Stuart J. Shankland

Podocyte Biology and Response to Injury

The visceral glomerular epithelial cell, also called podocyte, is a terminally differentiated cell that lines the outer aspect of the glomerular basement membrane (GBM). It therefore forms the final barrier to protein loss, which explains why podocyte injury is typically associated with marked

Hematopoietic Stem Cells Contribute to the Regeneration of Renal Tubules after Renal Ischemia-Reperfusion Injury in Mice

Ischemia-reperfusion injury (I/R injury) is a common cause of acute renal failure. Recovery from I/R injury requires renal tubular regeneration. Hematopoietic stem cells (HSC) have been shown to be capable of differentiating into hepatocytes, cardiac myocytes, gastrointestinal epithelial cells, and vascular endothelial cells during tissue repair. The current study tested the hypothesis that murine HSC can contribute to the regeneration of renal tubular epithelial cells after I/R injury. HSC isolated from male Rosa26 mice that express beta-galactosidase constitutively were transplanted into female nontransgenic mice after unilateral renal I/R injury. Four weeks after HSC transplantation, beta-galactosidase-positive cells were detected in renal tubules of the recipients by X-Gal staining. PCR analysis of the male-specific Sry gene and Y chromosome fluorescence in situ hybridization confirmed the presence of male-derived cells in the kidneys of female recipients. Antibody co-staining showed that beta-galactosidase was primarily expressed in renal proximal tubules. This is the first report to show that HSC can differentiate into renal tubular cells after I/R injury. Because of their availability, HSC may be useful for cell replacement therapy of acute renal failure.

Proteinuria in diabetic kidney disease: A mechanistic viewpoint

Proteinuria is the hallmark of diabetic kidney disease (DKD) and is an independent risk factor for both renal disease progression, and cardiovascular disease. Although the characteristic pathological changes in DKD include thickening of the glomerular basement membrane and mesangial expansion, these changes per se do not readily explain how patients develop proteinuria. Recent advances in podocyte and glomerular endothelial cell biology have shifted our focus to also include these cells of the glomerular filtration barrier in the development of proteinuria in DKD. This review describes the pathophysiological mechanisms at a cellular level which explain why patients with DKD develop proteinuria.

Induction of TRPC6 Channel in Acquired Forms of Proteinuric Kidney Disease

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.

Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27(Kip1).

Proliferation and apoptosis are increased in many types of inflammatory diseases. A role for the cyclin kinase inhibitor p27(Kip1) (p27) in limiting proliferation has been shown. In this study, we show that p27(-/-) mesangial cells and fibroblasts have strikingly elevated rates of apoptosis, not proliferation, when deprived of growth factors. Apoptosis was rescued by restoration of p27 expression. Cyclin A-cyclin-dependent kinase 2 (CDK2) activity, but not cyclin E-CDK2 activity, was increased in serum-starved p27(-/-) cells, and decreasing CDK2 activity, either pharmacologically (Roscovitine) or by a dominant-negative mutant, inhibited apoptosis. Our results show that a new biological function for the CDK inhibitor p27 is protection of cells from apoptosis by constraining CDK2 activity. These results suggest that CDK inhibitors are necessary for coordinating the cell cycle and cell-death programs so that cell viability is maintained during exit from the cell cycle.

Urinary Podocyte Loss Is a More Specific Marker of Ongoing Glomerular Damage than Proteinuria

Podocyte loss contributes to the development of glomerulosclerosis. Although podocyte detachment has been recognized as a new mechanism of podocyte loss in glomerular diseases, its time course and relationship to disease activity are not known. Urinary excretion of viable podocytes was quantified in two models of transient glomerular injury, i.e., rats with puromycin aminonucleoside-induced nephrosis (PAN) and mesangioproliferative nephropathy (anti-Thy 1.1 nephritis model), as well as in a model of continuous glomerular injury, i.e., hypertensive nephropathy (5/6-nephrectomy model), and in aging rats. The number of glomerular Wilms tumor (WT)-1-positive podocytes and the glomerular expression of cell-cycle proteins in vivo were assessed. Urinary podocyte loss occurred in both primary (PAN) and secondary (anti-Thy 1.1 nephritis) in parallel to the onset of proteinuria. However, subsequently proteinuria persisted despite remission of podocyturia. In continuous glomerular injury, i.e., after 5/6-nephrectomy, podocyturia paralleled the course of proteinuria and of systemic hypertension, whereas no podocyturia became detectable during normal aging (up to 12 mo). Despite podocyte detachment of varying degrees, no decrease in glomerular podocyte counts (i.e., WT-1 positive nuclei) was noted in either disease model. Podocyturia in the PAN and anti-Thy 1.1 nephritis model was preceded by entry of glomerular podocytes into the cell cycle, i.e., cyclin D1, cdc2, and/or proliferating cell nuclear antigen (PCNA) expression. Podocyturia is a widespread phenomenon in glomerular disease and not simply a reflection of proteinuria because it is limited to phases of ongoing glomerular injury. The data suggest that podocyturia may become a more sensitive means to assess the activity of glomerular damage than proteinuria.

Inducible rodent models of acquired podocyte diseases

Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded.

Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors are believed to have a major role. Here, we show that deletion of the Von Hippel–Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor α subunits (HIFs). Using gene expression profiling, we identified de novo expression of the HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and humans with RPGN. The course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease.

Direct in vivo inhibition of the nuclear cell cycle cascade in experimental mesangial proliferative glomerulonephritis with Roscovitine, a novel cyclin-dependent kinase antagonist.

Glomerular injury is characterized by mesangial cell (MC) proliferation and matrix formation. We sought to determine if reducing the activity of cyclin-dependent kinase 2 (CDK2) with the purine analogue, Roscovitine, decreased MC proliferation in vitro and in vivo. Roscovitine (25 microM) inhibited FCS-induced proliferation (P < 0.0001) in cultured MC. Rats with experimental mesangial proliferative glomerulonephritis (Thy1 model) were divided into two groups. A prevention group received daily intraperitoneal injections of Roscovitine in DMSO (2.8 mg/kg) starting at day 1. A treatment group received daily Roscovitine starting at day 3, when MC proliferation was established. Control Thy1 rats received DMSO alone. MC proliferation (PCNA +/OX7 + double immunostaining) was reduced by > 50% at days 5 and 10 in the Roscovitine prevention group, and at day 5 in the treatment group (P < 0.0001). Early administration of Roscovitine reduced immunostaining for collagen type IV, laminin, and fibronectin at days 5 and 10 (r = 0.984; P < 0.001), which was associated with improved renal function (urinary protein/creatinine, blood urea nitrogen, P < 0.05). We conclude that reducing the activity of CDK2 with Roscovitine in experimental glomerulonephritis decreases cell proliferation and matrix production, resulting in improved renal function, and may be a useful therapeutic intervention in disease characterized by proliferation.

TLR4 Links Podocytes with the Innate Immune System to Mediate Glomerular Injury

Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN; mice transgenic for thymic stromal lymphopoietin [TSLP], with or without deletion of the Fcgamma receptor IIb). Expression of TLR1 through 9 and TLR11 mRNA was detectable in whole kidneys and in isolated glomeruli of wild-type mice, with TLR3 and TLR4 having the highest absolute levels of expression. TLR1, 2, and 4 were increased in TSLP transgenic mice and even higher in TSLP transgenic FcgammaRIIb-deficient mice. TLR5 through 9 and 11 were upregulated to similar degrees in TSLP transgenic and TSLP transgenic FcgammaRIIb-deficient mice. Immunohistochemical studies of nephritic glomeruli localized TLR4 protein to podocytes. Cultured podocytes also expressed TLR4, and stimulation with TLR4-specific ligands resulted in a marked induction of chemokines; this was reduced by specific knockdown of TLR4 with siRNA. Fibrinogen, a potential endogenous TLR4 ligand, was shown to induce a similar profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered by the deposition of immune complexes.