Biomedical subjects

Stuart J Shankland

Publications and source records attributed to Stuart J Shankland.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Cyclin I protects podocytes from apoptosis.

The limited regenerative capacity of the glomerular podocyte following injury underlies the development of glomerulosclerosis and progressive renal failure in a diverse range of kidney diseases. We discovered that, in the kidney, cyclin I is uniquely expressed in the glomerular podocyte, and have constructed cyclin I knock-out mice to explore the biological function of cyclin I in these cells. Cyclin I knock-out (-/-) podocytes showed an increased susceptibility to apoptosis both in vitro and in vivo. Following induction of experimental glomerulonephritis, podocyte apoptosis was increased 4-fold in the cyclin I -/- mice, which was associated with dramatically decreased renal function. Our previous data showed that the Cdk inhibitor p21(Cip1/Waf1) protects podocytes from certain apoptotic stimuli. In cultured cyclin I -/- podocytes, the level of p21(Cip1/Waf1) was lower at base line, had a shorter half-life, and declined more rapidly in response to apoptotic stimuli than in wild-type cells. Enforced expression of p21(Cip1/Waf1) reversed the susceptibility of cyclin I -/- podocytes to apoptosis. Cyclin I protects podocytes from apoptosis, and we provide preliminary data to suggest that this is mediated by stabilization of p21(Cip1/Waf1).

Animals↗

PubMed2006

Glial cell line-derived neurotrophic factor and its receptor ret is a novel ligand-receptor complex critical for survival response during podocyte injury.

Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair.

Animals↗

PubMed2006

The renin-angiotensin system in glomerular podocytes: mediator of glomerulosclerosis and link to hypertensive nephropathy.

The renoprotective effects of pharmacologic inhibition of angiotensin II extend beyond the blood pressure-lowering effects alone, consistent with the observation that angiotensin II is produced locally within the kidney and mediates tissue injury through a series of nonhemodynamic effects. Podocytes are terminally differentiated epithelial cells that contribute to the filtration barrier of the kidney, but also safeguard against the development of glomerulosclerosis. Mounting evidence demonstrates that podocytes are not only a local source of angiotensin II production, but are also vulnerable to its deleterious effects, thus fueling the future development of glomerular scarring. In this review article, we explore the role of a local angiotensin system as a mediator of podocyte injury and discuss its potential link to hypertensive renal disease.

Angiotensin II↗

PubMed2006

Podocyte injury and targeting therapy: an update.

PURPOSE OF REVIEW: Podocyte injury is a central event in the development of glomerulosclerosis. This review highlights contributions from the past year to our understanding of mechanisms of podocyte injury and implications for potential treatment strategies of glomerular disease. RECENT FINDINGS: Rearrangement of the actin cytoskeleton, the backbone linking the slit diaphragm, apical domain and sole plate, serves as a common denominator during foot process effacement. Reports on the role of synaptopodin and CDK5 on actin dynamics as well as cathepsin L and B7.1 in subsequent cell migration have expanded our understanding of the podocyte response to injury. Mounting evidence supports an expanding role of the slit diaphragm in signal transduction to mediate downstream cellular responses, including prosurvival effects of the integral proteins nephrin and CD2AP. The discovery that TRPC6 localizes to the slit diaphragm and identification of specific mutations of the transport channel in kindreds of familial focal segmental glomerulosclerosis implicate a causal role for aberrant calcium signaling in podocyte injury. Disruption of the dystroglycan complex, which anchors the podocyte to the underlying basement membrane, in states of foot process effacement may have implications for the recent finding of viable podocytes in the urine in glomerular disease. SUMMARY: The resurgence of research in podocyte biology over the past decade underscores the importance of this unique cell in preserving glomerular structure and function. A greater understanding of the complex signaling mechanisms governing podocyte biology in health and disease will ultimately lead to novel therapeutic avenues for treating disorders of the podocyte.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2005

Therapeutics in renal disease: the road ahead for antiproliferative targets.

Discovery into the molecular basis of renal disease is occurring at an unprecedented rate. With the advent of the NIH Roadmap, there is a greater expectation of translating this knowledge into new treatments. Here, we review the therapeutic strategy to preserve renal function in proliferative renal diseases by directly inhibiting the mitogenic pathways within renal parenchymal cells that promote G0 to G1/S cell-cycle phase progression. Reductionist methodologies have identified several antiproliferative molecular targets, and promising preclinical testing of leading small-molecule drugs to modulate these targets has now led to landmark clinical trials. Yet, this advancement into targeted therapy highlights important differences between the therapeutic goals of molecular nephrology versus molecular oncology and, by extension, the poorly understood role of alternative target activity in drug efficacy. Systems research to clarify these issues should accelerate the development of this promising therapeutic strategy.

Animals↗

PubMed2005

Cell cycle and glomerular disease: a minireview.

Globally, glomerular diseases are a leading cause of chronic and end-stage renal disease. In the mature glomerulus, under normal conditions, glomerular cells have a low turnover rate. However, in disease, a variety of pathophysiological stimuli can lead to disturbances in glomerular cell biology, including toxins, immune-mediated stresses, metabolic derangements, drugs, infections, hemodynamic changes, growth factors, and cytokines. Not only does the form of injury govern the histologic and clinical manifestations of disease, but also the nature of the response to injury. This response to injury is largely cell-type specific, and the glomerulus represents a rare microcosm of the larger organism in which one can study the cellular responses of three very distinct cell types: mesangial cells, visceral epithelial cells or podocytes, and endothelial cells. These cells can undergo several cell fates in response to injury, including proliferation, de-differentiation, hypertrophy, senescence, apoptosis, or necrosis. The regulation of these responses occurs at the level of the cell cycle, coordinated by positive regulators, cyclins and cyclin-dependent kinases, and negative regulators, cyclin-dependent kinase inhibitors. There is now a large body of literature confirming the importance of cell cycle regulatory proteins in the glomerular cellular response to injury. The recent advances in cell cycle biology in diseases of the mesangial cell and the podocyte are the focus of this minireview.

Animals↗

PubMed2005

The cyclin-dependent kinase inhibitor p21 limits murine mesangial proliferative glomerulonephritis.

BACKGROUND: Mesangial cell (MC) proliferation underlies increased matrix accumulation in glomerulonephritis (GN), and the resolution of MC proliferation occurs largely through apoptosis. Proliferation and apoptosis are controlled by specific cell cycle proteins, where cyclin-dependent kinase (CDK) inhibitors such as p21 bind target cyclin-CDK complexes. However, the role of p21 in acute mesangial proliferative GN is not known. This study was conducted to test the hypothesis that p21 regulates MC proliferation and apoptosis in anti-MC serum-induced GN. METHODS: Age and sex matched wild-type (p21+/+) and p21-deficient (p21-/-) mice were injected with sheep anti-MC serum. Renal function (BUN, urinary albumin excretion), histology, DNA synthesis (BrdU. Ki-67) and apoptosis (TUNEL) were quantified at day 6 and day 12 (n = 6-8/time point). RESULTS: In p21+/+ mice, anti-MC-serum induced mild MC proliferative GN, and glomerular p21 expression was increased. Renal function was worse in nephric p21-/- mice. PAS and silver staining revealed that p21-/- mice had typical features of MC proliferative GN with focal segmental tuft necrosis, focal mesangiolysis and focal mesangial hypercellularity. Occasional features of podocyte injury (swelling, vacuolization) were noted. Double immunostaining confirmed increased mesangial cell DNA synthesis in nephritic p21-/- mice at day 6. In contrast, there was no difference in glomerular apoptosis in nephritic p21+/+ and p21-/- mice at each time point. Glomerular lesions were accompanied by severe glomerular and tubulointerstitial fibrosis in p21-/- mice. CONCLUSIONS: This data shows that the CDK-inhibitor p21 regulates the MC proliferative response to immune-mediated injury. In contrast, p21 does not alter the apoptotic response, resulting in a delayed resolution in nephritic p21-/- mice.

Animals↗

PubMed2005

Protection of endothelial cells by dextran sulfate in rats with thrombotic microangiopathy.

The characteristic features of thrombotic microangiopathy (TMA) include glomerular and peritubular capillary endothelial cell injury in association with loss of heparan sulfate proteoglycans on the cell surface and thrombus formation, followed by subsequent ischemic tubulointerstitial damage. It therefore was hypothesized that dextran sulfate (DXS) may protect the kidney against endothelial damage in a model of TMA. TMA was induced in rats by renal artery perfusion of an antiglomerular endothelial antibody, followed by the administration of DXS or vehicle. Renal damage was assessed by histologic analysis and measurements of blood urea nitrogen and creatinine. Whereas control rats developed severe renal failure with extensive glomerular and tubular injury, administration of DXS significantly protected renal function and preserved the glomerular endothelium and peritubular capillaries. The beneficial effect of DXS could be attributed to the ability of DXS to protect endothelial cells from coagulation and complement activation, as demonstrated by the histologic analysis. In addition, binding of the administered DXS to the surface of the glomerular endothelium was confirmed in TMA rats, suggesting that DXS acts as a "repair coat" of injured glomerular endothelium. In conclusion, DXS protects the kidney from experimental TMA. This protection may be mediated by DXS's binding directly to the surface of glomerular endothelium and amelioration of coagulation, complement activation, and cellular matrix loss.

Animals↗

PubMed2005

Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins.

Nephrotic-range proteinuria is due to glomerular diseases characterized by podocyte injury. Glucocorticoids are the standard of care for most forms of nephrotic syndrome. However, the precise mechanisms underlying the beneficial effects of glucocorticoids on podocytes, beyond its general immunosuppressive and anti-inflammatory effects, are still unknown. This study tested the hypothesis that the synthetic glucocorticoid dexamethasone directly reduces podocyte apoptosis. Growth-restricted immortalized mouse podocytes in culture were exposed to puromycin aminonucleoside (PA) to induce apoptosis. Our results showed that dexamethasone significantly reduced PA-induced apoptosis by 2.81-fold. Dexamethasone also rescued podocyte viability when exposed to PA. PA-induced apoptosis was associated with increased p53 expression, which was completely blocked by dexamethasone. Furthermore, the inhibition of p53 by the p53 inhibitor pifithrin-alpha protected against PA-induced apoptosis. Dexamethasone also lowered the increase in the proapoptotic Bax, which was increased by PA, and increased expression of the antiapoptotic Bcl-xL protein. Moreover, the decrease in p53 by dexamethasone was associated with increased Bcl-xL levels. Podocyte apoptosis induced by PA was caspase-3 independent but was associated with the translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. AIF translocation was inhibited by dexamethasone. These results show that PA-induced podocyte apoptosis is p53 dependent and associated with changes in Bcl-2-related proteins and AIF translocation. The protective effects of dexamethasone on PA-induced apoptosis were associated with decreasing p53, increasing Bcl-xL, and inhibition of AIF translocation. These novel findings provide new insights into the beneficial effects of corticosteroids on podocytes directly, independent of its immunosuppressive effects.

Active Transport, Cell Nucleus↗

PubMed2005

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 Wilm's 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.

Aging↗

PubMed2005

Cellular response to injury in membranous nephropathy.

The pathogenesis of membranous nephropathy (MN) involves in situ formation of subepithelial immune deposits that produce glomerular injury by damaging and/or activating podocytes through complement-dependent processes. C5b-9 formation and insertion into podocyte cell membranes causes glomerular injury in MN. C5b-9 in sublytic quantities stimulates podocytes to produce proteases, oxidants, prostanoids, extracellular matrix components, and cytokines including TGF-beta. C5b-9 also causes alterations of the cytoskeleton that lead to abnormal distribution of slit diaphragm protein and detachment of viable podocytes that are shed into Bowman's space. These events result in disruption of the functional integrity of the glomerular basement membrane and the protein filtration barrier of podocytes with subsequent development of massive proteinuria. Complement components in proteinuric urine also induce tubular epithelial cell injury and mediate progressive interstitial disease in MN. Measurements of urinary C5b-9 or podocyte excretion in the urine may be useful in the diagnosis of MN and as measures of disease activity and response to therapy. Recent studies of cell-cycle proteins and DNA damage in podocytes have clarified why podocytes fail to proliferate in response to C5b-9-mediated injury and podocyte loss in MN, resulting in the development of glomerular sclerosis and renal failure. Improved understanding of the role of complement in the pathogenesis of MN and of the cellular response to C5b-9 attack creates several new opportunities for therapeutic intervention that may benefit patients with MN in the future.

Complement Membrane Attack Complex↗

PubMed2005

Mechanical stretch induces podocyte hypertrophy in vitro.

BACKGROUND: Increased intraglomerular pressure is a final pathway toward glomerulosclerosis in systemic hypertension, diabetes, and focal segmental glomerulosclerosis (FSGS). Increased intraglomerular pressure causes stress-tension, or stretch, on resident glomerular cells. However, the effects of stretch on podocyte growth, and the mechanisms that underlie this, have not been elucidated. METHODS: To test the hypothesis that stretch alters podocyte growth, cultured mouse podocytes were exposed to cyclic mechanical stretch created by vacuum; control cells were grown under similar conditions, but not exposed to stretch. Proliferation (cell cycle phases) and hypertrophy (forward light scatter) were measured in stretched and control podocytes by flow cytometry. The role of the cyclin-dependent kinase (CDK) inhibitors, p21 and p27, was examined by stretching podocytes isolated from p21 and p27 knockout (-/-) mice, and the role of specific signaling pathways was assessed by Western blot analysis and blocking studies. RESULTS: Our results showed that stretch reduced cell cycle progression in wild-type and single p27-/- podocytes and induced hypertrophy in these cells in all phases of the cell cycle at 24, 48, and 72 hours. In contrast, stretch did not induce hypertrophy in single p21-/- and double p21/p27-/- podocytes. Stretch-induced hypertrophy required cell cycle entry, and was prevented by specifically blocking extracellular signal-regulated kinase 1/2 (Erk1/2) or Akt. Although stretch increased p38 activation, inhibition of this pathway had no effect on hypertrophy. CONCLUSION: Mechanical stretch induces hypertrophy in podocytes in vitro in all phases of the cell cycle. This effect is cell cycle dependent, and requires p21, Erk1/2, and Akt. Stretch may play a role in podocyte injury when intraglomerular pressure is increased.

Animals↗

PubMed2005

Limitation of podocyte proliferation improves renal function in experimental crescentic glomerulonephritis.

BACKGROUND: Many forms of glomerular diseases are characterized by injury to the glomerular visceral epithelial cell, or podocyte, which usually results in depletion of podocyte number. However, in diseases where podocyte proliferation occurs there is a rapid decline in renal function. The consequences of inhibiting podocyte proliferation on renal function have not been fully established. At the level of the cell cycle, cyclin-dependent kinase 2 (CDK2) is required for proliferation. METHODS: To determine if decreasing podocyte proliferation improves renal function, CDK2 activity was reduced with the purine analogue roscovitine in mice with antibody-induced experimental glomerulonephritis. Nephritic animals given vehicle, dimethyl sulfoxide (DMSO), served as control. Blood urea nitrogen (BUN), proteinuria, and renal histology were assessed at days 5 and 14 of disease. RESULTS: Inhibiting CDK2 activity resulted in a marked decrease in glomerular DNA synthesis [5-bromo-2'-deoxyridine (BrdU) staining] in Roscovitine-treated animals at day 5 of nephritis (P < 0.05 versus control). This was associated with a significant decrease in BUN and glomerulosclerosis at day 14 (P < 0.01 versus control) and a decrease in the accumulation of the extracellular matrix protein laminin (P < 0.01 versus control). CONCLUSION: Inhibiting podocyte proliferation in experimental glomerulonephritis is associated with improvement in renal function and histology, suggesting that inhibiting CDK2 activity is a potential therapeutic target for glomerular diseases characterized by podocyte proliferation.

Animals↗

PubMed2005

ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo.

BACKGROUND: Podocytes are terminally differentiated and highly specialized epithelial cells. The factors governing podocyte differentiation are poorly understood. We tested the hypothesis that all-trans retinoic acid (ATRA), a vitamin A derivative, induces podocyte differentiation in vitro and in vivo. METHODS: We tested the effects of ATRA on podocytes. Primary rat, primary mouse, and immortalized mouse podocytes were exposed to ATRA (1, 5, 10, 20, 40, 50, 80, 160, and 200 micromol/L) or control (ethanol) for 72 hours. Cell morphology was examined by electron microscopy, the expression of podocyte specific proteins was measured by immunoflourescence and Western blot analysis, cell number and apoptosis were measured by 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst staining, respectively. To determine if ATRA alters podocyte differentiation in vivo, experimental injury was induced in C57BL6 mice using the antiglomerular antibody. Animals were given either daily intraperitoneal ATRA (16 mg/kg) or vehicle (corn oil). For end points, we measured proteinuria, podocyte-specific protein immunostaining, and proliferation [proliferating cell nuclear antigen (PCNA)] at days 5 and 14 (N= 5/group/time point). RESULTS: ATRA induced podocyte process formation in vitro, and significantly increased the expression of nephrin and podocin. This coincided with a reduction in proliferation. ATRA also significantly prevented the decrease in staining for synaptopodin, nephrin, and podocin in experimental animals (P < 0.05 vs. control). This was accompanied by reduced proteinuria and decreased podocyte proliferation (P < 0.05 vs. control). CONCLUSION: ATRA induces podocyte differentiation in vitro and in vivo and alters the expression of certain podocyte-specific proteins. Further studies are ongoing to delineate the mechanism of this effect.

Animals↗

PubMed2005

p27(Kip1) Knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency.

BACKGROUND: High glucose up-regulates the mesangial cell expression of p27(Kip1), an inhibitor of cyclin-dependent kinases/cyclin complexes. Previous in vitro studies using cultured mesangial cells from p27(Kip1-/-) mice demonstrated that these cells do not undergo high glucose-mediated cellular hypertrophy. Since glomerular hypertrophy is an early feature of diabetic nephropathy and may precede the development of glomerulosclerosis, interference with p27(Kip1) expression may attenuate diabetic nephropathy. However, it is unclear whether deletion of p27(Kip1) protects the kidneys of diabetic nephropathy in vivo. METHODS: Type 1 diabetes mellitus was induced in p27(Kip1+/+), p27(Kip1+/-), and p27(Kip1-/-) mice by injection of streptozotocin (STZ). Mice were studied for 6 weeks. Animals injected with citrate buffer only served as controls. At the end of the experiments, urine was collected, albuminuria was determined with an enzyme-linked immunosorbent assay (ELISA), and blood glucose concentrations were measured. Kidneys were perfusion-fixed for quantitative morphologic analysis with glutaraldehyde and for immunohistochemical studies with formaldehyde. Glomerular cell number and volume were analyzed. Glomerulosclerosis, tubulointerstitial, and vascular damage indices were semiquantitatively assessed according to standard methodology. Quantitative glomerular parameters (cell numbers and volumes of endothelial, mesangial, and epithelial cells) were measured on semithin sections. Expression of transforming growth factor-beta1 (TGF-beta1), laminin, and collagen type IV were determined by immunohistochemical staining. RESULTS: In contrast to animals only injected with citrate buffer, mice that received STZ developed hyperglycemia. There was no significant difference in the degree of hyperglycemia among p27(Kip1+/+), p27(Kip1+/-), and p27(Kip1-/-) mice. Diabetic p27(Kip1+/+), but not control p27(Kip1+/+) animals, developed albuminuria. Albuminuria was significantly reduced in diabetic p27(Kip1+/-) and more profoundly in p27(Kip1-/-) animals. Diabetic p27(Kip1+/+) mice revealed a significant increase in mean glomerular volume at 6 weeks. The volumes of mesangial and endothelial cells and podocytes all increased, whereas cell numbers were reduced, consistent with cell hypertrophy. Glomerular, endothelial, mesangial and podocyte hypertrophy were reduced in diabetic p27(Kip1+/-) and p27(Kip1-/-) animals. Diabetic p27(Kip1) (+/+) animals had significantly increased glomerulosclerosis, tubulointerstium, and vascular damage indices compared to nondiabetic p27(Kip1+/+) controls. Diabetic p27(Kip1-/-) mice exhibited significantly less structural damage than diabetic wild-type animals. Diabetic p27(Kip1+/-) animals revealed intermediate glomerulosclerosis, tubulointerstium, and vascular damage values. Immunohistological stainings demonstrated increases in TGF-beta1, collagen type IV, and laminin expression in kidneys of diabetic p27(Kip1+/+) animals compared to nondiabetic p27(Kip1+/+) controls. Staining intensity for type IV collagen and laminin, but not for TGF-beta1, was significantly lower in diabetic p27(Kip1-/-) mice. CONCLUSION: Deletion of p27(Kip1) attenuates the functional and morphologic features of diabetic nephropathy. Although deletion of p27(Kip1) abolished some parameters of diabetic glomerular hypertrophy, the significant reduction of TGF-beta1 expression in the tubulointerstitium indicates that other protective mechanisms could be operative. The p27(Kip1) gene is haplo-insufficient because diabetic p27(Kip1)+/- mice exhibited an intermediate degree of functional and structural renal injury. Our data shows that p27(Kip1) plays an important role in diabetic nephropathy.

Albuminuria↗

PubMed2005

The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis.

BACKGROUND: Reduced podocyte number is a critical determinant in the development of glomerulosclerosis. Transforming growth factor-beta1 (TGF-beta1) induces podocyte apoptosis, but the cell cycle events are not known. The cyclin-dependent kinase (CDK) inhibitor p21 increases in podocytes in diseases where TGF-beta increases. Accordingly, we studied the role of p21 in podocyte apoptosis. METHODS: Immortalized and primary p21+/+ and p21-/- mouse podocytes were used. Apoptosis was measured by Hoechst 33342 staining and caspase-3 activity following the exposure to TGF-beta1 or puromycin aminonucleoside. p21 and specific Bcl-2-related family proteins levels were measured by Western blot analysis. To prove a role for p21, we reconstituted p21 expression in p21-/- podocytes utilizing an adenovirus vector. RESULTS: TGF-beta1 increased the protein levels of p21 in p21+/+ podocytes, and this coincided with apoptosis. In contrast, TGF-beta1 did not induce apoptosis in p21-/- podocytes. Restoring p21 expression increased apoptosis in p21-/- podocytes following exposure to TGF-beta1. TGF-beta1 increased the protein levels of an anti-apoptotic Bcl-2 in p21-/- podocytes, but not in p21+/+ podocytes. Moreover, TGF-beta1 did not increase Bcl-2 expression in p21-/- podocytes in which p21 expression was restored. Finally, puromycin aminonucleoside also induced apoptosis in p21+/+ podocytes, but not in p21-/- podocytes. CONCLUSION: Podocyte apoptosis induced by TGF-beta1 and puromycin aminonucleoside requires p21, and Bcl-2 plays a crucial role downstream of p21 in mediating this effect. These results suggest that p21 may play a critical role in the decrease in podocyte number in disease status accompanied by increased TGF-beta1.

Animals↗

PubMed2005

Mechanical strain increases SPARC levels in podocytes: implications for glomerulosclerosis.

Glomerular capillary hypertension is a final common pathway to glomerulosclerosis. Because podocyte loss is an early event in the development of glomerulosclerosis, it is logical that the deleterious effects of glomerular capillary hypertension involve podocyte injury. Yet, the mechanisms by which elevated intraglomerular pressure is translated into a maladaptive podocyte response remain poorly understood. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein activated in various disease states of the podocyte and accelerates renal injury, as evidenced by the milder course of experimental diabetic nephropathy in SPARC-null mice compared with diabetic SPARC wild-type mice. Accordingly, we tested the hypothesis that mechanical strain activates SPARC in podocytes and thus is a putative mediator of podocyte injury in states of intraglomerular capillary hypertension. Conditionally immortalized mouse podocytes were subjected to 10% cyclical stretch while nonstretched cells served as controls. SPARC levels were measured in whole cell lysate and cell media. Immunostaining was performed for SPARC in an experimental model of glomerular capillary hypertension. Our results demonstrate cyclical stretch of podocytes markedly increased SPARC levels in cell lysate, through activation of p38, as well as secreted SPARC. Relevance was shown by demonstrating increased podocyte staining for SPARC in the uninephrectomized spontaneously hypertensive rat. In conclusion, we have made the novel observation that mechanical forces characteristic of states of glomerular capillary hypertension lead to increased levels of SPARC in podocytes. We speculate that the increase in SPARC may be maladaptive and lead to a progressive reduction in podocyte number, thus fueling the future development of glomerulosclerosis.

Animals↗