Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice.

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS-/-) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS-/- than in STZ-WT GECs. Additionally, genes in STZ-eNOS-/- GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS-/- mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS-/- GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.

Protein S Protects against Podocyte Injury in Diabetic Nephropathy.

Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.

Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury.

Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. In vivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease.

Transcriptomic analysis uncovers novel synergistic mechanisms in combination therapy for lupus nephritis.

A recent clinical study showed that combination therapy consisting of mycophenolate mofetil, tacrolimus and steroids was shown to be more effective in achieving complete remission in patients with severe forms of lupus nephritis than conventional therapy consisting of intravenous cyclophosphamide and steroids. To explore the underlying molecular and cellular mechanisms of increased efficacy of the combination therapy regimen, we employed a mouse model of lupus nephritis, MRL/lpr mice, and treated them with monotherapies of prednisone, mycophenolate mofetil, or tacrolimus, or with their combination. Consistent with previous clinical findings, combination therapy markedly improved renal outcome compared to the monotherapies in mice with lupus nephritis. Transcriptomic analysis of their kidneys revealed distinct molecular pathways that were differentially regulated in combination therapy versus monotherapies. Combination therapy not only provided additive immunosuppressive effects, but also induced gene expression and molecular pathways to confer enhanced renoprotection. Specifically, combination therapy inhibited TLR7 expression in the kidneys of mice with lupus nephritis; combination of tacrolimus and mycophenolate mofetil led to better stabilization of the podocyte actin cytoskeleton through the reciprocal regulation of RhoA and Rac1 activities. Combination therapy strongly suppressed the IL-6/Stat3 pathway. These findings were further validated in renal biopsy samples from patients with lupus nephritis before and after treatments with mycophenolate mofetil, tacrolimus or combination therapy. Thus, our study further supports the earlier clinical finding and further provides insights into the molecular basis for increased efficacy of combination therapy.

Retinoic acid improves nephrotoxic serum-induced glomerulonephritis through activation of podocyte retinoic acid receptor α.

Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum-induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes.

Reduction in podocyte SIRT1 accelerates kidney injury in aging mice.

Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1RNAi) and control mice that express shRNA for luciferase (Pod-LuciRNAi). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1RNAi mice compared with aged Pod-LuciRNAi mice. Although podocyte-specific markers decreased in aged mice compared with the young controls, the decrease was further exacerbated in aged Pod-Sirt1RNAi compared with Pod-LuciRNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1RNAi mice than Pod-LuciRNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1RNAi glomeruli were associated with reduced activation of the transcription factors peroxisome proliferator-activated receptor (PPAR)-α coactivador-1 (PGC1α)/PPARγ, forkhead box O (FOXO)3, FOXO4, and p65 NF-κB, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.

Knockdown of RTN1A attenuates ER stress and kidney injury in albumin overload-induced nephropathy.

Our previous studies have suggested a critical role of reticulon (RTN)1A in mediating endoplasmic reticulum (ER) stress in kidney cells of animal models and humans with kidney diseases. A large body of evidence suggests that proteinuria itself can cause tubular cell injury leading to the progression of kidney disease. In the present study, we determined whether RTN1A mediates proteinuria-induced tubular cell injury through increased ER stress. We found that incubation of HK2 cells with human serum albumin induced the expression of RTN1A and ER stress markers, whereas knockdown of RTN1A expression attenuated human serum albumin-induced ER stress and tubular cell apoptosis in vitro. In vivo, we found that tubular cell-specific RTN1 knockdown resulted in a significant attenuation of tubular cell ER stress, apoptosis, and renal fibrosis in a model of albumin overload nephropathy. Based on these findings, we conclude that RTN1A is a key mediator for proteinuria-induced tubular cell toxicity and renal fibrosis.

RTN1 mediates progression of kidney disease by inducing ER stress.

Identification of new biomarkers and drug targets for chronic kidney disease (CKD) is required for the development of more effective therapy. Here we report an association between expression of reticulon 1 (RTN1) and severity of CKD. An isoform-specific increase in the expression of RTN1A is detected in the diseased kidneys from mice and humans, and correlates inversely with renal function in patients with diabetic nephropathy. RTN1 overexpression in renal cells induces ER stress and apoptosis, whereas RTN1 knockdown attenuates tunicamycin-induced and hyperglycaemia-induced ER stress and apoptosis. RTN1A interacts with PERK through its N-terminal and C-terminal domains, and mutation of these domains prevents this effect on ER stress. Knockdown of Rtn1a expression in vivo attenuates ER stress and renal fibrosis in mice with unilateral ureteral obstruction, and also attenuates ER stress, proteinuria, glomerular hypertrophy and mesangial expansion in diabetic mice. Together, these data indicate that RTN1A contributes to progression of kidney disease by inducing ER stress.

Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys.

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference-mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm-mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling to preserve glomerular function and podocyte viability in adult mice.

Krüppel-like factor 6 regulates mitochondrial function in the kidney.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.

BAMBI elimination enhances alternative TGF-ß signaling and glomerular dysfunction in diabetic mice.

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-ß type I receptor family and a negative modulator of TGF-ß kinase signaling, and BAMBI(-/-) mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-ß overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI(-/-) mice developed more albuminuria, with a widening of foot processes, than BAMBI(+/+) mice, along with increased activation of alternative TGF-ß pathways such as extracellular signal-related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI(-/-) mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI(-/-) mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI(+/+) or BAMBI(-/-) mice with TGF-ß resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI(-/-) mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-ß signaling may therefore be of interest in diabetic nephropathy.

Reduced Krüppel-like factor 2 expression may aggravate the endothelial injury of diabetic nephropathy.

Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, has endoprotective effects. In streptozotocin-induced diabetic rats, we found that glomerular Klf2 expression was reduced in comparison with nondiabetic rats. However, normalization of hyperglycemia by insulin treatment increased Klf2 expression to a level higher than that of nondiabetic rats. Consistent with this, we found that Klf2 expression was suppressed by high glucose but increased by insulin in cultured endothelial cells. To determine the role of KLF2 in streptozotocin-induced diabetic nephropathy, we used endothelial cell-specific Klf2 heterozygous knockout mice and found that diabetic knockout mice developed more kidney/glomerular hypertrophy and proteinuria than diabetic wild-type mice. Glomerular expression of Vegfa, Flk1, and angiopoietin 2 increased, but expression of Flt1, Tie2, and angiopoietin 1 decreased, in diabetic knockout mice compared with diabetic wild-type mice. Glomerular expression of ZO-1, glycocalyx, and eNOS was also decreased in diabetic knockout compared with diabetic wild-type mice. These data suggest knockdown of Klf2 expression in the endothelial cells induced more endothelial cell injury. Interestingly, podocyte injury was also more prominent in diabetic knockout compared with diabetic wild-type mice, indicating a cross talk between these two cell types. Thus, KLF2 may play a role in glomerular endothelial cell injury in early diabetic nephropathy.

Glomerular endothelial cell injury and cross talk in diabetic kidney disease.

Diabetic kidney disease (DKD) remains a leading cause of new-onset end-stage renal disease (ESRD), and yet, at present, the treatment is still very limited. A better understanding of the pathogenesis of DKD is therefore necessary to develop more effective therapies. Increasing evidence suggests that glomerular endothelial cell (GEC) injury plays a major role in the development and progression of DKD. Alteration of the glomerular endothelial cell surface layer, including its major component, glycocalyx, is a leading cause of microalbuminuria observed in early DKD. Many studies suggest a presence of cross talk between glomerular cells, such as between GEC and mesangial cells or GEC and podocytes. PDGFB/PDGFRß is a major mediator for GEC and mesangial cell cross talk, while vascular endothelial growth factor (VEGF), angiopoietins, and endothelin-1 are the major mediators for GEC and podocyte communication. In DKD, GEC injury may lead to podocyte damage, while podocyte loss further exacerbates GEC injury, forming a vicious cycle. Therefore, GEC injury may predispose to albuminuria in diabetes either directly or indirectly by communication with neighboring podocytes and mesangial cells via secreted mediators. Identification of novel mediators of glomerular cell cross talk, such as microRNAs, will lead to a better understanding of the pathogenesis of DKD. Targeting these mediators may be a novel approach to develop more effective therapy for DKD.

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis.

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7-like 2-dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-ß1 administration upregulated SHROOM3 expression in a ß-catenin/TCF7L2-mediated manner, while SHROOM3 in turn facilitated canonical TGF-ß1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell-specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-ß1 signaling and contributes to allograft injury.

In vivo RNA interference models of inducible and reversible Sirt1 knockdown in kidney cells.

The silent mating type information regulation 2 homolog 1 gene (Sirt1) encodes an NAD-dependent deacetylase that modifies the activity of well-known transcriptional regulators affected in kidney diseases. Sirt1 is expressed in the kidney podocyte, but its function in the podocyte is not clear. Genetically engineered mice with inducible and reversible Sirt1 knockdown in widespread, podocyte-specific, or tubular-specific patterns were generated. We found that mice with 80% knockdown of renal Sirt1 expression have normal glomerular function under the basal condition. When challenged with doxorubicin (Adriamycin), these mice develop marked albuminuria, glomerulosclerosis, mitochondrial injury, and impaired autophagy of damaged mitochondria. Reversal of Sirt1 knockdown during the early phase of Adriamycin-induced nephropathy prevented the progression of glomerular injury and reduced the accumulation of dysmorphic mitochondria in podocytes but did not reverse the progression of albuminuria and glomerulosclerosis. Sirt1 knockdown mice with diabetes mellitus, which is known to cause mitochondrial dysfunction in the kidney, developed more albuminuria and mitochondrial dysfunction compared with diabetic mice without Sirt1 knockdown. In conclusion, these results demonstrate that our RNA interference-mediated Sirt1 knockdown models are valid and versatile tools for characterizing the function of Sirt1 in the kidney; Sirt1 plays a role in homeostatic maintenance of podocytes under the condition of mitochondrial stress/injury.

Role of transcription factor acetylation in diabetic kidney disease.

Nuclear factor (NF)-κB and signal transducer and activator of transcription 3 (STAT3) play a critical role in diabetic nephropathy (DN). Sirtuin-1 (SIRT1) regulates transcriptional activation of target genes through protein deacetylation. Here, we determined the roles of Sirt1 and the effect of NF-κB (p65) and STAT3 acetylation in DN. We found that acetylation of p65 and STAT3 was increased in both mouse and human diabetic kidneys. In human podocytes, advanced glycation end products (AGEs) induced p65 and STAT3 acetylation and overexpression of acetylation-incompetent mutants of p65 and STAT3 abrogated AGE-induced expression of NF-κB and STAT3 target genes. Inhibition of AGE formation in db/db mice by pyridoxamine treatment attenuated proteinuria and podocyte injury, restored SIRT1 expression, and reduced p65 and STAT3 acetylation. Diabetic db/db mice with conditional deletion of SIRT1 in podocytes developed more proteinuria, kidney injury, and acetylation of p65 and STAT3 compared with db/db mice without SIRT1 deletion. Treatment of db/db mice with a bromodomain and extraterminal (BET)-specific bromodomain inhibitor (MS417) which blocks acetylation-mediated association of p65 and STAT3 with BET proteins, attenuated proteinuria, and kidney injury. Our findings strongly support a critical role for p65 and STAT3 acetylation in DN. Targeting protein acetylation could be a potential new therapy for DN.

Diabetic nephropathy in a nonobese mouse model of type 2 diabetes mellitus.

A large body of research has contributed to our understanding of the pathophysiology of diabetic nephropathy. Yet, many questions remain regarding the progression of a disease that accounts for nearly half the patients entering dialysis yearly. Several murine models of diabetic nephropathy secondary to Type 2 diabetes mellitus (T2DM) do exist, and some are more representative than others, but all have limitations. In this study, we aimed to identify a new mouse model of diabetic nephropathy secondary to T2DM in a previously described T2DM model, the MKR (MCK-KR-hIGF-IR) mouse. In this mouse model, T2DM develops as a result of functional inactivation of insulin-like growth factor-1 receptor (IGF-1R) in the skeletal muscle. These mice are lean, with marked insulin resistance, hyperinsulinemia, hyperglycemia, and dyslipidemia and thus are representative of nonobese human T2DM. We show that the MKR mice, when under stress (high-fat diet or unilateral nephrectomy), develop progressive diabetic nephropathy with marked albuminuria and meet the histopathological criteria as defined by the Animal Models of Diabetic Complications Consortium. Finally, these MKR mice are fertile and are on a common background strain, making it a novel model to study the progression of diabetic nephropathy.

Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis-associated glomerular injury.

Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated ß1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained ß1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of ß1 integrin.