Ceramides are found in high concentrations within a cell membrane and are component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer of the cell membrane.

[0035] Cell ceramides typically have long N-acyl chains ranging from 16 to 26 carbons in length (Merrill et al., Methods, 36:207-224, 2005; Merrill et al., J. Biol. Chem., 277:25843- 25846, 2002; Pettus et al., Rapid Commun. Mass Spectrom., 17: 1203-1211, 2003). However, in many studies short-chain analogs (N-acetylsphingosine, or C2-ceramide, N- hexanoylsphingosine, or C6-ceramide, and N-octanoylsphingosine, or C8-ceramide) have been used in experiments because these are more water soluble than long-chain ceramides.

[0036] A major metabolite of ceramide is ceramide 1-phosphate (C1P), which is generated through direct phosphorylation of ceramides by ceramide kinase (CerK).

[0037] In any of the method described herein, the ceramide is selected from the group consisting of C1P, C2, C6 and C8. In some embodiments, the ceramide is C16:0 C1P.

[0038] Timing of Administration and Dosage [0039] In some embodiments, one or more administrations of a ceramide described herein are carried out over a therapeutic period of, for example, about 1 week to about 18 months (e.g., about 1 month to about 12 months, about 1 month to about 9 months or about 1 month to about 6 months or about 1 month to about 3 months). In some embodiments, a subject is administered one or more doses of a ceramide described herein over a therapeutic period of, for example, about 1 month to about 12 months (52 weeks) (e.g., about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months). In some embodiments, a subject is administered one or more doses of the ceramide to maintain insulin signaling in a podocyte, maintain a reduced level of albuminuria and/or proteinuria the subject and/or preserve podocyte function in a mammalian subject. The term "maintain a reduced level of albuminuria and/or proteinuria" or "preserve podocyte function" as used herein means that the reduced level of albuminuria and/or proteinuria in the subject resulting from an initial dose of the ceramide does not increase by more than about 1% to about 5% over the course of about 6 months, about 9 months about 1 year, about 18 months, about 2 years, or over the course of the patient's life. Methods of determining the level of albuminuria and/or proteinuria in a subject can be done by methods known in the art such as urinalysis.

[0040] In addition, it may be advantageous to administer multiple doses of the ceramide or space out the administration of doses, depending on the therapeutic regimen selected for a particular human subject. In some embodiments, the ceramide is administered periodically over a time period of one year (12 months, 52 weeks) or less (e.g., 9 months or less, 6 months or less, or 3 months or less). In this regard, the ceramide is administered to the human once every about 3 days, or about 7 days, or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks, or 14 weeks, or 15 weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19 weeks, or 20 weeks, or 21 weeks, or 22 weeks, or 23 weeks, or 6 months, or 12 months.

[0041] In some embodiments, a dose of the ceramide described herein comprise between about 1 to about 500 milligrams (e.g., between about 1 to about 400 milligrams or about 3 to about 300 milligrams) of ceramide per kilogram of body weight (mg/kg). For example, the dose of ceramide may comprise at least about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, or about 49 mg/kg, or about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 450 mg/kg, or about 500 mg/kg. Ranges between any and all of these endpoints are also contemplated, e.g., about 1 mg/kg to about 100 mg/kg, about 3 mg/kg to about 300 mg/kg, about 3 mg/kg to about 100 mg/kg, about 5 mg/kg to about 50 mg/kg, about 3 mg/kg to about 75 mg/kg, about 1 mg/kg to about 50 mg/kg, about 100 mg/kg to about 300 mg/kg, about 50 mg/kg to about 200 mg/kg, or about 200 mg/kg to about 300 mg/kg.

[0042] Pharmaceutical Compositions

[0043] In some embodiments, a ceramide described herein is formulated together with a pharmaceutically effective diluents, carrier, solubilizer, emulsifier, preservative, and/or adjuvant. Pharmaceutical compositions include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0044] Preferably, formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of a ceramide are provided.

[0045] In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;

hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or

pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A. R. Genrmo, ed.), 1990, Mack Publishing Company.

[0046] In some embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL

SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the ceramide. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. In certain embodiments of the invention, the composition may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution.

[0047] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by, e.g., filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. [0048] Combination Therapies

[0049] Treatment of a pathology by combining two or more agents that target the same pathogen or biochemical pathway or biological process sometimes results in greater efficacy and diminished side effects relative to the use of a therapeutically relevant dose of each agent alone. In some cases, the efficacy of the drug combination is additive (the efficacy of the combination is approximately equal to the sum of the effects of each drug alone), but in other cases the effect is synergistic (the efficacy of the combination is greater than the sum of the effects of each drug given alone). As used herein, the term "combination therapy" means that two or more agents are delivered in a simultaneous manner, e.g., concurrently, or wherein one of the agents is administered first, followed by the second agent, e.g., sequentially.

[0050] In some embodiments, the methods described herein optionally comprise the step of administering a standard of care therapeutic for the treatment of a renal disorder or complication, nephropathy (e.g,. diabetic nephropathy), diabetes, dyslipidemia, hypertension and/or obesity.

[0051] In some embodiments, co-administration of a ceramide described herein with a standard of care therapeutic may allow lowering of the necessary dosage of the standard of care therapeutic such that co-administration, for examples, decreases side effects or improves blood glucose levels control. Co-administration may also prevent, treat or lessen one or more symptoms or features of metabolic syndrome, or reduce the risk of diabetes-related health complications.

[0052] In some embodiments, a ceramide described herein is administered in combination with common anti-diabetic drugs such as sulphonylureas (e.g., glicazide, glipizide), glitazones (e.g., rosiglitazone, pioglitazone), prandial glucose releasing agents (e.g., repaglinide, nateglinide), acarbose, insulin, biguanides, such as, for example metformin (Glucophage®, Bristol-Myers Squibb Company, U.S.; Stagid®, Lipha Sante, Europe); sulfonylurea drugs, such as, for example, gliclazide (Diamicron®), glibenclamide, glipizide (Glucotrol® and Glucotrol XL®, Pfizer), glimepiride (Amaryl®, Aventis), chlorpropamide (e.g., Diabinese®, Pfizer), tolbutamide, and glyburide (e.g., Micronase®, Glynase®, and Diabeta®); glinides, such as, for example, repaglinide (Prandin® or NovoNorm®; Novo Nordisk), ormitiglinide, nateglinide (Starlix®), senaglinide, and BTS-67582; DPP-IV inhibitors such as vildagliptin and sitagliptin; insulin sensitizing agents, such as, for example, glitazones, a thiazolidinedione such as rosiglitazone maleate (Avandia®, Glaxo SmithKline), pioglitazone (Actos®., Eli Lilly, Takeda), troglitazone, ciglitazone, isaglitazone, darglitazone, englitazone; glucagon-like peptide I (GLP-1) receptor agonists, such as, for example, Exendin-4 (1-39) (Ex-4), Byetta.TM. (Amylin

Pharmaceuticals Inc.), CJC-1 131 (Conjuchem Inc.), NN-221 I (Scios Inc.), GLP-1 agonists such as those described in International Patent Publication No. WO 98/08871; agents that slow down carbohydrate absorption, such as, for example, a-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose, and emiglltate); agents that inhibit gastric emptying, such as, for example, glucagon- like peptide 1, cholescystokinin, amylin, and pramlintide; glucagon antagonists, such as, for example, quinoxaline derivatives (e.g., 2-styryl-3-[3-(dimethylamino)propylmethylaminol-6, 7- dichloroquinoxaline, Collins et al., Bioorganic and Medicinal Chemistiy Letters 2(9):91 5-91 8, 1992), skyrin and skyrin analogs (e.g., those described in WO 94/14426), 1-phenyl pyrazole derivatives (e.g., those described in U.S. Pat. No. 4,359,474), substituted disllacyclohexanes (e.g., those described in U.S. Pat. No. 4,374,130), substituted pyridines and biphenyls (e.g., those described in WO 98/04528), substituted pyridyl pyrroles (e.g., those described in U.S. Pat. No. 5,776,954), 2,4-diaryl-5-pyridylimidazoles (e.g., those described in International Patent

Publication Nos. WO 98/21957, WO 98/22108, WO 98/22109, and U.S. Pat. No. 5,880,139), 2,5-substituted aryl pyrroles (e.g., those described in International Patent Publication No. WO 97/1 6442 and U.S. Pat. No. 5,837,719), substituted pyrimidinone, pyridone, and pyrimidine compounds (e.g., those described in International Patent Publication Nos. WO 98/24780, WO 98/24782, WO 99/24404, and WO 99/32448), 2-(benzirnidazol-2-ylthio)-l-(3,4- dihydroxyphenyl)-l-ethanones (see Madsen et aL, J. Med. Chem. 41:5151-5157, 1998), alkylidene hydrazides (e.g., those described in International Patent Publication Nos. WO

99/01423 and WO 00/39088), glucokinase activators, such as, for example, those described in International Patent Publication Nos. WO 00/58293, WO 01/44216, WO 01/83465, WO

01/83478, WO 01/85706, and WO 01/85707 and other compounds, such as selective ADP- sensitive K⁺ channels activators (e.g., diazoxide), hormones (e.g., cholecytokinin, GRP- bombesin, and gastrin plus EGF receptor ligands; see Banerjee et al. Rev Diabet Stud, 2005 2(3): 165-176); peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist (e.g., pioglitazone; see Ishida et al., Metabolism, 2004, 53(4), 488-94); antioxydants (e.g., 1-bis-o- hydroxycinnamoylmethane, curcuminoid bis-demethoxycurcumin; see Srivivasan et al., J Pharm Pharm Sci. 2003, 6(3): 327-33), W International Patent Publication Nos. O 00/69810, WO 02/00612, WO 02/40444, WO 02/40445, WO 02140446, and the compounds described in International Patent Publication Nos. WO 97/41097 (DRF-2344), WO 97/41119, WO 97/41120, WO 98/45292, WO 99/19313 (NN622/DRF-2725), WO 00/23415, WO 00/23416, WO 00/23417, WO 00/23425, WO 00/23445, WO 00/23451, WO 00/41121, WO 00/50414, WO 00/63153, WO 00/63189, WO 00/63190, WO 00/63191, WO 00/63192, WO 00/63193, WO 00/63196, WO 00/63209, WO 2006/131836 and WO 2006/120574, as well as U.S. Pat. No. 6,967,019, U.S. Pat. No. 7,101,845, U.S. Pat. No. 7,074,433, U.S. Pat. No. 6,992,060, and U.S. Pat. No. RE39,062,; and the compounds referred to in the public domain such as T-174, GI- 262570, YM-440, MCC-555, JTT-501, AR-H039242, KRP-297, GW-409544, CRE-16336, AR- H049020, LY510929, MBX-102, CLX-0940, and GW-501516.

[0053] Additional examples of agents that can be co-administered with a ceramide described herein are compounds for stimulating pancreatic beta-cell neogenesis and/or regeneration of islets. Examples of compounds currently used or in development which have a positive effect on islet number (i.e. beta-cells) include Byetta™ (exendin-4 inhibitor), vildagliptin (Galvus™, dipeptidylpeptidase inhibitor), Januvia™ (sitagliptin phosphate) and extracts from Gymnema sylvestrae leaf (Pharma Terra). The ceramide(s) described herein may also be administered with biomolecules related to cell regeneration such as β-cellulin, plant extracts from Beta vulgaris or Ephedra herba, and nicotinamide (see Banerjee et al. Rev Diabet Stud, 2005 2(3): 165-176).

[0054] Additional examples of agents that can be co-administered with a ceramide described herein include sodium-glucose co-transporter 2 (SGLT2) inhibitors including, but not limited to, empagliflozin, canagliflozin, dapagliflozin and ipragliflozin.

[0055] Additional compounds or agents that may be administered in combination with a ceramide described herein include compounds capable of inducing pancreatic beta-cell growth or insulin producing cell growth and/or insulin production. Such compounds include, but are not limited to: glucagon-like peptide- 1 (GLP-1) and long-acting, DPP-IV-resistant GLP-1 analogs thereof, GLP-1 receptor agonists, gastric inhibitory polypeptide (GIP) and analogs thereof (e.g., which are disclosed in U.S. Patent Publication No. 20050233969), dipeptidyl peptidase IV (DPP- IV) inhibitors, insulin preparations, insulin derivatives, insulin-like agonists, insulin

secretagogues, insulin sensitizers, biguanides, gluconeogenesis inhibitors, sugar absorption inhibitors, renal glucose re-uptake inhibitors, β3 adrenergic receptor agonists, aldose reductase inhibitors, advanced glycation end products production inhibitors, glycogen synthase kinase-3 inhibitors, glycogen phosphorylase inhibitors, antilipemic agents, anorexic agents, lipase inhibitors, antihypertensive agents, peripheral circulation improving agents, antioxidants, diabetic neuropathy therapeutic agents, and the like.

[0056] In some embodiments, a ceramide described herein is administered in combination with a standard of care therapeutic for preventing or treating a renal disorder such as

nephropathy, or an associated disorder or complication. Examples of such known compounds include but are not limited to: ACE inhibitor drugs (e.g. captopril (Capoten®), enalapril

(Innovace®), fosinopril (Staril®), lisinopril (Zestril®), perindopril (Coversyl®), quinapril (Accupro®), trandanalopril (Gopten®), lotensin, moexipril, ramipril); RAS blockers; angiotensin receptor blockers (ARBs) (e.g. Olmesartan, Irbesartan, Losartan, Valsartan, candesartan, eprosartan, telmisartan, etc); protein kinase C (PKC) inhibitors (e.g. ruboxistaurin); inhibitors of AGE-dependent pathways (e.g. aminoguanidine, ALT-946, pyrodoxamine (pyrododorin), OPB- 9295, alagebrium); anti-inflammatory agents (e.g. clyclooxigenase-2 inhibitors, mycophenolate mophetil, mizoribine, pentoxifylline), GAGs (e.g. sulodexide (U.S. Pat. No. 5,496,807));

pyridoxamine (U.S. Pat. No. 7,030,146); endothelin antagonists (e.g. SPP 301), COX-2 inhibitors, PPAR-γ antagonists and other compounds like amifostine (used for cisplatin nephropathy), captopril (used for diabetic nephropathy), cyclophosphamide (used for idiopathic membranous nephropathy), sodium thiosulfate (used for cisplatin nephropathy), tranilast, etc. (Williams and Tuttle (2005), Advances in Chronic Kidney Disease, 12 (2):212-222; Giunti et al. (2006), Minerva Medica, 97:241-62).

[0057] Additionally, the methods described herein may also include co-administration of at least one other therapeutic agent for the treatment of another disease directly or indirectly related to diabetes and/or renal disorder complications, including but not limited to: dyslipidemia, hypertension, obesity, neuropathy, inflammation, and/or retinopathy, etc. Such additional therapeutic agents include, but are not limited to, corticosteroids; immunosuppressive medications; antibiotics; antihypertensive and diuretic medications (such as thiazide diuretics and ACE-inhibitors or β-adrenergic antagonists); lipid lowering agents such as bile sequestrant resins, cholestyramine, colestipol, nicotinic acid, and more particularly drugs and medications used to reduce cholesterol and triglycerides (e.g. fibrates (e.g. Gemfibrozil®) and HMG-CoA inhibitors such as Lovastatin®, Atorvastatin®, Fluvastatin®, Lescol®, Lipitor®, Mevacor®, Pravachol®, Pravastatin®, Simvastatin®, Zocor®, Cerivastatin®, etc); compounds that inhibit intestinal absorption of lipids (e.g. ezetiminde); nicotinic acid; and Vitamin D. [0058] Additional examples of agents that can be co-administered with a ceramides described herein include immunomodulating agents or immunouppressants (such as those that are used by type 1 diabetics who have received a pancreas transplant and/or kidney transplant (when they have developed diabetic nephropathy) (see Vinik Al et al. Advances in diabetes for the millennium: toward a cure for diabetes. Med Gen Med 2004, 6: 12)), anti-obesity agents, and appetite reducers (including, but not limited to, Xenical™ (Roche), Meridia™ (Abbott), Acomplia™ (Sanofi-Aventis), and sympathomimetic phentermine), agents that are used to treat hyperkalemia and/or to reduce the risk of ventricular fibrillation caused by hyperkalemia (e.g. calcium gluconate, insulin, sodium bicarbonate, β ₂-selective catacholamine such as salbutamol (albuterol, Ventolin®), and polystyrene sulfonate (Calcium Resonium, Kayexalate)).

[0059] As used herein, the term "concomitant" or "concomitantly" as in the phrases

"concomitant therapeutic treatment" or "concomitantly with" includes administering a first agent in the present of a second agent. A concomitant therapeutic treatment method includes methods in which the first, second, third or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step- wise by different actors. For example, one actor may administer to a subject a first agent and as a second actor may administer to the subject a second agent and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and/or additional agents) are after administration in the presence of the second agent (and/or additional agents). The actor and the subject may be the same entity (e.g. a human). Preferably the first agent is a ceramide described herein. The second agent may be selected from the standard of care therapeutics described herein.

EXAMPLES

Example 1 - Materials and Methods

[0060] Human podocyte culture and treatment. A human podocyte cell line transfected with a thermosensitive SV40-T construct were cultured as previously described (14). Briefly, human podocytes were initially grown at 33°C until 75-80% confluence in RPMI media (Corning) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). After shifting podocytes to 37°C for 14 days, they become growth arrested, differentiated and, at the 14th day, they are ready for experiments. Stable SMPDL3b overexpression (SMP OE) or SMPDL3b knock down (SMP KD) podocytes cell lines were previously described (15). Human podocytes were serum starved for 24h before treatments.

[0061] Stimulation with insulin (Sigma- Aldrich) was performed in concentrations 0, 0.1 and 1 nM for 30 min at 37°C. Untreated podocytes were served as a control. Exogenous pre-treatment with 100 uM recombinant C IP C16:0 (Avanti Polar Lipids) was performed for 1 h at 37°C.

[0062] Isolation of Plasma Membranes. Cells were collected in homogenization media (15 mM KC1, 1.5 mM MgCl₂, 10 mM HEPES, 1 mM DTT) and incubated on ice for 5 min. Then cell pellets were homogenized 5 times using insulin syringes and incubated with 2.5 M Sucrose solution for 250 nM final concentration. Cell pellets were exposed to several steps of

centrifugation: to separate nuclear fraction (l,000xg, 5 min, 4°C); to separate mitochondria and endoplasmic reticulum fraction (10,000xg, 15 min, 4°C); and to separate plasma membrane fraction and cytosol fraction (100,000xg, lh, 8°C). Resulting membrane pellets were collected and resuspended in 50 μΐ of homogenization media with following loading for the Western blot analysis. Cytosol fraction supernatants were concentrated in 3-4 times using 3K concentrators (Vivaspin 500), 20,000xg, 30 min, 4°C with following loading for the Western blot analysis. Na/K-ATPase was used as a marker of plasma membrane fraction, and MEK-1/2 was used as a marker of cytosol fraction.

[0063] Western Blot and Used Antibodies. Cells were collected in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease and phosphatase inhibitors) and incubated on ice for 30 min. Proteins were loaded into 4-20% SDS-PAGE gels (BioRad) and transferred to PDVF membranes (Millipore). The following primary antibodies were used: polyclonal rabbit anti phospho-Cavl (1: 1000, Cell Signaling), polyclonal rabbit anti total Cavl (1: 1000, Cell Signaling), polyclonal rabbit anti insulin receptor β-subunit (1: 100, Cell Signaling), polyclonal rabbit anti Na/K-ATPase (1: 1000, Cell Signaling), polyclonal rabbit anti MEK-1/2 (1: 1000, Cell Signaling), polyclonal rabbit anti phospho-AKT Ser473 (1: 1000, Cell Signaling), polyclonal rabbit anti total AKT (1: 1000, Cell Signaling), polyclonal rabbit anti phospho-p70S6K Thr389 (1: 1000, Cell Signaling), polyclonal rabbit anti total p70S6K (1: 1000, Cell Signaling), monoclonal mouse anti GAPDH (1: 10000, Calbiochem), monoclonal mouse anti GFP (1: 1000, Clontech), polyclonal rabbit anti FLAG (1:5000, Sigma- Aldrich). HRP-conjugated secondary antibodies (Promega Corp.) were used at 1: 10000. Membranes were visualized with ECL (Amersham Pharmacia Biotech.) or SuperSignal West Pico Chemiluminescent Substrate

(ThermoScientific).

[0064] Isolation of plasma membranes. PM and cytosolic fraction of podocytes expressing SMPDL3b wild type and mutants were utilized to determine if SMPDL3b phosphodiesterase and phosphatase activity affect the subcellular localization of SMPDL3b, IR, Cav-1, CERK.

Preparation of membrane pellets will be performed by ultracentrifugation of cell pellets suspended in homogenization media (15mM KC1, 1.5 mM MgCl₂, lOmM HEPES, ImM DTT) supplemented with protease inhibitors. Effective separation was verified with WB for Na-K ATPase.

[0065] Lipid rafts isolation. Lipid rafts isolation allows for the determination of whether SMPDL3b affects IRA and IRB lipid raft localization. For lipid raft isolation, lysates were centrifuged at lOOOx g for 10 min. The resulting supernatant was collected and mixed with equal volume of 70% OptiPrep in basic buffer (20 mM Tri-HCl, pH 7.8, 250 mM sucrose), placed on the bottom of the Ultra-clear tube (Sigma), where 5 ml of 30%, 2 ml of 5% and 1 ml of 0% basic buffer was added. Gradients were centrifuged for 4 h at 170,000x g (37,100 rpm), and the appropriate fraction transferred to a protein concentration unit (Centricon-10, 2 ml capacity) and spun at less than 5000x g for 100 min at 4°C. Fraction concentrates were collected for SDS- PAGE gel and Western blot of flotillin-1 (rabbit polyclonal anti-human, 1:200, Santa Cruz Biotechnology) as described previously (Fornoni et al., Science translational medicine.

2011;3(85):85ra46).

[0066] Transient Transfection and HEK293 Cell Culture. HEK293 cells were cultured in DMEM (Gibco) with L-glutamine, supplemented with 10% FBS (Gibco) and 1%

penicillin/streptomycin (Gibco). HEK293 cells were transfected with FuGENE-6 (Promega Corp.). HEK293 cells were grown until 50-60% confluence, transfected and incubated in DMEM (10% FBS, 1% penecilin/streptomycin) for 48 h. For immunoprecipitation (IP) assay, a full- length human insulin receptor A or B-GFP-pRC/CMV and human insulin receptor A or B- FLAG-pRC/CMV vectors (Susztak et al., Diabetes. 2006;55(l):225-33), human caveolin-l-GFP- PS 100010 vector (OriGene), human SMPDL3b-FLAG-pCMV6-Entry vector (OriGene) were used for proteins over-expression in HEK293 cells. Empty FLAG-pcDNA3.0 vector or empty GFP-pcDNA3.0 vector were used as negative control. [0067] Co-lmmunoprecipitation ( Co-IP). For Co-IP of FLAG and GFP fusion proteins, HEK293 cells were grown on a 10 cm dish, co-transfected at a confluence of approximately 100% for 48 h, and starved for 24 h in FBS free media. Cells were pelleted by centrifugation at 1500xg for 5 min at 4°C and washed twice with ice-cold PBS. For cell lysis, the pellet was resuspended in 900 μΐ of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease and phosphatase inhibitors) and incubated on ice for 30 min. The cell lysate was cleared by centrifugation at 20000xg for 15 min at 4°C. 800 μΐ of cell extract was incubated with 30 μΐ agarose beads coated with anti-FLAG-M2 antibody (Sigma- Aldrich) at 4°C overnight. Beads were collected by centrifugation for 1 min at lOOOxg and washed 5 times with 1 ml of lysis buffer without protease and phosphatase inhibitors for 10 min on a rotator at 4°C. Bound proteins were eluted by boiling agarose beads in 100 μΐ Laemmli buffer at 95°C for 5 min and analyzed by standard SDS gel electrophoresis and Western blot detection of FLAG (Sigma- Aldrich) and GFP (Clontech).

[0068] Immunofluorescence staining, Confocal Microscopy and Co-localization Analysis. HEK293 cells cultured in chamber slides were fixed with 4% paraformaldehyde and 2% sucrose for 10 min at room temperature and permeabilized with 0.3% Triton X-100. The expression constructs with different fluorescent tags for determination of different isoforms of insulin receptor (IRA-dTomato, IRB-dTomato) and plasma membrane lipid rafts (Myr-Palm-mCFP) (17). To detect caveolin-1 in HEK293 cells we used total caveolin-1 antibodies (CellSignaling). Fluorescence detection was performed using Alexa Flour secondary antibodies (Invitrogen).

[0069] Laser scanning confocal microscopy was performed using a Leica SP5 Inverted microscope (60x wet objective). The following settings were used: for dTomato fluorescence, excitation wavelength 588 nm, for mCFP fluorescence, excitation wavelength 458 nm, for GFP fluorescence, excitation wavelength 488 nm.

[0070] Co-localization of IRA/IRB , IRA/Cav 1 , IRB/Cav 1 , IRA/Palm-mCFP, IRB/Palm- mCFP fluorescence was quantified using the "Just Another Co-localization Plugin" (JACoP) of Fiji ImageJ Software. To exclude signals originating from the cytoplasm, the analysis was limited to the plasma membrane by using the "region of interest" feature. Measurements were repeated 10 times on different cells. [0071] Lipid Content Determination. To determine total cholesterol mass, differentiated podocytes were grown in 9 cm plates. Podocytes were washed two times with IxPBS, followed by cellular lipid extraction with hexane-isopropanol 3:2 (v/v). Total cholesterol content was measured directly using the Amplex Red Cholesterol Assay Kit (Invitrogen) following manufacturer instructions and normalizing to cell protein content using BCA method.

Sphingolipid analysis was performed using electrospray ionization/tandem mas spectrometry on a Thermo Finningan TSQ 7000 triple quadropole mass spectrometer (18).

[0072] Podocyte cell culture (Example 7). Human podocytes were plated at 33°C, cultured and then thermoshifted at 37°C for 14 days to achieve terminal differentiation. After terminal differentiation, cells were serum starved in 0.2% FBS for 24 hours prior to any analysis. For experiments designed to test insulin-signaling, serum starved, normal human podocytes were treated with insulin (1 to 100 nmol, Sigma) for 15 minutes prior to the collection of cell lysates for the analysis of phosphorylated and total AKT and p70S6K. For CD treatment, human podocytes were serum starved for 24h before treatment with CD (5mM/mL, lh, Sigma).

Untreated cells will serve as controls.

[0073] Co-Immunoprecipitation (Co-IP) and competitive IP (Example 7). Co-IP experiments will be performed following previous published protocols (Wei et al., Nat Med. 2011;17(8):952- 60). Briefly, HEK cells will be transfected with 1 μg of a plasmid containing a GFP-SMPDL3b and FLAG-empty vector, FLAG-IRA/IRB or FLAG-cavl. In addition, in order to discriminate preferential IP between IRA and IRB, cells will be transfected with FLAG-Cavl or FLAG- SMPDL3b and GFP-IRA/YFP-IRB. FLAG- S MP mutated constructs generated in this application will also be studied. For competitive IP experiments, HEK cells will be transfected with increasing amounts of GFP-tagged SMPDL3b cDNA or a plasmid containing a GFP-tag without any fused cDNA (empty vector) together with ^g of each, FLAG-IRA/IRB or FLAG- cav-1 in HEK cells. For endogenous Co-IP experiments glomeruli was isolated from 10

C57B1/6J mice by sieving technique and IP SMP.

[0074] Ceramide-l-phosphate-phosphatase assay. 48 hours after transfection, cells were collected, centrifuged and pellets resuspended in reaction buffer (200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10 mM HEPES, pH7.4) with protease inhibitors. Homogenates were centrifuged (600 g, 10 min) and the nuclear pellet resuspended in reaction buffer. C6-NBD-ceramide-l- phosphate was added as a complex with defatted bovine serum albumin (BSA) in volume of 20 μΐ (2 μg protein) at 37°C for 10 min. Lipids were extracted according to Bligh and Dyer and lipids resolved by TLC using chloroform:methanol:acetic acid: 15 niM CaCl, (60:35:2:4, v/v/v/v) as the developing solvent. NBD-lipids were identified by appropriate standards and quantified as described (65).

[0075] Phosphodiesterase activity assay. Generation of p-nitrophenol from bis-p- nitrophenolphosphate by phosphodiesterase activity will be measured as absorbance at 405 nm in 96-well plates with 100 μΐ reaction volume. Kinetic measurements will be carried out using 325 ng/ml enzyme purified from HEK293T cells transfected with wild type, mutated and truncated forms of SMPDL3b in the presence of different buffers adjusted to the indicated pH in the presence of 1 mM substrate. For determination of the impact of point mutations, enzymes were incubated with HEPES buffer (20 mM [pH 7.8]) in the presence of 1 mM substrate. For measurement of cell-associated enzymatic activity, cells were incubated in 96-well plates with isotonic Tris-buffered saline (TBS) in the presence of 1 mM substrate at 37°C and 5% C0₂. Apoptosis analysis. Apoptosis in cells expressing different SMPDL3b mutants was measured using the Caspase-3 Fluorometric Assay Kit (Bio Vision). Data was normalized for protein content and expressed as fold change of caspase-3 activity in treatments versus controls.

Treatment with staurosporine will be utilized as positive control. Both unstimulated and TNF stimulated cells was utilized, as the contribution of lipids to TNF-induced podocyte injury was demonstrated previously.

[0076] Immunofluorescence studies for IRA/IRB subcellular localization. WT, SMP OE and SMP KD cells was fixed as described above and stained with an anti-Insulin receptor (beta- subunit) clone CT-3, mouse monoclonal (Millipore 1:300) and with FM-143 dye (Molecular Probes) as a marker for PM to determine if SMPDL3b affect the subcellular localization of total IR. To determine whether SMPDL3b affects the distribution of IRA and IRB, HEK cells transfected with exogenous proteins with a different fluorescent tags was utilized in conjunction with a Myr-Palm-mCFP construct to identify cholesterol enriched PM domains and quantified as described(39). Human IR-A and IR-B constructs with different color tags were obtained and utilized: GFP, YFP (Venus), red (Tomato) and CFP (Cerulean).

[0077] Statistical analysis. Statistical analysis will be implemented using GraphPad Prism Software 5. Analysis of Variance (ANOVA) followed by the Bonferroni' s posttest or Student's t- test will be utilized to analyze results. [0078] Breeding strategy to generate mice with a podocyte-specific deletion of Smpdl3b, assessment of the geno- and phenotype. Mice in which Exon 2 of Smpdl3b is flanked by loxP sites were purchased from the International Knockout Mouse Consortium (B6N;B6N- SMPDL3btmla(EUCOMM)Wtsi/H). Mice carrying a Cre-recombinase transgene specifically expressed in podocytes, B6.Cg-Tg(NPHS2-cre)295Lbh/J were purchased from Jackson laboratories. Heterozygous Smpdl3b floxed (Smpdl3b fl/+) mice were already mated with Podocin-Cre Tg/Tg mice to generate double-heterozygous Podocin-Cre Tg/+; Smpdl3b-fl/+ mice (pSmpdl3b-fl/+ mice). The latter have been intercrossed to generate mice with a homozygous deletion of Smpdl3b in podocytes (Podocin-Cre Tg/Tg; Smpdl3b-fl/fl, shortly named pSmpdl3b fl/fl mice thereafter). Genotyping will be performed by PCR on DNA isolated from tail biopsies of the weanlings. The following three experimental groups were assessed and followed up to 12 months: (1) pSmpdl-+/+, (2) pSmpdl-fl/+ and (3) pSmpdl-fl/fl.

[0079] Breeding strategy to generate db/db mice and C1P treatment. Male and female db/+ mice (B6.BKS(D)-Leprdb/J) were bred to generate db/db mice. Mice were treated with exogenous C1P starting at 12 weeks of age. 30 mg/kg C1P was injected daily for 12 additional weeks. The following six experimental groups were assessed: (1) +/+ - C1P , (2) +/+ + C1P, (3) db/+ - C1P , (4) db/+ + C1P, (5) db/db - C1P, and (6) db/db + C1P.

[0080] Breeding strategy to generate db/db mice with a podocyte-specific deletion of Smpdl3b. pSmpdl3b-fl/+ mice were generated as described and then crossed to db/+ mice (B6.BKS(D)- Leprdb/J) to generate pSmpdl3b-fl/+; db/+ mice. Double heterozygous mice were crossed to generate double mutant mice, pSmpdl3b-fl/fl; db/db mice. The following nine experimental groups were utilized: (1) pSmpdl3b-+/+;db/+, (2) pSmpdl3b-fl/+;db/+, (3) pSmpdl3b-fl/fl;db/+, (4) pSmpdl3b-+/+ db/+, (5) pSmpdl3b-fl/+;db/+, (6) pSmpdl3b-fl/fl;db/+, (7) pSmpdl3b- +/+;db/db, (8) pSmpdl3b-fl/+;db/db and (9) pSmpdl3b-fl/fl; db/db. Phenotypical analysis of mouse mutants will include the following parameters, which have been established previously (Pedigo et al., The Journal of clinical investigation. 2016). Urinary albumin and creatinine will be determined as described by using mouse specific albumin- specific ELISA and creatinine companion kits (Bethyl Laboratories). Serology: Blood samples collected from mice at baseline and at sacrifice will be analyzed for CBC, lipid panel, AST, ALT, Alkaline Phosphatase, GGT, and BUN. [0081] Molecular analysis of glomeruli. Glomeruli will be isolated by sieving technique followed by hand-picking in PBS under a lOx light microscope. Glomerular expression of Smpdl3b will be analyzed in all mouse models by QRT-PCR and/or Western blot. Histological analysis of glomeruli and quantification of mesangial expansion will be performed.

[0082] Podocyte ultrastructure: For transmission electron microscopy, tissue will be perfused and fixed with 2.5% glutaraldehyde, incubated with Os04, counterstained with 1% tannic acid, dehydrated in a graded series of ethanol and embedded in Epon 812. Ultrathin sections will be studied. Foot process (FP) effacement will be determined as podocyte FP numbers per μιη of GBM in 15 glomerular loops of at least four mice per group. Glomerular basement membrane (GBM) thickness will be assessed by direct measurements from electron micrographs at 3 specified points along each of 10 randomly selected glomerular capillaries and an average GBM thickness will be calculated. An increase representing more than 25% over age-matched controls will be considered as GBM thickening according to the guidelines provided by the "Animal Models of Diabetic Complications Consortium". Podocyte density will be estimated with a relatively new developed method (Venkatareddy et al., J Am Soc Nephrol. 2014;25(5): 1118-29). Podocyte numbers and apoptosis will be assessed by immunofluorescent labeling with rabbit anti-Wilms' Tumor 1 (WT1; Santa Cruz) and mouse anti-synaptopodin (Mundel et al., The Journal of cell biology. 1997;139(l): 193-204). WTl-positive nuclei will be counted in 50 consecutive glomerular cross-sections per animal as reported previously (Guzman et al., Diabetes. 2013). To assess podocyte apoptosis, glomerular sections will be stained for WT1 and the Click-iT Tunnel Assay (Invitrogen) will be used and analyzed as previously described (Bayracki et al., Pediatr Transplant. 2009;13(2):240-3, Hudkins et al., J Am Soc Nephrol.

2010;21(9): 1533-42). Lysates obtained from isolated glomeruli will be utilized in parallel to determine active caspase 3 as described for in vitro studies. Mesangial expansion will be determined as area of Periodic Acid Schiff staining and quantified by Image J.

[0083] Determination oflNSR localization and function. The localization of IR and Cav-1 to podocyte plasma membranes will be studied by immunofluorescence in frozen tissue biopsies. The degree of Akt phosphorylation will be studied by immunofluorescence as we have previously described (Tejada et al., Kidney Int. 2008;73(12): 1385-93). Primary culture of podocytes will be performed and characterized from microdissected glomeruli. Example 2 - SMPDL3b Qverexpression suppresses the IRB signaling and facilitates IRA signaling.

[0084] Podocyte insulin sensitivity is an established fact (1). It has been demonstrated that human podocytes as well as pancreatic β-cells express both insulin receptor isoforms (2). As SMPDL3b is upregulated in podocytes in the setting of DKD (3), and the insulin receptor (IR) has a key role as a modulator of podocyte function (4-6), the ability of podocytes to respond to insulin stimulation depending on different SMPDL3b expression level was evaluated. Human WT podocytes phosphorylate AKT (Ser473) in response to insulin stimulation (0, 0.1 and 1 nM). However, overexpression of SMPDL3b in podocytes resulted in a complete abrogation of AKT phosphorylation under insulin treatment (Figure 1A) and augmentation of the IRA signaling, leading to increased phosphorylation of p70S6K (Figure IB). These data demonstrate that SMPDL3b facilitates the IRA signaling and suppresses the IRB signaling in podocytes.

Example 3 - SMPDL3b Qverexpression Affects the Expression and Localization of the Insulin Receptor.

[0085] The amount of the total IR protein (Figure 2A), phosphorylated or total Cav-1 (Figure 2B) remains unchanged in SMP OE cells when compared to wild type podocytes (WT). To estimate if SMPDL3b affects localization of the IR or Cav-1 at the plasma membrane, experiments were performed where the plasma membrane fraction (Na/K-ATPase positive) of podocytes were separated from their cytosolic fraction (MEK-1/2 positive). A significant reduction of the PM localization of the IR in SMP OE podocytes was observed when compared to WT (Figure 2C). At the same time, SMPDL3b does not affect the PM localization of the total or phosphorylated Cav-1 (Figure 2D).

Example 4. SMPDL3b Binds IRA and IRB and Affects Co-localization of the Insulin Receptor Isoforms, Caveolin-1 and Lipid Rafts.

[0086] Given the observed effect of SMPDL3b expression level in podocytes in response to insulin stimulation, the interaction of SMPDL3b with IRA and/or IRB by immunoprecipitation experiments of exogenous proteins co-transfected in HEK293 cells was investigated. In adipocytes and myocytes it is established that the IR interacts with caveolin 1 (Cav-1) and can modulate the insulin signaling (7, 8). Results demonstrated that SMPDL3b interacted not only with both isoforms of the IR, but also with Cav-1 (Figure 3 A). Empty-GFP vector was used as a negative control. To determine if SMPDL3b affects IRA/Cav-1 and/or IRB/Cav-1 interaction, co-immunoprecipitation experiments in HEK293 cells was performed. Results demonstrated that Cavl-GFP can immunoprecipitate both IRA-FLAG and IRB-FLAG (Figure 3B). Empty-GFP vector was used as a negative control. Surprisingly, it was demonstrated that SMPDL3b augments the IRA/Cav-1 interaction, while suppressing the IRB/Cav-1 interaction (Figure 3B).

[0087] One possibility to explain the difference in interaction between the IR isoforms and Cav-1 is the different localization of the IRA/IRB and Cav-1 at the plasma membrane in the presence of SMPDL3b overexpression. To test whether different isoforms of the insulin receptor are co-localized, or whether IRA/IRB and Cav-1 exhibit a distinct distribution in vitro, HEK293 cells were co-transfected with plasmids containing tagged receptor isoforms with monomeric fluorescent proteins dTomato or mCFP and Cav-l-GFP plasmid in the presence of SMPDL3b overexpression. To get a quantitative assessment of the co-localization degree, a Pearson's coefficient analysis using JACoP by ImageJ was performed and the results are provided in Table 1.

Table 1.

Control SMPDL3b+

iRA/IRB O.S4±0.07 n/a

!RA/Cavl 0.82+0.07 0.85+0.09

iR8/Cav1 0.85±0.05 0.75±0.06*

!RA LR 0.79+0.10 0.77+0.12

!RB/LR 0.87+0.05 0.78±0.11

[0088] Analysis of these images showed an approximately 80% co-localization when differently tagged IRA and IRB isoforms were co-expressed (data not shown),, which supports the hypothesis that isoforms of the IR have different localization at the plasma membrane.

Results demonstrated that SMPDL3b abrogates the co-localization of IRB and Cav-1, but maintains the co-localization of IRA and Cav-1 (data not shown).

[0089] To test whether or not both IR isoforms are located within cholesterol-enriched plasma membrane microdomains (lipid rafts), either IRA-dTomato or IRB-dTomato was co-expressed with Myr-Palm-mCFP. Myr-Palm-mCFP is a monomeric CFP variant fused with an amino acid string that allows lipid modification by myristoylation/palmitoylation and results in the co- localization with cholesterol-enriched membrane domains (9). Expression of IRA/Myr-Palm- mCFP or IRB/Myr-Palm-mCFP resulted in high degree of co-localization (data not shown), which suggested that both IR isoforms are located with cholesterol-enriched membrane domains. In the presence of SMPDL3b replacement of the IRB from cholesterol-enriched membrane domains was observed. SMPDL3b does not affect co-localization of IRA with lipid rafts.

Example 5 - SMPDL3b Overexpression Caused Cholesterol Accumulation and Decreased Ceramide- 1 -Phosphate (CIP) Production in Podocytes.

[0090] Intracellular accumulation of cholesterol is one of the major determinants of cellular insulin signaling (10, 11) and multiple studies have shown that insulin resistance correlates with microalbuminuria in patients with type 1 diabetes (12) or type 2 diabetes (13).

[0091] To demonstrate if SMPDL3b overexpression causes changes in lipid content, multiple lipid analysis was performed. Oil red O staining in podocytes showed significant elevation of lipid droplets in SMP OE cells compared to WT (Figure 4A), demonstrating increased total cholesterol content, but not amount of triglycerides or phospholipids (Figure 4B). Using electrospray ionization/tandem mas spectrometry, it was shown that the amount of total ceramide (Figure 5A) and amount of total CIP (Figure 5B) was significantly decreased in SMP OE podocytes compared to WT cells. Interestingly, SMP OE podocytes as well as WT podocytes demonstrated the prevalence of C16:0 CIP species (Figure 5C). No effect of SMPDL3b overexpression on amount of total sphingomyelin (Figure 5D), total sphingosine (Figure 5E), or total sphingosine- 1 -phosphate (Figure 5F) was observed.

[0092] Taken together, these results support the significant role of SMPDL3b as a regulator of insulin signaling in human podocytes.

Example 6 - CIP Replacement Rescuing AKT Phosphorylation in SMPDL3b Overexpression Podocytes.

[0093] Since CIP C16:0 species showed the most significant changes in human podocytes, whether exogenous CIP substitution would restore AKT phosphorylation in SMP OE podocytes was investigated. Podocytes were pre-treated with lOOuM of a recombinant CIP C16:0 for lh and then stimulated with InM of human insulin for 30 min. CIP pre-treatment did significantly restore AKT (Ser473) phosphorylation in SMP OE podocytes (Figure 7) compared to WT cells. Overall, these data suggest that SMPDL3b-dependence decreases endogenous CIP production and may cause insulin resistance of SMP OE podocytes.

Example 7 - CIP replacement in vivo improved kidney function in an animal model of diabetes [0094] As shown in the previous Examples, CIP replacement in vitro restored insulin signaling and Akt phosphorylation in podocytes. The experiment in this Example was designed to determine whether CIP replacement in vivo would improve kidney function in a mouse experimental model of diabetes.

[0095] The db/db mouse model that has been demonstrated to be associated with insulin resistance in prior studies (Tejada T et al, Kidney International 2008) was selected. It was determined that kidney cortex isolated from diabetic db/db mice are characterized by a decreased ceramide content when compared to db/+ mice. This was due to decreased ceramide 16:0, which is the most abundant ceramide species found in the kidney. Although CIP species was not detected in kidney cortex, based on the in vitro data provided herein that SMPDL3b

overexpression was associated with CIP deficiency (primarily C16-C1P), CIP was administered to the diabetic animal (daily intraperitoneal administration of 30 mg/kg C16:0 CIP for a period of four weeks) to determine if CIP administration was safe and effective in reducing

albuminuria, which is the first sign of renal impairment in diabetes. CIP was determined to be safe (Figure 7C) and partially protect from albuminuria. These data establish that treatment and or prevention strategies with CIP, as well as its analogues or derivatives, protect from the development of diabetic nephropathy in affected patients.

Example 8 - SMPDL3b Functions as a ceramide- 1 -phosphate (CIP) phosphatase

[0096] Electrospray ionization/tandem mass- spectrometry analysis of sphingolipids in human podocytes was performed. Results indicated that SMP OE podocytes have the same amount of total sphingomyelin and ceramide (Figure 8 A,B), they are characterized by a reduction of CIP (Figure 8C), and unchanged S IP (Figure 8D). Assessment of C6 ceramide production by TLC in SMPDL3b or empty vector transfected cells exposed to increasing uM concentrations of CIP demonstrated that SMP OE cells had a dose dependent increase in ceramide production (n=3, Dr Futerman lab). Reduction in total CIP is not linked to SMPDL3b phosphodiesterase activity, as the total CIP content does not vary in HEK cells transfected with the SMPDL3b

phosphodiesterase mutant SMPH135A or wild type SMP (Figure 8F).

Example 9 - pSMPDL3b-fl/fl mice are phenotypically normal and may be protected from DKD.

[0097] Kidney weight/body weight (Figure 9A) and mesangial expansion (Figure 9B), glomerulosclerosis were analyzed by Picrousirius Red (Figure 9C) and albuminuria (Figure 9D) from pSmpdl3b-+/+, and pSmpdl3b-fl/fl. Homozygous pSmpdl3b-fl/fl mice were viable and did not show albuminuria or histological abnormalities at 9 months when compared to pSmpdl3b- +/+ littermates. Urine collected from an ongoing colony of 20 weeks old pSMPDL3bfl/fl;db/db mice demonstrated that pSMPDL3b-fl/fl;db/db mice have less albuminuria than pSmpdl3b- +/+;db/db mice (Figure 9E).

[0098] References

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[00107] 9. Zacharias DA, et al., .Science (New York, NY). 2002;296(5569):913-6.

[00108] 10. Saltiel AR, Kahn CR., Nature. 2001;414(6865):799-806.

[00109] 11. White MF, Kahn CR., The Journal of biological chemistry. 1994;269(l): l-4.

[00110] 12. Ekstrand AV, et al., .Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

1998;13(12):3079-83.

[00111] 13. Parvanova Al, et al., Diabetes. 2006;55(5): 1456-62.

[00112] 14. Saleem MA, et al., Journal of the American Society of Nephrology : JASN. 2002;13(3):630-8.

[00113] 15. Fornoni A, et al., Science translational medicine. 2011;3(85):85ra46-85ra46. [00114] 16. Leibiger B et al., Molecular cell. 2001;7(3):559-70. [00115] 17. Uhles S, et al, The Journal of Cell Biology. 2003;163(6): 1327-37. [00116] 18. Bielawski J, et al., Methods (San Diego, Calif). 2006;39(2):82-91.

Claims

What is claimed is:

1. A method of restoring insulin signaling in a cell that overexpresses

sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) comprising contacting the cell with a ceramide in an amount effective to restore insulin signaling in the cell.

2. The method of claim 1, wherein the cell is a podocyte.

3. The method of claim 1, wherein the ceramide is C1P.

4. The method of claim 1, wherein the ceramide is C16:0 C1P.

5. The method of claim 1, wherein the contacting occurs in vitro.

6. The method of claim 1, wherein the contacting occurs in vivo.

7. A method of restoring insulin signaling in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to restore insulin signaling in the subject.

8. The method of claim 7, wherein the subject is suffering from a disorder associated with impaired insulin signaling.

9. The method of claim 8, wherein the disorder associated with impaired insulin signaling is selected from the group consisting of diabetes, pre-diabetes, obesity, insulin resistance, polycystic ovary syndrome, diabetes related macrovascular complications, and diabetes related microvascular complications.

10. The method of claim 1, wherein the subject is suffering from diabetic

nephropathy.

11. The method of claim 7, wherein the ceramide is C1P.

12. The method of claim 11, wherein the ceramide is C16:0 C1P.

13. A method of treating a disorder associated with impaired insulin signaling in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to restore insulin signaling in the subject.

14. The method of claim 13, wherein the disorder is a proteinuric kidney disease.

15. The method of claim 13, wherein the disorder is diabetic nephropathy.

16. The method of claim 13, wherein the ceramide is C1P.

17. The method of claim 13, wherein the ceramide is C16:0 C1P.

18. A method of treating a disorder associated with albuminuria or proteinuria in a mammalian subject in need thereof comprising administering a ceramide to the subject in an amount effective to improve proteinuria or albuminuria in the subject.

19. The method of claim 18, wherein the disorder associated with albuminuria is selected from the group consisting of proliferative glomerulonephritis and nonproliferative glomerulonephritis .

20. The method of claim 18, wherein the disorder associated with proteinuria is diabetic nephropathy

21. The method of claim 18, wherein the ceramide is C1P.

22. The method of claim 18, wherein the ceramide is C16:0 C1P.

23. The method of any of the preceding claims, wherein the ceramide is administered parenterally.

24. The method of claim 23, wherein the ceramide is administered by intraperitoneal injection.

25. The method of claim 23, wherein the ceramide is administered by subcutaneous injection.

26. The method of any one of claims 1-22, wherein the ceramide is administered orally.

27. The method claim 7, 13, or 18, wherein the ceramide is administered for a period of at least 4 weeks.

28. The method of claim 7, 13 or 18, wherein the ceramide is administered at a dose ranging from about 3 mg/kg to about 300 mg/kg.