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PubMed1995

Development of renal podocytes cultured under medium perifusion.

BACKGROUND: In the past, podocytes have been described as highly susceptible to dedifferentiation under cell culture. Whether this process resulted from insufficient culture conditions or whether it was a consequence of missing cellular interactions remained unclear. A further reason could be that podocytes within the maturing kidney are irreversibly growth-arrested at a very early point of development because proliferating cells have been detected at the S-shaped body stage but not at the capillary loop stage or in the maturing glomeruli. These were important reasons that hindered the establishment of podocyte cell culture systems. EXPERIMENTAL DESIGN: The aim of our present study was to culture podocytes under the most organotypic conditions possible to maintain typical cellular characteristics. Cortex explants of neonatal rabbit kidneys consisting of nephrogenic tissue were used as a source for podocytes. No serum additives were given for the whole culture period of 13 days. An organ-specific environment was obtained by keeping the podocytes within the surrounding renal tissue and by ensuring a permanent exchange of medium. RESULTS: mAb were used to characterize podocytes and the other glomerular cell types. Cultured podocytes and parietal cells of Bowman's capsule were identified by EnPo 1. Ks 19.2.105, a marker for cytokeratin 19, was used to discriminate among these epithelial cells because cytokeratin 19 is expressed by the parietal cells of Bowman's capsule but not by podocytes. The Ab EC1 specifically detected endothelial cells. Glomerular endothelium cultured under medium perifusion expressed these typical Ag and thus could be unequivocally discriminated. Furthermore, by means of the proliferation marker Ki-67, it could be demonstrated that glomerulus-like structures developed under culture by proliferation of visceral and parietal cells of Bowman's capsule. CONCLUSIONS: A culture model is presented that allows the maintenance of developing podocytes within the organ-specific tissue environment and under permanent medium perifusion.

Animals↗
PubMed2006

The relationship among nephrin, podocin, CD2AP, and alpha-actinin might not be a true 'interaction' in podocyte.

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or alpha-actinin-4 mRNA by using reconstructed RNA interference vector--psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of alpha-actinin-4, whereas alpha-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

Actinin↗
PubMed2006

FSGS-associated alpha-actinin-4 (K256E) impairs cytoskeletal dynamics in podocytes.

Mutations in the ACTN4 gene, encoding the actin crosslinking protein alpha-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E alpha-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E alpha-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E alpha-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on alpha-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type alpha-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E alpha-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E alpha-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E alpha-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E alpha-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E alpha-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.

Actinin↗
PubMed2006

Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo.

A decline in podocyte number correlates with progression to glomerulosclerosis. A mechanism underlying reduced podocyte number is the podocyte's relative inability to proliferate in response to injury. Injury by the podocyte toxin puromycin aminonucleoside (PA) is mediated via reactive oxygen species (ROS). The precise role of ROS in the pathogenesis of PA-induced glomerulosclerosis remains to be determined. We sought to examine whether PA-induced ROS caused podocyte DNA damage, possibly accounting for the podocyte's inability to proliferate in response to PA. In vitro, podocytes were exposed to PA, with or without the radical scavenger 1,3-dimethyl-2-thiourea (DMTU). In vivo, male Sprague-Dawley rats were divided into experimental groups (n = 6/group/time point): PA, PA with DMTU, and control, killed at days 1.5, 3, or 7. DNA damage was measured by DNA precipitation, apurinic/apyrimidinic site, Comet, and 8-hydroxydeoxyguanosine assays. Cell cycle checkpoint protein upregulation (by immunostaining and Western blotting), histopathology, and biochemical parameters were examined. DNA damage was increased in cultured podocytes that received PA, but not PA with DMTU. PA exposure activated specific cell cycle checkpoint proteins, with attenuation by DMTU. DNA repair enzymes were activated, providing evidence for attempted DNA repair. The PA-treated animals developed worse proteinuria and histopathologic disease and exhibited more DNA damage than the DMTU pretreated group. No significant apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. A mechanism underlying the lack of podocyte proliferation following PA-induced injury in vitro and in vivo may be ROS-mediated DNA damage, with upregulation of specific cell cycle checkpoints leading to cell cycle arrest.

Animals↗
PubMed2006

Antioxidants ameliorate the expression of vascular endothelial growth factor mediated by protein kinase C in diabetic podocytes.

BACKGROUND: The increased production of reactive oxygen species (ROS) may be involved in the onset or development of diabetic vascular complications. The release of ROS from podocytes plays a role in the pathogenesis of glomerular damage in various experimental glomerular diseases. Although it is assumed that the podocyte injury also plays an important role in diabetic nephropathy, the mechanism is still unknown. METHODS: Using a differentiated mouse podocyte cell line, we investigated: (1) whether a high level of ambient glucose increases the level of ROS, (2) whether the protein kinase C (PKC) pathway is involved in a high-glucose-induced generation of ROS and vascular endothelial growth factor (VEGF) and (3) whether antioxidants ameliorate PKC-mediated VEGF expression in diabetic milieu. RESULTS: Intracellular ROS generation was significantly higher in high glucose than in control conditions in cultured podocytes. High ambient glucose also increased VEGF mRNA and protein expression. The high-glucose-induced increases in ROS and VEGF mRNA and protein by podocytes were effectively inhibited by pretreatment with various antioxidants and were completely restored by PKC inhibition. The results show that cultured mouse podocytes produce ROS in response to high glucose, and that PKC is involved in high-glucose-induced ROS and VEGF production by podocyte. CONCLUSION: Increased ROS in podocytes may play a role in the pathogenesis of podocyte injury in diabetic nephropathy.

Animals↗
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

Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.

BACKGROUND: Human immunodeficiency virus (HIV)-1-associated nephropathy (HIVAN) is characterized by collapsing focal and segmental glomerulosclerosis (FSGS) and microcystic tubular dilatation. HIV-1 infection is also associated with other forms of nephropathy, including mesangial hyperplasia. Since HIV-1 gene products are detected in podocytes and other renal cells, it remains uncertain whether podocyte-restricted HIV-1 gene expression can account for the full spectrum of renal lesions involving nonpodocytes. METHODS: To define the role of podocyte-restricted HIV-1 gene expression in the progression of HIVAN, we generated transgenic mice that express nonstructural HIV-1 genes selectively in podocytes. RESULTS: Four of the seven founder mice developed proteinuria and nephropathy. In a subsequently established transgenic line, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected mRNAs for vif, vpr, nef, and spliced forms of tat and rev, but not vpu, in the kidney. In situ hybridization localized HIV-1 RNA to the podocyte. Transgenic mice on FVB/N genetic background exhibited cuboidal morphology of podocytes with reduced extension of primary and foot processes at 2 weeks of age. After 3 weeks of age, these mice developed massive and nonselective proteinuria with damage of podocytes and other glomerular cells and, after 4 weeks of age, collapsing FSGS and microcystic tubular dilatation. In marked contrast, transgenic mice with C57BL/6 genetic background showed either normal renal histology or only mild mesangial expansion without overt podocyte damage. CONCLUSION: The present study demonstrates that podocyte-restricted expression of HIV-1 gene products is sufficient for the development of collapsing glomerulosclerosis in the setting of susceptible genetic background.

AIDS-Associated Nephropathy↗
PubMed2005

TGF-beta concentration specifies differential signaling profiles of growth arrest/differentiation and apoptosis in podocytes.

Podocyte depletion occurs in most progressive glomerular diseases and is thought to result from podocyte loss while the remaining podocytes are unable to proliferate. The underlying mechanisms for podocyte growth arrest/differentiation and depletion remain poorly understood but may involve TGF-beta, which is typically upregulated in injured glomeruli. The TGF-beta are multifunctional cytokines that regulate growth, differentiation, and apoptosis in most cells. Determinants of functional specificity of TGF-beta signaling in cell-cycle control and apoptosis remain poorly understood. Using a unique system of conditionally immortalized podocytes, it is demonstrated that autocrine TGF-beta2 induces G0/G1 arrest and differentiation under nonpermissive culture through Smad3-dependent induction of the cyclin-dependent kinase inhibitor p15(Ink4b) (Cdkn2b). When exposed to recombinant TGF-beta1 (or TGF-beta2), nonpermissive culture podocytes switch to G2/M arrest and apoptosis, selectively at advanced TGF-beta concentrations and specifically in association with suppression of Cdkn2b and activation of proapoptotic p38 mitogen-activated protein kinase. Thus, distinct signaling profiles activated in a concentration-dependent manner by TGF-beta were identified. Autocrine TGF-beta2/Smad3/Cdkn2b signaling in podocytes specifies G0/G1 arrest associated with podocyte differentiation, whereas increasing TGF-beta concentrations beyond a critical threshold induces G2/M block and apoptosis associated with selective p38 mitogen-activated protein kinase activation and with suppression of Cdkn2b. In summary, the results suggest a new functional requirement of TGF-beta2 in growth arrest and differentiation of murine podocytes in vitro and demonstrate that a critical TGF-beta concentration threshold may specify a molecular switch to proapoptotic signaling profiles and apoptosis.

Animals↗
PubMed2005

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome.

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.

Actins↗
PubMed2006

Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling.

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.

Acute-Phase Proteins↗
PubMed2002

Regulation of adhesive interaction between podocytes and glomerular basement membrane.

Glomerular filtration depends on well-orchestrated cell-cell and cell-matrix contacts of glomerular podocytes. Over the last years critical constituents of these contacts have been identified via molecular approaches. Podocyte cell-matrix interactions have been shown to be mediated in part by alpha(3)beta(1)-integrin heterodimers. Disturbances of integrin matrix interaction lead to detachment of podocytes in vitro, corresponding to the critical event of foot process retraction and glomerular basement membrane (GBM) denudation in vivo. Further, dystroglycan-mediated matrix attachment appears to play a critical role for podocyte foot process architecture. Downstream signaling events are currently elucidated concentrating mainly on integrin-dependent cascades and their consequences for podocyte adhesion and proliferation. An activation of the integrin-linked kinase in podocyte damage in vivo and in vitro makes this molecule a particularly interesting candidate for integrin-mediated inside-out and outside-in signaling in podocytes. Podocyte cell-cell interaction has been characterized in a few studies in vitro, indicating the slit diaphragm to be a modified adherens junction. The structural link between the cell-matrix and cell-cell contacts is maintained by the actin cytoskeleton, which may also enable cross-talk between these two cell contact sites. Examining podocyte function in tissue culture, animal models and human expression studies should allow further detailed dissection of the molecular pathways responsible for maintenance and failure of the glomerular filtration barrier.

Animals↗
PubMed2004

Alteration of podocytes in a murine model of crescentic glomerulonephritis.

Recent observations suggest a central role of podocytes in crescent formation. In experimental glomerulonephritis podocytes disrupt the parietal epithelial layer and attach on its basement membrane, thus forming bridges between the tuft and Bowman's capsule, and they are a major constituent of crescents. In order to explain these findings we hypothesize that inflammation triggers motility in podocytes. In the present study we asked whether podocytes display alterations which suggest a migratory behavior in glomerulonephritis. Glomerulonephritis was induced in mice by injection of a rabbit serum against the glomerular basement membrane. The kidneys were perfusion-fixed 6 days later and examined by light and electron microscopy as well as by immunohistochemistry. In glomerulonephritis the apical cytoplasm of podocytes displayed numerous actin-containing microprotrusions. Cortactin, a protein involved in the regulation of actin polymerization, was predominantly expressed in foot processes of podocytes in control mice. It was redistributed to the cell body in glomerulonephritis. In untreated mice betal-integrin was restricted to the foot processes. In glomerulonephritis it was additionally found in the cytoplasm and in the apical cell membrane. Recycling of integrins is a crucial event in initiation of cell migration. ICAM-1 and CD44, the ligation of which induces migratory behaviors, were absent from healthy podocytes but expressed by some podocytes in glomerulonephritis. Thus, in glomerulonephritis podocytes display some characteristic features of migrating cells. This might explain their ability to break through the parietal epithelium and to become a constituent of early crescents.

Actins↗
PubMed1997

Podocytes in metanephric organ culture express characteristic in vivo phenotypes.

Podocytes outgrown from isolated glomeruli in vitro have failed to express fully differentiated in vivo phenotypes. In an attempt to determine whether podocytes in metanephric culture accomplish terminal differentiation, as observed in vivo, we investigated expression of their characteristic phenotypic features in rat metanephric organ cultures using immunohistochemistry and electron microscopy. Rat metanephroi were harvested on embryonic day 12.5 and cultured on transmembrane filters for 9 days. Morphological examination revealed two maturation stages when the podocytes resembled those of the S-shaped body stage and maturational stages of glomeruli in vivo. Electron microscopy revealed that, firstly podocytes lost their intercellular contacts and, simultaneously, the tight junctions shifted into close proximity to cell bases, followed by foot process development. Immunohistochemistry demonstrated that the tight junction protein, ZO-1, and specific podocytic markers, pp44, 5-1-6, podocalyxin and vimentin were expressed in a cell maturity-dependent manner, as observed in newborn rat kidneys. Furthermore, glomerular basement membrane components, collagen type IV and laminin, were expressed in the glomerular center. Our findings that cell maturity-dependent expression of structural and functional phenotypes in podocytes in metanephric culture was the same as that observed in developing kidneys in vivo indicate that podocyte differentiation during glomerulogenesis may be operated by an intrinsic property, such as programmed cell fate. Furthermore, these highly differentiated podocytes in vitro may provide clues that will help to establish a podocyte culture system.

Animals↗
PubMed2005

Potential role of angiotensin-converting enzyme inhibitors and statins on early podocyte damage in a model of type 2 diabetes mellitus, obesity, and mild hypertension.

BACKGROUND: Experimental findings suggest that the obese Zucker rat (OZR) is a model of type 2 diabetes-related nephropathy with several metabolic abnormalities. However, the exact mechanisms by which these factors cause early glomerulosclerosis and proteinuria remain unclear. Furthermore, structural abnormalities and regulation of podocytes have recently emerged as prominent underlying factors in proteinuria. The aim of this study was to evaluate the potential role of angiotensin-converting enzyme inhibitors and statins on early podocyte damage in an experimental model of type 2 diabetes mellitus. METHODS: We used OZR to evaluate some of the pathogenic mechanisms and the effects of two drugs, an angiotensin-converting enzyme (ACE) inhibitor (quinapril) and a statin (atorvastatin), involved in the development of proteinuria and especially podocyte damage. We studied glomerular and tubulointerstitial injury by assessing inflammation mediators (murine monoclonal antibody against CD68 [ED1+], interleukin-8 [IL-8], interferon-gamma-inducible protein 10 [IP-10]) and podocyte damage markers using desmin staining and electron microscopy. RESULTS: Glomerular lesions were correlated with cholesterol (r = 0.676), proteinuria (r = 0.804), triglycerides (r = 0.593), insulin (r = 0.345), creatinine (r = 0.266), and glucose (r = 0.245). In addition, podocytes from OZR showed positive staining for desmin. Use of the ACE inhibitor quinapril normalized proteinuria, cholesterol levels, glomerular lesions, and podocyte morphology. In contrast, atorvastatin ameliorated but did not normalize renal damage, with a partial reduction in desmin staining and podocyte morphology. Treatment with both drugs resulted in only a slight reduction in IL-8 and IP-10 in the tubulointerstitium. CONCLUSIONS: In the OZR, cholesterol was an important determinant of renal injury. Most notably, glomerulosclerosis in the OZR Is characterized by early podocyte damage and tubulointerstitial injury. In addition, our findings showed that quinapril primarily normalized podocyte morphology, whereas atorvastatin ameliorated renal lesions through the diminution of lipids and by its lipid-independent pleiotropic effect.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2002

Transforming growth factor-beta1 is up-regulated by podocytes in response to excess intraglomerular passage of proteins: a central pathway in progressive glomerulosclerosis.

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. In rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGF-beta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

Cell cycle regulation and differentiation in the human podocyte lineage.

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (approximately 20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells), cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.

Biomarkers↗
PubMed2003

Characterization of angiotensin II-receptor subtypes in podocytes.

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.

Animals↗
PubMed1998

Angiotensin II modulates cellular functions of podocytes.

Angiotensin II modulates cellular functions of podocytes. The aim of this study was to examine the effects of angiotensin II (Ang II) on membrane voltage (Vm) and cytosolic calcium activity ([Ca2+]i) of rat podocytes. To approach better the in vivo situation, we have developed an experimental approach that allows podocytes to be studied in the intact microdissected glomerulus. Ang II depolarized podocytes in the glomerulus (EC50 15 nM, N = 49). Like podocytes in the glomerulus, podocytes in short-term culture also depolarized in response to Ang II (10 nM, N = 5). Ang II increased [Ca2+]i in podocytes in culture (EC50 3 nM, N = 229). In a solution with reduced extracellular [Ca2+] (10 microM), Ang II-mediated [Ca2+]i increase was significantly reduced by 60% +/- 20% (N = 12). Flufenamate, an inhibitor of nonselective ion channels, inhibited Ang II-mediated increase of [Ca2+]i (IC50 20 microM, N = 29). The Ang subtype 1 (AT1) receptor antagonist losartan inhibited both Ang II-mediated depolarization and [Ca2+]i increase in podocytes (N = 5 to 35). Our results support the concept that Ang II might influence podocyte function directly via an AT1 receptor.

Angiotensin II↗