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PubMed1997

Podocytes are the major source of IL-1 alpha and IL-1 beta in human glomerulonephritides.

To address the question of in situ production of IL-1 alpha and IL-1 beta in proliferative and non-proliferative forms of human glomerulonephritis (GN), we performed immunocytochemical and in situ hybridization studies on renal biopsies from patients with mesangial IgA-GN (N = 38), idiopathic membranous GN (MGN; N = 12), minimal change disease (MCD; N = 9), focal segmental glomerulosclerosis (FSGS; N = 5) and acute endocapillary GN (AGN; N = 3). Normal kidneys (N = 10) served as controls. Concomitantly, the expression of IL-1 receptor type I (IL-1 RI), IL-1 receptor type II (IL-1 RII) and of IL-1 receptor antagonist (IL-1 RA) was analyzed. Antibodies against antigens expressed on podocytes (PP-44), endothelial cells (CD31) and monocytes/macrophages (CD11b, CD14, CD68) were applied to attribute the expression of IL-1/IL-1 related peptides to intrinsic glomerular and/or blood-derived infiltrating cells. Our results demonstrate that IL-1 RII is constitutively expressed on endothelial cells, and its expression can be induced in proximal tubular cells and in the interstitium. In diseased glomeruli podocytes are capable of producing IL-1 alpha/beta. In MGN and MCD/FSGS, the expression of both IL-1 forms is particularly noted in early stages of the disease and is not only accompanied by a marked reactivity for IL-1 RI, but also for IL-1 RA. In segmental sclerosing lesions in FSGS and in IgA-GN with marked glomerular proliferation and/or sclerosis, a reduced expression of the PP-44 antigen and a diminished ability of podocytes to produce IL-1/IL-1 related peptides are noted. These results suggest that intrinsic glomerular production of IL-1 may be of relevance for the protection of glomeruli from continuing injury.

Acute Disease↗
PubMed1999

Antibody induced injury to podocytes with proteinuria and foot process swelling in a transgenic (T16) mouse.

T16 mice contain a human 3' untranslated sequence of the Thy 1.1 gene. Unlike normal mice they express Thy 1.1 protein on the podocytes which was immuno-localized to podocyte apical and basal plasma membranes and filtration slit. When monoclonal anti-Thy 1.1 antibody (OX7) was injected in nonproteinuric heterozygous mice there was rapid podocyte foot process swelling and proteinuria. Immunofluorescence showed granular glomerular OX7 binding at one hour. Progressive loss of pedicels occurred with 17.9 +/- 2.5, 14.4 +/- 1.1 and 10.5 +/- 3.5 per 10 nm glomerular basement membrane (GBM) remaining 1, 6 and 24 hours, respectively, after 1 mg OX7, vs 32.2 +/- 2.0 in T16 mice given saline. Twenty-four hour proteinuria was OX7 dose-dependent, peaked at 1-3 days and reduced to near basal levels 9-11 days thereafter. Proteinuria was nonselective except at very low doses (0.1 mg OX7) where microalbuminuria was seen. F(ab')2 OX7 administration also caused proteinuria in T16 mice. One milligram F(ab')1 OX7 caused diffuse foot process swelling without manifest proteinuria in T16 mice. Anti-Thy 1.1 IgM monoclonal antibody did not produce the effects of OX7 in T16 mice. Foot process swelling was not modified by histamine or 5-hydroxytryptamine antagonists. OX7 did not cause complement activation or leucocyte infiltration, hence glomerular injury appeared to be mediated directly by the antibody.

Albuminuria↗
PubMed1998

NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate.

Reactive oxygen species contribute to glomerular damage and proteinuria. In this study, we show that cultured human podocytes produce superoxide in response to extracellular adenosine triphosphate (ATP), and we identified the oxidases involved in this process. Adenosine triphosphate (10-4 M for 4 hr) raised superoxide production from 1.28 +/- 0.15 to 2.67 &/- 0.34 nmol/mg protein/min. Studies with podocyte homogenates revealed activation of both nicotinamide adenine dinucleotide (NADH; from 2.65 +/- 0.23 to 7.43 +/- 0.57) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidases [from 1.74 +/- 0.13 to 4.05 +/- 0.12 (nmol O2/mg protein/min)] by ATP. Activity of xanthine-oxidases was low and unchanged by ATP. Activation of the plasma-membrane bound NAD(P)H oxidases by ATP was time and dose dependent. Reverse transcribed-polymerase chain reaction (RT-PCR) studies with primers derived from monocyte sequences amplified mRNA for the NADPH oxidase subunits p22phox, p47phox, gp91phox, and p67phox, and the latter was transiently increased by ATP. Experiments with actinomycin D and cycloheximide suggested that ATP modulates enzyme activity at the transcriptional and translational levels. In conclusion, NAD(P)H dependent, membrane associated oxidases represent the major superoxide source in human podocytes. Activation of NAD(P)H oxidase by ATP might be secondary to increased mRNA expression of the NADPH oxidase subunit gp67phox.

Adenosine Triphosphate↗
PubMed2001

Podocyte injuries exacerbate mesangial proliferative glomerulonephritis.

BACKGROUND: From the observations of morphology seen in early phases of the experimental models of the irreversible mesangial proliferative glomerulonephritis, we hypothesized that podocyte injury is one of the important factors in bringing upon irreversible glomerular alterations. To verify this hypothesis, we investigated whether podocyte injury induced by puromycin aminonucleoside (PAN) injection affects the mesangial alterations of anti-Thy 1.1 glomerulonephritis. METHODS: Female Wistar rats were injected with 0.5 mg monoclonal antibody (mAb) 1-22-3 five days after the injection of 10 mg or 5 mg/100 g body weight (BW) of puromycin aminonucleoside (PAN), and sacrificed at 7 days or 8 weeks after the mAb 1-22-3 injection. RESULTS: Consecutive injections of 10 mg/100 g BW of PAN and mAb 1-22-3 caused the irreversible mesangial alteration with persistent proteinuria (at week 8, proteinuria 100.3 +/- 57.8 mg/24 h, matrix score 1.13 +/- 0.52, collagen type I score 2.04 +/- 0.53, mRNA for collagen type I 227 +/- 79% to the group with a single injection of 1-22-3). Although single injection of 5 mg/100 g BW of PAN was not capable of inducing abnormal proteinuria, consecutive injections of 5 mg/100 g BW of PAN and mAb 1-22-3 also caused irreversible mesangial alteration and persistent proteinuria. CONCLUSIONS: Podocyte injury might be an important factor that exacerbates mesangial proliferation and mesangial matrix expansion. The irreversible mesangial alterations caused by consecutive injections of PAN and mAb 1-22-3 may be a novel model that could be used to analyze the mechanism of progressive mesangial alteration.

Animals↗
PubMed2001

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains.

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Animals↗
PubMed1999

Nephrin is specifically located at the slit diaphragm of glomerular podocytes.

We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.

Cell Membrane↗
PubMed1995

Molecular cloning, expression, and characterization of podocalyxin-like protein 1 from rabbit as a transmembrane protein of glomerular podocytes and vascular endothelium.

Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxin-like protein base upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N- and O-linked glycosylation, and a potential glycosaminoglycan attachment sites. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.

Amino Acid Sequence↗
PubMed1983

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Acetylgalactosamine↗
PubMed1994

Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

Adult↗
PubMed1982

The detection of effaced podocytes by high resolution light microscopy.

Fifteen cases of renal minimal change disease and five normal glomeruli were studied by high resolution light microscopy (HRLM) and transmission electron microscopy (TEM). Effacement of podocytes usually is accompanied by aggregation of microfilaments in the cytoplasm of the epithelial cells parallel to the glomerular basement membrane (GBM). The aggregated microfilaments were detected as a dense blue line by high resolution light microscopy. The presence of this line appears to be a reliable sign for the detection of diffusely effaced podocytes by light microscopy. However, when effacement of podocytes was not accompanied by the condensation of microfilaments, the detection of this change by HRLM was more difficult.

Cytoskeleton↗
PubMed2005

P-Cadherin is decreased in diabetic glomeruli and in glucose-stimulated podocytes in vivo and in vitro studies.

BACKGROUND: Proteinuria is a cardinal feature of glomerular disease, including diabetic nephropathy, and the glomerular filtration barrier acts as a filter, restricting protein excretion in urine. We tested whether the expression of P-cadherin, a molecule known to be located at the slit diaphragm, was altered by diabetes in vivo and by high glucose in vitro. METHODS: In vivo, 24 Sprague-Dawley rats were injected with diluent [control (C), n=8] or streptozotocin intraperitoneally and the latter were left untreated (DM, n=8) or treated with insulin (DM+I, n=8) for 6 weeks. In vitro, immortalized mouse podocytes were cultured in media with 5.6 mM glucose (LG), LG+19.4 mM mannitol (LG+M) or 25 mM glucose (HG) with or without protein kinase C (PKC) inhibitor (10(-7) M calphostin C or 10(-6) M GF 109203X). Reverse transcription-polymerase chain reaction, western blotting for P-cadherin mRNA and protein expression, respectively, were performed with sieved glomeruli and cell lysates, and immunofluorescence staining was undertaken with renal tissue. RESULTS: Twenty-four hour urinary albumin excretion was significantly higher in DM compared with C and DM+I rats (P<0.05). Glomerular P-cadherin mRNA expression was significantly lower in DM (1.36+/-0.20x10(-2) attm/ng RNA) than in C rats (2.61+/-0.33x10(-2) attm/ng RNA) (P<0.05). P-Cadherin protein expression, assessed by western blot and immunofluorescence staining, was also decreased in DM compared with C and DM+I glomeruli. HG significantly reduced P-cadherin mRNA and protein expression in cultured podocytes by 42% and 62%, respectively (P<0.05), and these decrements were ameliorated by PKC inhibitor. CONCLUSIONS: Diabetes in vivo and exposure of podocytes to HG in vitro reduced P-cadherin mRNA and protein expression, and PKC was involved in the regulation of HG-induced down-regulation of P-cadherin. These findings suggest that the decrease in P-cadherin expression is connected with the early changes of diabetic nephropathy and, thus, may contribute to the development of proteinuria.

Animals↗
PubMed2001

Update in podocyte biology.

Knowledge of podocyte biology is growing rapidly. Podocytes are crucially involved in most hereditary diseases affecting the glomerulus, which all exhibit podocyte-specific defects, that is, foot process effacement and protein leakage. Efforts to understand molecular mechanisms causing these derangements are increasingly successful and will allow a better targeting of interventions to halt the progression of chronic renal disease.

Epithelial Cells↗
PubMed2003

Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats.

The foot processes forming the slit diaphragm are disrupted in diseases associated with proteinuria. Although they are often repairable, regulators for the repairing process remain unknown. By extrapolating from the fact that vitamin A is essential for the nephrogenesis, this study examined whether or not injured podocytes in the middle of the repairing process require retinaldehyde dehydrogenase type 2 (RALDH2), one of the key enzymes to produce all-trans-retinoic acid (ATRA). RALDH2 was dramatically upregulated in podocytes of puromycin aminonucleoside-induced nephrosis (PAN nephrosis) rats. On day 5 of PAN nephrosis, RALDH2 showed the remarkable induction, whereas glomerular expression levels of nephrin and midkine, one of the ATRA target genes, were downregulated. Daily administration of ATRA ameliorated proteinuria, which was accompanied by the improvement in the effacement of the foot processes and by the induction of nephrin and midkine. In contrast, recovery from PAN nephrosis was delayed in rats fed with a vitamin A-deficient diet. Consistently, the promoter region of human nephrin gene (NPHS1) contained three putative retinoic acid response elements (RARE) and showed the enhancer activity in response to ATRA in a dose-dependent manner. This transcriptional activation was regulated through the receptors for retinoids because BMS-189453, an antagonist to the retinoid receptors, counteracted it in a dose-dependent manner. In conclusion, active metabolites of vitamin A, especially ATRA produced by RALDH2 play relevant roles during the repairing process of injured podocytes. The results obtained from PAN nephrosis rats might be applicable to human renal diseases.

Aldehyde Oxidoreductases↗
PubMed2003

Podocyte expression of hypoxia-inducible factor (HIF)-1 and HIF-2 during glomerular development.

The heterodimeric transcription factors, hypoxia-inducible factor (HIF)-1 and HIF-2, are essential for the maintenance of cellular oxygen homeostasis. In response to hypoxia, stabilized HIF-1alpha and HIF-2alpha proteins bind HIF-1beta and initiate expression of genes that alleviate hypoxic stress, including those promoting neovascularization. Both HIF-1 and HIF-2 stimulate transcription of vascular endothelial growth factor (VEGF), a crucial regulator of vascular development. Because VEGF is highly expressed by metanephric podocytes and collecting ducts, developing mouse kidney was examined for the presence and distribution of HIF-1alpha, HIF-2alpha, and HIF-1beta. The expression of HIF-1alpha and HIF-2alpha mRNAs in newborn mouse kidney was confirmed by RT-PCR and Northern blot analysis. By in situ hybridization, HIF-1alpha and HIF-2alpha mRNAs were highly expressed in the nephrogenic zone of newborn kidney cortex and in the medulla. Particularly intense hybridization was found in podocytes of developing glomeruli and in medullary collecting ducts. Both HIF-1 and HIF-2 heterodimers were identified in newborn kidney lysates by immunoprecipitation with HIF-1alpha, HIF-2alpha, and HIF-1beta antibodies and Western blots. Immunofluorescence analysis of the hypoxia marker, pimonidazole, showed that collecting ducts and many developing tubules undergo severe hypoxia in developing kidney. Immunohistochemistry of newborn kidney demonstrated widespread expression of HIF-1beta protein in nuclei of glomeruli and all tubular segments, whereas HIF-2alpha protein expression was more restricted and localized chiefly to podocytes of developing glomeruli and developing tubules. HIF-1alpha and HIF-2alpha protein and VEGF mRNA were all strongly induced in embryonic kidneys maintained in hypoxic organ cultures. Collectively, these data suggest that HIF stabilization, by hypoxia and/or by other means, may be critical for VEGF production and kidney vascular development.

Animals↗
PubMed2004

The major podocyte protein nephrin is transcriptionally activated by the Wilms' tumor suppressor WT1.

NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms' tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter >10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(-KTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(+KTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.

Animals↗
PubMed2002

Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton.

Nephrin is an Ig-like transmembrane protein. It is a major component of the podocyte slit diaphragm and is essential for maintaining normal glomerular permeability. CD2-associated protein (CD2AP) is also necessary for normal glomerular permeability and is a putative nephrin adapter molecule. Here, we document that nephrin and CD2AP are linked to the actin cytoskeleton. As detected by Western blot analysis, nephrin and CD2AP were both insoluble when cell membranes from normal rat glomeruli were extracted with 0.5% Triton X-100 (TX-100) at 4 degrees C in the presence of divalent cations, but they were solubilized when the extraction included potassium iodide (KI) to depolymerize F-actin. In addition, a small fraction of the solubilized nephrin and CD2AP was recovered in the low-density fractions of OptiPrep flotation gradients, which indicates that a portion of nephrin, possibly associated with CD2AP, resides in a cholesterol- or sphingolipid-rich region of the plasma membrane. Immunofluorescent staining of unfixed sections of normal rat kidney for nephrin, CD2AP, and F-actin was unaltered by treatment with TX-100 but was greatly diminished by addition of KI. Nephrin staining was slightly reduced by cholesterol depletion with methyl-beta-cyclodextrin in the presence of TX-100 but was nearly absent after addition of KI. These results document that nephrin anchors the slit diaphragm to the actin cytoskeleton, possibly by linkage to CD2AP, and that nephrin traverses a relatively cholesterol-poor region of the podocyte plasma membrane. In addition, a small pool of actin-associated nephrin and CD2AP resides in lipid rafts, possibly in the cholesterol-rich apical region of the podocyte-foot processes.

Actins↗
PubMed1998

Monoclonal antibody P-31 recognizes a novel intermediate filament-associated protein (p250) in rat podocytes.

The visceral glomerular epithelial cells (GECs) or podocytes of the renal glomerulus constitute a highly specialized epithelium. To study the nature of podocytes, we established mouse monoclonal antibodies against GEC. Clone P-31 reacted exclusively with the cytoplasm of GEC by immunofluorescence. Immunoblot analysis with P-31 showed that a single band of 250 kDa was detectable in a glomerular lysate. The 250-kDa polypeptide (p250) was recovered from Triton X-100-insoluble fractions of isolated glomeruli, suggesting that this molecule is associated with the cytoskeleton. Immunogold staining with P-31 demonstrated that the gold particles were located at the intersections of vimentin-type intermediate filaments of podocytes. In developing kidney, this protein first appeared in immature GECs during the S-shaped body stage. In puromycin aminonucleoside nephrosis, p250 was dramatically increased in glomeruli where enhanced desmin expression was observed in GECs. These results indicate that p250 is a novel intermediate filament-associated protein and plays a role in the organization of the intermediate filament network in both normal and diseased conditions.

Animals↗
PubMedDate not supplied

Podocyte phenotypes as defined by expression and distribution of GLEPP1 in the developing glomerulus and in nephrotic glomeruli from MCD, CNF, and FSGS. A dedifferentiation hypothesis for the nephrotic syndrome.

Glomerular epithelial protein 1 (GLEPP1) is a podocyte receptor membrane protein tyrosine phosphatase located on the apical cell membrane of visceral glomerular epithelial cell (VGEC) foot processes. Double label immunofluorescence, immunoelectron microscopy, and peroxidase immunohistochemistry were used to map the GLEPP1 distribution in the developing glomerulus and in minimal-change nephropathy (MCN), congenital nephrotic syndrome of the Finnish type, and focal-segmental glomerulosclerosis (FSGS). In MCN GLEPP1 was shifted away from the glomerular basement membrane on the apical cell membrane of effaced foot processes. These data are compatible with the previously suggested concept that MCN can be considered a form of dedifferentiation of the podocyte phenotype. Similarly, changes seen in congenital nephrotic syndrome of the Finnish type can be considered a consequence of failure to complete normal podocyte development. In FSGS glomeruli GLEPP1 was frequently absent from VGECs, even when no sclerosis was detectable in that glomerulus. Therefore, in FSGS, VGECs may lose GLEPP1, and this loss appears to occur in the absence of scarring and may, therefore, precede the scarring process. We speculate that a changed VGEC phenotype that does not express GLEPP1 might have properties similar to the early undifferentiated VGEC developmental phenotype. GLEPP1 distribution pattern and absence from glomeruli of individuals with nephrotic syndrome may, therefore, represent a useful phenotypic marker.

Animals↗