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PubMedDate not supplied

The podocyte: from bench to bedside.

The understanding of the molecular structure of the podocyte has increased considerabely in recent 5 years by genetic research in inherited glomerulopathies. Injury to the podocyte often leads to a reorganisation of the slit diaphragm and to foot process effacement, which can be noted in all forms of the nephrotic syndrome. Since the recent progress in our understanding of the molecular biology of the podocyte, it has become obvious that the podocyte is crucial in the pathogenesis of not only genetic but also of many acquired glomerulopathies such as focal segmental glomerulosclerosis, membranous glomerulopathy and diabetic nephropathy. In this review we provide a summary of the recent physiological and molecular insights into the podocyte and of its significance in different glomerulopathies.

Angiotensin II↗
PubMed2002

The number of podocyte slit diaphragms is decreased in minimal change nephrotic syndrome.

The pathophysiology of proteinuria in acquired kidney diseases is mostly unknown. Recent findings in genetic renal diseases suggest that glomerular epithelial cells (podocytes) and the slit diaphragm connecting the podocyte foot processes play an important role in the development of proteinuria. In this work we systematically evaluated the podocyte slit pores by transmission electron microscopy in two important nephrotic diseases, minimal change nephrotic syndrome (MCNS) and membranous nephropathy (MN). As controls, we used kidneys with tubulointerstitial nephritis (TIN). Effacement of podocyte foot processes was evident in proteinuric kidneys. However, quite normal looking foot processes and slit pores with varying width were also observed. Careful analysis of slit pores revealed, that the proportion of the pores spanned by the linear image of slit diaphragm, was reduced by 39% in kidneys from MCNS patients (1265 pores analyzed) compared with TIN samples (902 pores analyzed, p = 0.0003). To enhance the detection rate of the slit diaphragms, the "empty" podocyte pores were further analyzed with tilting series from -45 to +45. This revealed the linear diaphragm image in 71% and 26% of the slits in TIN and MCNS kidneys, respectively (p = 0.0003). In contrast to findings in MCNS, no significant reduction of the slit diaphragms were seen in MN kidneys compared with the controls. The results suggest that MCNS is associated with disruption of glomerular slit diaphragms.

Adolescent↗
PubMed2001

Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis.

Although experimental crescentic glomerulonephritis starts with an endocapillary inflammation, the crescents themselves seem to originate from the proliferation of parietal epithelial cells (PEC). In this study, an attempt was made to disclose a link between the two processes by a morphologic analysis of early stages of the disease. Mice were immunized with rabbit IgG in complete Freund's adjuvant on day -6. At day 0, they received an intravenous injection of a rabbit antiglomerular basement membrane serum. On days 3, 6, and 10, the kidneys were fixed by vascular perfusion for examination by light and electron microscopy. On day 3, morphologic alterations affected mainly the endocapillary compartment; most podocytes appeared to be intact. On day 6, alterations of podocytes were widespread, including foot process effacement and prominent microvillous transformation, and some crescents were found. On day 10, crescents were found in 40% of glomeruli. The most surprising finding was podocytes that adhered to both the glomerular basement membrane and the parietal basement membrane, thus forming bridges between the tuft and Bowman's capsule. Those podocyte bridges were sparse on day 3 but were regularly encountered on days 6 and 10 in glomeruli without crescents and also as a component of crescents. They were interposed between PEC and later between the cells of a crescent without formation of junctional connection with these cells. It is proposed that the spreading of podocytes on the parietal basement membrane represents a lesion of the parietal epithelium and that this process initiates the proliferation of PEC to form a crescent.

Animals↗
PubMed2002

Human podocytes express angiopoietin 1, a potential regulator of glomerular vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is abundantly expressed by podocytes, but its role in glomeruli is unknown. Angiopoietins are endothelial cell growth factors that function in concert with VEGF but have not previously been observed in human glomeruli. Angiopoietin 1 (Ang1) acts via the endothelial receptor Tie2 to promote maturation and stabilization of blood vessels, resisting angiogenesis and opposing some actions of VEGF. Ang1, Ang2, Tie2, and VEGF expression in normal human renal cortex was examined with immunofluorescence and immunohistochemical analyses. High-power, multiple-color, immunofluorescence analyses and additional antibodies (specific for particular components of the glomerular filtration barrier) were used to determine the exact locations of Ang1 and Tie2 in the glomerular capillary wall. Immuno-electron-microscopic analysis of rat glomeruli was used to further localize endothelial Tie2 expression. RNA and protein extracted from human glomeruli, cultured human podocytes, and cultured human endothelial cells were analyzed for Ang1, Ang2, and Tie2 by using reverse transcription-PCR and Western blotting. Ang1 was detected in podocytes in normal glomeruli and, with VEGF, was concentrated in podocyte foot processes. Tie2 was demonstrated on glomerular capillary endothelial cells, particularly on the abluminal surface. Reverse transcription-PCR and Western blotting analyses confirmed the expression of Ang1 and Tie2 in glomeruli and of Ang1 in cultured podocytes. These findings suggest a role for Ang1 in the maintenance of the glomerular endothelium and make it a good candidate to be a regulator of the actions of VEGF on glomerular permeability, resisting angiogenesis while allowing the induction of high permeability to water and small solutes.

Angiopoietin-1↗
PubMed1997

Mouse glomerular epithelial cells in culture with features of podocytes in vivo express aminopeptidase A and angiotensinogen but not other components of the renin-angiotensin system.

The binding of antibodies to podocytic antigens such as the Heymann antigen or aminopeptidase A may lead to the induction of a membranous glomerulonephritis in several species. To study the possible future interactions of antibodies with antigens on these podocytes, epithelial cells from isolated mouse glomeruli were cultured. By indirect immunofluorescence, the cells were positive for cytokeratin, vimentin, desmin, and the ZO-1 protein, a component of the tight junction complex. When rat monoclonal antibodies were used, the cells were also positive for the hydrolases aminopeptidase A and dipeptidyl peptidase IV, and they stained with ASD-33, a monoclonal antibody that recognized an epitope only present on the cell membranes of mouse podocytes. They were negative for the von Willebrand factor and did not stain with a monoclonal antibody (ASD-13) that binds to endothelial cells of glomeruli and peritubular capillaries. By electron microscopy, the cells showed tight junctions but lacked Weibel Palade bodies (endothelium), desmosomes, and cilia (parietal epithelium). The mRNA expression of several components of the renin-angiotensin system was also examined, and some factors indirectly coupled to the renin-angiotensin system component angiotensin II in this podocytic culture by RT-PCR analysis. mRNA Expression for the angiotensin II degrading hydrolase aminopeptidase A and angiotensinogen was found, but this was not found for any other component of this system, such as renin, angiotensin-converting enzyme, or the angiotensin II receptors AT1a, AT1b, and AT2. Low mRNA expression for dipeptidyl peptidase IV was observed. In addition, expression of the growth factors transforming growth factor-beta and interleukin-7, and the extracellular matrix components fibronectin, laminin B2, perlecan, and collagen IV alpha 1, was observed. Given these characteristics, a glomerular epithelial cell culture with features of podocytes in vivo that will allow future studies on the interaction of anti-aminopeptidase A monoclonal antibodies and angiotensin II with aminopeptidase A was established. This is of interest in light of the observation that injection of mice with anti-aminopeptidase A antibodies causes an acute albuminuria.

Aminopeptidases↗
PubMed2004

[Angiotensin II-induced podocyte apoptosis: role of the MAPK subtypes].

OBJECTIVE: To evaluate the role of the MAPK subtypes (p38MAPK, ERK and JNK) in ANG II-induced apoptosis of cultured human podocytes. METHODS: The cultured podocytes were incubated in media containing either vehicle, SB202190(5 micromol/L, an inhibitor of p38MAPK), PD98059 (1 micromol/L, an inhibitor of ERK), SP600125 (5 micromol/L, an inhibitor of JNK), ANG II (10(-8)mol/L) with or without SB202190 PD98059 and SP600125 for 18 hours; the cells were assayed for apoptosis by morphologic staining with H-33342 and propidium iodide and DNA fragmentation assays; the cell proteins were probed for phosphorylated MAPKs to determine the activation of specific MAPK subtypes. RESULTS: ANG II promoted podocyte apoptosis in a time- and dose-dependent manner; ANG II stimulated p38MAPK, but inhibited JNK; SB202190 inhibited both ANG II-induced podocyte apoptosis and p38MAPK phosphorylation; Inhibition of ERK by PD98059 had no effect on ANG II-induced cell apoptosis. CONCLUSION: ANG II-induced apoptosis through stimulation of p38MAPK and inhibition of JNK in human podocytes.

Angiotensin II↗
PubMed2004

[Podocyte dysfunction and proteinuria].

Podocytes play a central role in the pathogenesis of several glomerular diseases. In recent years, this has been revealed by molecular analysis of a number of rare hereditary renal diseases. Podocytes contain three domains: the domain bound to the glomerular basement membrane (GBM), the domain of the slit diaphragms and the apical domain. The slit diaphragms are situated basolaterally between the pedicles and form together with the GBM a mechanism for the selective filtration of blood to primary urine. The apical cell membrane forms a negatively charged layer which prevents adhesion to the adjacent cell membranes, thus keeping the slit diaphragms and urinary space open. Many podocyte diseases are characterised by foot process effacement, which causes the loss of slit diaphragms, and could lead to podocyte loss. Specific abnormalities have been discovered in the three domains of the podocyte to which a number of glomerular diseases can be attributed.

Humans↗
PubMed1977

A scanning and transmission electron microscopic comparison of puromycin aminonucleoside-induced nephrosis to hyperalbuminemia-induced proteinuria with emphasis on kidney podocyte pedicel loss.

Ultrastructural changes in the visceral epithelium and proximal tubules of rats were studied by scanning and transmission electron microscopy during the onset and progression of puromycin aminonucleoside nephrosis (PAN)-induced proteinuria. These changes were compared with those that occur during a similar degree of proteinuria induced by intraperitoneal injections of albumin. With the onset of proteinuria and oliguria, PAN rats exhibit loss of podocyte pedicels and podocyte major processes, an increase in pinocytotic activity, and an accumulation of cytoplasmic vacuoles and granules of variable size, shape, and electron density. Loss of podocyte pedicels involves a gradual decrease in pedicel height beginning at the pedicel tip and progressing down the pedicel arm, formation of nublike protrusions and interpedicel microbridges (35 to 45 nm. in width and 40 to 60 nm. in length) along the pedicel's base, the merging of microbridges to form more extensive regions of interpedicel contact, and a gradual broadening and retraction of pedicels. In response to hyperalbuminemia-induced proteinuria, kidney podocytes exhibit reactions during PAN, however, the podocyte pedicels, slit pores, and major processes of rats with hyperalbuminemia-induced proteinuria remain discrete. The loss of pedicels and major processes during PAN, therefore, apparently results from the effects of puromycin aminonucleoside per se rather than from the proteinuria associated with this disease. The proximal tubules of rats with hyperalbuminemia-induced proteinurea exhibit the same characteristic changes as PAN rat proximal tubules (i.e., loss of brush border, dilated lumina, abnormally thin walls, and accumulation of periodic acid-Schiff positive electron-dense luminal casts and cytoplasmic protein absorption droplets). The significance of these ultrastructural findings during PAN and hyperalbuminemia-induced proteinurea are discussed in terms of the etiology of PAN.

Albumins↗
PubMed1996

Macrophagic cells outgrowth from normal rat glomerular culture: possible metaplastic change from podocytes.

One representative of a number of severe lesions that occur outside the glomerular capillaries and involve podocytes is crescentic glomerulonephritis. The question of whether the crescent-forming cells are derived from glomerular epithelial cells or monocytes/macrophages is highly controversial and has not yet been clarified. To investigate the pathophysiology of podocytes in crescentic glomerulonephritis, we attempted to establish methods for culturing cells confirmed to be derived from podocytes, focusing particularly on the relationship between podocytes and macrophages. Nonadherent cells of unknown origin that grew from normal rat isolated glomerular cultures increased in number, reaching a total of 3.5 x 10(5)/ml on Day 11. They showed several characteristics of macrophages, the expression of specific antigens and enzymes, morphology, and production of H2O2. They expressed Fx1A but lacked the expressions of Thy1.1 or factor VIII. A morphologic kinetic study on Days 3 to 11 of culture showed that the cells with foot processes on the glomerular basement membrane changed into macrophagic cells (MC) and migrated from the glomeruli. Immunofluorescence double staining indicated that the cells that migrated from the glomerular surface on Day 8 were both anti-podocalyxin- and ED-1-positive. Furthermore, immunoelectron microscopy revealed that the ED-1-positive cells were located on the glomerular basement membrane. Pretreatment with anti-macrophages and -Thy1.1 antibodies, both with complement, did not reduce the number of MC, whereas pretreatment with puromycin aminonucleoside predominantly reduced the number of MC. A predominant decrease in the number of glomerular macrophages by gamma-irradiation did not result in a reduction of the number of MC. MC derived from glomerular cultures of bone marrow chimeric rats expressed the la antigen originated from recipient, which indicates that MC is not derived from bone marrow cells. Macrophage colony-stimulating factor accelerated the speed of the change into MC, and granulocyte-macrophage colony-stimulating factor dramatically enhanced its degree with increase of cell number on Day 8. We concluded that podocytes change into MC in normal rat glomerular culture and that the change is enhanced by colony-stimulating factors. The results provide a completely new insight into the origin of crescent-forming cells.

Animals↗
PubMed2005

T-/H-cadherin (CDH13): a new marker for differentiating podocytes.

Cadherin molecules are known to be involved in various biological processes other than cell adhesion such as morphogenesis, cell-cell communication, cell recognition or cell signalling. While the classical cadherin molecule is characterized by an extracellular moiety, a transmembrane region and a variable cytoplasmic domain, T-/H-cadherin differs from this pattern due to the absence of a transmembrane region and a cytoplasmic domain, respectively. Its extracellular moiety is bound to the apical cell membrane by a glycosyl-phosphatidyl-inositol anchor and localized to lipid raft domains. As its molecular function and expression pattern is still not fully understood, we used a newly generated anti-T-/H-cadherin antiserum to study immunohistochemically the expression of T-/H-cadherin during the differentiation of foetal human glomeruli. At the early capillary loop stage a strong apical signal comes up for visceral epithelial cells of Bowman's capsule, which begin to differentiate towards podocytes. At the advanced capillary loop stage, when podocytes have become part of the glomerular filtration barrier, the expression pattern, however, becomes more distinct and most likely restricted to the foot processes of the podocytes. We thus postulate a functional role of T-/H-cadherin for the differentiation of the podocytes and the formation of the glomerular capillary network.

Adult↗
PubMed2006

Podocyte-associated proteins FAT, alpha-actinin-4 and filtrin are expressed in Langerhans islets of the pancreas.

Nephrin is a crucial podocyte molecule in the kidney glomerular filtration barrier and it is also expressed in Langerhans islet beta cells of the pancreas. Recently, genetic mapping of proteinuric kidney disease genes and animal models have revealed further important molecules for the kidney filtration function including alpha-actinin-4, podocin, FAT, and NEPH1. This study was addressed to explore the pancreatic expression of the podocyte molecules podocin, FAT, alpha-actinin-4, NEPH1, NEPH2, filtrin/NEPH3, synaptopodin and CD2 associated protein (CD2AP). The mRNA and protein expressions were studied by RT-PCR and immunoblotting, and localization in the pancreas was investigated by immunofluorescence. Of the nephrin-associated podocyte proteins, filtrin/NEPH3, FAT, and alpha-actinin-4 were found to be expressed in the pancreas at the gene and protein level and localized to Langerhans islets. Immunoreactivity with the podocin antibody was detected mostly in the exocrine pancreas. NEPH1 and synaptopodin expression was detected only at the mRNA level. Further studies are needed to unravel the functional role of these podocyte-associated molecules in the pancreatic Langerhans islets.

Actinin↗
PubMed2005

Messenger RNA expression of glomerular podocyte markers in the urinary sediment of acquired proteinuric diseases.

BACKGROUND: Podocyte slit diaphragm plays an important role in the control of glomerular permeability. We hypothesize that studying the gene expression profile of podocyte in urinary sediment may provide diagnostic and prognostic information on acquired proteinuric diseases. METHODS: We studied 28 patients who required kidney biopsy for acquired proteinuric diseases (diabetic glomerulosclerosis, 9 cases; IgA nephropathy, 10 cases; minimal change disease, 5 cases; membranous nephropathy, 5 cases). We also studied 10 cases of diabetic microalbuminuria and 9 healthy controls. The mRNA expressions of nephrin (NephRNA), podocin (PodRNA) and synaptopodin (SynRNA) in urinary sediment were measured by real time quantitative PCR. After recruitment, all patients were followed for at least 12 months. RESULTS: There were significant differences in the NephRNA and PodRNA in the urinary sediment between diagnosis groups (p<0.005). On the other hand, SynRNA was only marginally significant between diagnosis groups (p<0.05). Although statistically significant, the degree of proteinuria had only modest correlations with the urinary expression of nephrin. After a median follow up for 23 months, there was a significant correlation between the rate of decline in renal function and NephRNA (r=0.559, p=0.001) and PodRNA (r=0.530, p=0.002), but not SynRNA (r=0.054, p=NS). The correlation remained statistically significant after multivariate analysis to adjust for the degree of proteinuria and initial renal function. CONCLUSIONS: Urinary mRNA expression of podocyte markers, such as nephrin and podocin, are significantly different between proteinuric disease categories. Further, NephRNA and PodRNA correlated with the rate of decline in renal function. Our results suggest that urinary podocyte gene expression may be a useful non-invasive tool which provides additional information for the management of proteinuric diseases.

Adult↗
PubMed2005

TRPC6 - a new podocyte gene involved in focal segmental glomerulosclerosis.

Hereditary kidney diseases have long been an enigma with respect to the identity of the mutated genes and the mechanisms by which they develop. Recently, the podocyte has been identified as a primary target in both genetic and acquired glomerular disorders. Mutations discovered by Winn et al. and Reiser et al. in the gene encoding TRPC6, a non-selective cation channel of the TRP family expressed in podocyte foot processes, have been shown to cause focal segmental glomerulosclerosis. It remains to be determined whether these mutations lead to (i) impaired channel function that initiates a new pathogenic mechanism or (ii) decreased ability of the podocyte to adapt to normal physiological challenges that account for disease development, as suggested for other late-onset autosomal-dominant podocyte disorders.

Glomerulosclerosis, Focal Segmental↗
PubMed2006

Urinary podocyte mRNA excretion in children with D+HUS: a potential marker of long-term outcome.

BACKGROUND: Diarrhea-associated hemolytic uremic syndrome (D+HUS) causes acute renal failure and may lead to podocyte loss. Objective. To determine if the urinary mRNA excretion of podocyte proteins is detectable in children with D+HUS and if it is a biomarker of a poor long-term outcome. METHODS: Patients were randomly selected from participants in the SYNSORB Pk trial. Urine samples were collected daily during the first week of hospitalization. Specimens were also obtained in healthy volunteers. Synaptopodin and nephrin mRNA levels were measured using real-time PCR. RESULTS: Fifteen children, aged 4.9+/-2.8 years, were studied. Patients were categorized based on urinary mRNA levels into normal (marker:GAPDH mean + SD) in controls. Twelve patients (80%) had increased urinary podocyte mRNA excretion; 11 (73%) had high synaptopodin and 5 (33%) had high nephrin mRNA levels. Follow-up data were available in 13/15 patients, all of whom had normal blood pressure, urinalysis, and serum creatinine concentration. CONCLUSION: The isolation of podocyte mRNA from routine urine samples is feasible in children with D+HUS. Most patients have podocyturia based on synaptopodin and nephrin mRNA excretion. Larger studies with extended follow-up are required to determine the relationship of these biomarkers to long-term renal prognosis in D+HUS.

Adolescent↗
PubMed2006

Glomerular podocytes contain neuron-like functional synaptic vesicles.

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.

Animals↗
PubMed2005

The role of Notch signaling in specification of podocyte and proximal tubules within the developing mouse kidney.

Notch genes encode transmembrane receptors that mediate intercellular interaction by binding to the ligands on the adjacent cells. Due to early embryonic lethality in mice deficient for some Notch pathway genes, the role of Notch signaling for kidney development has not yet been defined. Using an antibody specific to the N-terminal end of gamma-secretase-cleaved Notch 1, we found evidence for Notch 1 activation in the comma-shaped and S-shaped bodies. We therefore cultured embryonic (E) day E12.5 mouse metanephroi in the presence of a gamma-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. Fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes. Distal tubules were present but at a reduced number, and this was accompanied by an increase in intervening, nonepithelial cells. By culturing day E14.5 metanephroi, we observed the formation of podocyte clusters after 3 days of DAPT treatment. These observations suggest that gamma-secretase activity, probably through activation of Notch, is not essential for podocyte formation beyond the stage of S-shaped body but is required for the proximal tubule and podocyte fates when S-shaped bodies are forming.

Animals↗
PubMed2026

Biallelic pathogenic variants in FLNB are associated with paediatric steroid-resistant nephrotic syndrome via podocyte cytoskeletal dysfunction.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a severe paediatric kidney disease and a leading cause of end-stage kidney disease in children, with a high genetic contribution. While over 80 monogenic causes of SRNS have been identified, a significant proportion of affected patients still lack a clear genetic diagnosis, indicating that additional causative genes remain to be discovered. METHODS: Through whole-exome sequencing of a paediatric SRNS cohort, we identified three probands carrying biallelic FLNB pathogenic variants. Sanger sequencing was performed for familial cosegregation verification and ACMG classification. Expression of Filamin B, Nephrin and Synaptopodin in renal tissues was assessed by immunohistochemistry/immunofluorescence. Wild-type and patient-derived variant FLNB plasmids were constructed and transfected into HEK293T cells and immortalised human podocytes (HPCs). The effects of these variants on protein expression, localisation and cytoskeletal organisation were assessed by western blotting and immunofluorescence. FLNB expression in HPCs was silenced using shRNA to evaluate the impact on podocyte marker proteins, cytoskeletal integrity and migratory capacity. A zebrafish flnb knockdown model was employed to validate its effects on renal development. RESULTS: All three probands presented with isolated SRNS without skeletal developmental abnormalities, and renal tissues showed significantly reduced Filamin B protein expression. In vitro, p.L117P and p.M1803L variants led to markedly reduced protein expression, while p.R470L and p.K2586R induced perinuclear aggregation of Filamin B accompanied by F-actin rearrangement. FLNB silencing led to downregulation of Nephrin and Synaptopodin, cytoskeletal disorganisation and impaired cell migration. Zebrafish flnb knockdown exhibited pericardial oedema, defective nephron development and abnormal podocyte foot processes. CONCLUSION: We report for the first time that biallelic FLNB pathogenic variants are associated with paediatric SRNS by disrupting Filamin B expression, cytoskeletal integrity and podocyte function, providing evidence that FLNB is a novel monogenic cause of SRNS.

Humans↗
PubMed2006

Podocyte proteoglycan synthesis is involved in the development of nephrotic syndrome.

Proteoglycans (PGs) are important for the glomerular barrier, for cell signaling, and for the anchorage of cells to the glomerular basement membrane. They are, however, complex macromolecules, and their production has not yet been thoroughly investigated in podocytes. In the present study, we studied the biosynthesis of PGs by highly differentiated human podocytes and in rats. The cells were treated with puromycin aminonucleoside (PAN; a nephrosis-inducing agent), steroids (used as primary treatment for nephrotic syndrome), or both. Analysis was made by TaqMan real-time PCR, Western blotting, and by metabolic labeling with (35)S and (3)H. We found that podocytes produce versican, syndecan-1, decorin, and biglycan together with the previously known PG syndecan-4, glypican, and perlecan. PAN treatment downregulated the mRNA and the protein expression of both versican (by 24 +/- 6%, P < 0.01, for mRNA and by 50% for protein) and perlecan (by 14 +/- 5%, P < 0.05, for mRNA and by 50% for protein). The decreased expression was confirmed by studying the glomerular gene expression in rats treated with PAN during a time course study. In addition, puromycin decreased the expression of enzymes involved in the glycosaminoglycan biosynthesis. Steroid treatment decreased perlecan (by 24 +/- 3%, P < 0.01) and syndecan-1 expression (by 30 +/- 4%, P < 0.01) but increased the expression of decorin 2.5-fold. The observed alterations of PG synthesis induced by PAN may lead to decreased glomerular anionic charge and disturbed podocyte morphology, factors that are important for the development of a nephrotic syndrome.

Animals↗