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PubMed1986

Heterogeneity of the podocyte membrane in rat kidney as revealed by ethanol dehydration of unosmicated specimens.

The ultrastructure of the podocyte membrane was studied by means of transmission electron microscopy of unosmicated tissue samples after acetone or ethanol dehydration and subsequent embedding in a polyester resin. The podocyte membrane in glutaraldehyde (GA)-fixed, acetone-dehydrated samples consisted of a relatively thick, clear layer (about 6 nm) abutted by the dark staining cytoplasm and a dark surface layer. In GA-fixed, ethanol-dehydrated samples a striking intramembranous pattern was observed in the podocyte cell membrane. The luminal podocyte membrane was regularly perforated by gaps about 25 nm wide. In grazing sections these gaps appeared round and were separated by a honeycomb pattern of intact membrane. The abluminal membrane, in contrast, generally maintained its continuity. The clear layer of the podocyte membrane was thinner in ethanol-dehydrated samples than in acetone-dehydrated ones. In tissue samples fixed with GA supplemented by ruthenium red, ethanol dehydration was not associated with cell-membrane perforations. Based on these observations as well as on biochemical data from the literature we suggest that in GA-fixed, unosmicated, acetone-dehydrated samples the structural integrity of the podocyte membrane is well preserved, while ethanol dehydration extracts some specific material from regularly distributed domains in the podocyte cell membrane.

Animals↗
PubMed2000

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes.

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.

Adult↗
PubMed1999

Polycations induce calcium signaling in glomerular podocytes.

BACKGROUND: The neutralization of the polyanionic surface of the podocyte by perfusion of kidneys with polycations, such as protamine sulfate, leads to a retraction of podocyte foot processes and proteinuria. This study investigates the effects of protamine sulfate or anionic, neutral, or cationic dextrans on the cytosolic calcium activity ([Ca2+]i) in podocytes. METHODS: [Ca2+]i was measured in single cultured differentiated mouse podocytes with the fluorescence dye fura-2/AM. RESULTS: Protamine sulfate caused a concentration-dependent and partially reversible increase of [Ca2+]i (EC50 approximately 1.5 micromol/liter). Pretreatment of the cells with heparin (100 U/liter) inhibited the protamine sulfate-mediated increase of [Ca2+]i. Like protamine sulfate, diethylaminoethyl dextran (DEAE-dextran) concentration dependently increased [Ca2+]i in podocytes (EC50 approximately 20 nmol/liter), whereas dextran sulfate or uncharged dextran (both 10 micromol/liter) did not influence [Ca2+]i. A reduction of the extracellular Ca2+ concentration (from 1 mmol/liter to 1 micomol/liter) partially inhibited the protamine sulfate and the DEAE-dextran-induced [Ca2+]i response. Flufenamate (100 micromol/liter) or Gd3+ (10 micromol/liter), which are known to inhibit nonselective ion channels, did not influence the [Ca2+]i increase induced by protamine sulfate. In the presence of thapsigargin (50 nmol/liter), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, both protamine sulfate and DEAE-dextran increased [Ca2+]i. CONCLUSIONS: The data indicate that polycations increase podocyte [Ca2+]i. The increase of [Ca2+]i may be an early event in the pathogenesis of protamine sulfate-mediated retraction of podocyte foot processes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Regulation of mouse podocyte process dynamics by protein tyrosine phosphatases rapid communication.

BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.

Actins↗
PubMed2000

Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus.

UNLABELLED: Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus. BACKGROUND: Knowledge about biological functions of podocytes in the glomerulus is limited because of its unique anatomical location. Here we introduce a new method for measuring the intracellular calcium activity ([Ca2+]i) in the podocyte in the intact glomerulus. METHODS: With the help of fluorescence high-resolution digital imaging and a recently developed ultraviolet laser-scanning microscope, [Ca2+]i was measured in fura-2-loaded glomeruli and single podocytes of intact microdissected rat glomeruli. RESULTS: Angiotensin II (Ang II) increased [Ca2+]i reversibly in a biphasic and concentration-dependent manner. In contrast to Ang II, bradykinin, thrombin, arginine vasopressin, and serotonin did not change [Ca2+]i in the glomerulus. At reduced extracellular Ca2+ activity, Ang II released [Ca2+]i from intracellular stores, but the second phase, corresponding to a Ca2+ influx from the extracellular space, was absent. The L-type Ca2+ channel blocker nicardipine did not influence the Ang II-mediated [Ca2+]i increase, and an increase of the extracellular K+ concentration did not change [Ca2+]i in the glomerulus. The angrotensin II type I (AT1) receptor antagonist losartan inhibited the Ang II-mediated [Ca2+]i increase. Confocal [Ca2+]i measurements using fura-2 or fluo-3 or fluo-4 on the single cell level show that some of the Ang II-mediated [Ca2+]i response originated from podocytes. Costaining with calcein allowed the identification of podocytes because of the characteristic morphology and location in relationship to the capillary network. CONCLUSIONS: These data suggest that podocytes in the intact glomerulus respond to Ang II with an increase of [Ca2+]i via an AT1 receptor.

Angiotensin II↗
PubMed2000

The role of podocyte injury in the pathogenesis of focal segmental glomerulosclerosis.

The podocyte has diverse functions including glomerular filtration, biosynthesis and maintenance of the glomerular capillary architecture. It discharges these functions by virtue of a unique morphology, an intimate relationship with the capillary wall, and diverse synthetic and membrane specializations. Despite the complex role that it plays in glomerular function, the clinical manifestations of podocyte dysfunction are limited to proteinuria and renal insufficiency. Recurrent focal segmental glomerulosclerosis (FSGS) in renal transplants provides a unique opportunity to study the pathogenesis of FSGS in human beings, because the patients are monitored carefully to identify the onset of disease, the recurrence is presumed to have the same etiology as the primary disease, renal biopsy is a tool to study the pathogenesis of the lesion, and therapeutic intervention provides a mechanism to test pathogenesis. Pathologic studies have identified a proliferative lesion of the podocytes as the first sign of recurrent disease. The glomerular lesions evolve to form segmental glomerular scars with time. These findings and studies in experimental models of FSGS implicate podocyte injury in the pathogenesis of the recurrent disease. The cellular lesion (similar to the proliferative lesion of podocytes in recurrent FSGS), seen early in the course of primary FSGS suggests that the pathogenic sequence in recurrent FSGS also applies to primary FSGS. A soluble circulating factor that increases glomerular permeability and correlates with recurrence of FSGS has been identified in the pretransplant serum of patients with end-stage FSGS, but the mechanism of podocyte injury by this factor remains speculative. In any case, podocytes in the cellular lesion undergo morphologic changes and lose specialized functions seen in the normal mature cell, and these structural and functional abnormalities cause the permeability changes associated with proteinuria and destruction of glomerular filtration surface by scarring associated with loss of glomerular function.

Animals↗
PubMed1998

Nonuniform microtubular polarity established by CHO1/MKLP1 motor protein is necessary for process formation of podocytes.

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385-397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248-258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.

Animals↗
PubMed2002

Effect of cerivastatin on proteinuria and urinary podocytes in patients with chronic glomerulonephritis.

BACKGROUND: We previously reported urinary podocytes to be a marker of glomerular injury. The aim of the present study was to determine whether cerivastatin, a newly developed, potent synthetic statin, affects proteinuria and urinary podocyte excretion in patients with chronic glomerulonephritis (CGN). METHODS: We randomly assigned 40 normotensive hypercholesterolemic patients with CGN to receive either cerivastatin 0.15 mg/day (n=20) or placebo (n=20). Subjects comprised 24 men and 16 women, with a mean age of 40.8+/-14.4 years; 27 had IgA nephropathy and 13 had non-IgA proliferative glomerulonephritis. Treatment was continued for 6 months. Plasma total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides, urinary protein excretion and the number of podocytes were measured before treatment and at 3 and 6 months after treatment. RESULTS: After 6 months, a significant reduction in total cholesterol (P<0.001), LDL-cholesterol (P<0.001) and triglycerides (P<0.05), and a significant increase in HDL-cholesterol (P<0.001) were observed in the group treated with cerivastatin. Urinary protein excretion decreased from 1.8+/-0.6 to 0.8+/-0.4 g/day, (P<0.01) in this group, and urinary podocyte excretion decreased from 1.6+/-0.6 to 0.9+/-0.4 cells/ml (P<0.01). However, placebo showed little effect on these lipid levels, urinary protein excretion and urinary podocyte excretion. The differences between the cerivastatin group and the placebo group were significant (cholesterol, P<0.001; LDL-cholesterol, P<0.001; triglycerides, P<0.05; HDL-cholesterol, P<0.001; urinary protein, P<0.01; and urinary podocytes, P<0.01). CONCLUSION: Statins such as cerivastatin may be beneficial for restoration of injured podocytes in patients with CGN and hypercholesterolaemia.

Adult↗
PubMed2002

Phenotypic change of glomerular podocytes in primary focal segmental glomerulosclerosis: developmental paradigm?

WT1 and Pax2 are transcription factors involved in kidney development and phenotypic regulation of glomerular epithelial cells. In primary focal segmental glomerulosclerosis (FSGS), the alteration of the podocyte is important for the development of the cellular lesion (CL) and glomerular scar formation. To investigate the contribution of WT1 and Pax2 to the development of the CL in primary FSGS, immunohistological studies were performed using renal biopsy specimens on the expression of WT1, Pax2 and cytokeratin (CK), which is an epithelial marker but never found in normal podocytes. The expression of WT1 was decreased in the CL compared with unaffected podocytes, but Pax2 and CK were expressed significantly in the CL and also in the cells morphologically recognized as podocytes in cases with CL. Our results suggest that re-expression of Pax2 resulting in phenotypic change of podocytes to a different epithelial form is important for the development of the CL in primary FSGS. Normal podocytes resemble mesenchymal cells since they express both vimentin and WT1. In contrast, epithelial cells including parietal epithelial cells of the Bowman's capsule express both Pax2 and CK. Therefore, the mechanism of phenotypic change of podocytes in the CL in primary FSGS might be mesenchymal-epithelial transformation and a developmental paradigm.

Adult↗
PubMed2004

Differential induction of podocyte heat shock proteins by prolonged single and combination toxic metal exposure.

Cadmium, mercury, and arsenite are among the most abundant toxic metals (TM) in our environment, and chronic TM exposure leads to injury to the kidney's glomerular filtration barrier. The small heat shock protein hsp25, highly expressed in glomerular podocytes, is induced during development of experimental nephrotic syndrome, and hsp25 overexpression can protect cultured podocytes from injury. Because little is known about the effect of multiple TM on podocytes, we measured the response of cultured podocytes to prolonged exposures to single and multiple TM. Podocyte viability declined by approximately 50% after 3 days of treatment with 20 microM cadmium, mercury, or arsenite, and 40 microM of any of these metals was lethal. The toxicity of equimolar concentrations of two or all three metals in combination was significantly altered compared to individual metal treatments. Single TM treatments induced only modest increases in the amounts of hsp25, alphaB-crystallin, and inducible hsp70. Toxic metal combinations induced greater stress protein accumulation, especially arsenite + cadmium or arsenite + cadmium + mercury treatments, the TM mixtures with the lowest toxicity. All TM treatments caused a rapid and sustained increase in hsp25 phosphorylation. The intracellular accumulation of cadmium was greater and that of mercury was less in cells treated with TM combinations than in cells treated with a single TM. Our results showed that multiple TM effects on podocyte viability were neither additive nor synergistic and that induction of heat shock proteins correlated with increased resistance to TM injury, suggesting that induction of small heat shock proteins may play an important role in preventing TM-induced podocyte injury.

Animals↗
PubMed2002

Two gene fragments that direct podocyte-specific expression in transgenic mice.

Transgenic manipulation of the glomerular visceral epithelial cell offers a powerful approach for studying the biology of this morphologically complex cell type. It has been previously demonstrated that an 8.3-kb and a 5.4-kb fragment of the murine Nphs1 (nephrin) promoter-enhancer drives lacZ expression in podocytes, brain, and pancreas of transgenic mice, recapitulating the expression pattern of the endogenous nephrin gene. In this present study, two truly podocyte-specific promoters were identified that drive transgene expression in podocytes without expression in extrarenal tissues in adult or embryonic mice. A 1.25-kb fragment driving a lacZ reporter gene (p1.25N-nlacF) was derived from murine Nphs1 promoter similar to a human NPHS1 promoter fragment previously reported. Transgenic mice were generated and beta-galactosidase (beta-gal) expression was analyzed. Four of twelve founder mice were found to express beta-gal in podocytes (33% penetrance). Expression in brain and pancreas was absent in all animals, suggesting that nephrin expression in these organs might be driven by distinct cis-regulatory elements that can be removed to obtain podocyte-specific expression. A 2.5-kb fragment derived from the human NPHS2 (podocin) gene was designed in a similar fashion to drive lacZ expression in transgenic mice (p2.5P-nlacF). Twelve of twlve NPHS2 mouse founder lines expressed beta-gal exclusively in podocytes (100% penetrance). Beta-gal activity was not observed extrinsic to the kidney in p1.25N-nlacF or p2.5P-nlacF mouse embryos at gestational time points between 8.5 d post coitus and birth. In conclusion, the 2.5-kb NPHS2 promoter fragment may be useful for podocyte-specific transgenic expression when extrarenal expression of a transgene is problematic.

Animals↗
PubMed2003

Role of 12-lipoxygenase in the stimulation of p38 mitogen-activated protein kinase and collagen alpha5(IV) in experimental diabetic nephropathy and in glucose-stimulated podocytes.

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is implicated in extracellular matrix (ECM) synthesis, but its role in podocytes has not been studied. This study tested whether 12-LO induction by diabetes or by high glucose (HG) in cultured podocytes alters glomerular basement membrane by activating signal transduction pathways culminating in ECM synthesis. Sprague-Dawley rats received an injection of diluent (control [C]) or streptozotocin 65 mg/kg (DM) and were killed at 1 or 4 mo. Glomerular 12-LO mRNA and protein levels were higher in DM than in C glomeruli at 1 and 4 mo, and 12-LO localized predominantly in podocytes. Glomerular p38 mRNA and protein were higher in DM at months 1 and 4, but phospho-p38 mitogen-activated protein (MAPK) was increased only at month 1. Glomerular collagen alpha5(IV)/glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio was increased in DM at month 1 but not at month 4, whereas collagen alpha5(IV) protein was higher at both 1 and 4 mo. Mouse podocytes were cultured in media with 25 mM glucose (HG) with or without the 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) or with 5.5 mM glucose + 19.5 mM mannitol (low glucose [LG+M]) for 10 d at 37 degrees C. 12-LO mRNA and protein levels were higher in HG than in LG+M as was the p38 MAPK/GAPDH mRNA ratio. Phospho-p38 MAPK protein but not total p38 MAPK was higher in HG compared with LG+M. Collagen alpha5(IV)/GAPDH mRNA ratio and protein were higher in HG than in LG+M. 12-LO inhibition by CDC decreased HG-induced phospho-p38 MAPK and the phospho-p38/total p38 MAPK ratio, collagen alpha5(IV)/GAPDH mRNA ratio, and collagen alpha5(IV) protein expression. In summary, diabetes in vivo and exposure of podocytes to HG in vitro stimulated 12-LO, p38 MAPK, and collagen alpha5(IV) mRNA and (activated) protein. 12-LO inhibition by CDC diminished the expression of podocyte phospho-p38 MAPK and collagen alpha5(IV) mRNA and protein. These findings implicate 12-LO and the p38 MAPK signaling pathway in the mediation of ECM synthesis by podocytes in diabetes.

Animals↗
PubMed2004

Podocyte flattening and disorder of glomerular basement membrane are associated with splitting of dystroglycan-matrix interaction.

The transmembrane component of the dystroglycan complex, a heterodimer of alpha- and beta-dystroglycan, was recently localized at the basal cell membrane domain of podocytes, and it was speculated that it serves as a device of the podocyte for maintaining the complex podocyte foot process architecture, and for regulating the exact position of its ligands, the matrix proteins laminin and agrin, in the glomerular basement membrane (GBM). The redistribution of dystroglycan in two experimental rat models of foot process flattening and proteinuria-i.e., podocyte damage induced by polycationic protamine sulfate perfusion, and reactive oxygen species (ROS)-associated puromycin aminonucleoside nephrosis-was examined. In both experimental diseases, aggregation and reduced density of alpha-dystroglycan by endocytosis by podocytes was observed. In in vitro solid-phase binding assays, protamine and ROS competed with the binding of alpha-dystroglycan with purified laminin and a recombinant C-terminal fragment of agrin that contains the dystroglycan-binding domain. These changes were associated with disorder of the fibrillar components of the lamina rara externa of the GBM, as confirmed quantitatively by fractal analysis. These results indicate that both polycation and ROS induce similar changes in the distribution of podocyte alpha-dystroglycan that involve competitive disruption of alpha-dystroglycan/matrix protein complexes, endocytosis of the liberated receptor by podocytes, and disorganization of the matrix protein arrangement in the lamina rara externa. This links functional damage of the dystroglycan complex with structural changes in the GBM.

Animals↗
PubMed2004

Human podocytes possess a stretch-sensitive, Ca2+-activated K+ channel: potential implications for the control of glomerular filtration.

Podocytes express many proteins characteristic of smooth muscle, such as actin and myosin. They also express receptors to several vasoactive agents, including acetylcholine and angiotensin II; these phenotypic properties suggest that podocytes are not static entities but may respond to physiologic stimuli. The electrophysiologic properties of a conditionally immortalized human podocyte cell line that expresses the specific podocyte proteins nephrin, podocin, and synaptopodin were examined by patch clamp. Channels that were highly K(+)-selective and had a conductance of 224 +/- 11.5 pS in symmetrical 150 mM K(+) solutions were identified. Channel activity was Ca(2+)- and voltage-dependent, being increased with an increase in Ca(2+) or depolarization, and inhibited by penitrem A. The conductance and voltage- and Ca(2+)-dependence suggest that this is the large-conductance calcium-activated K(+) channel, BK (KCNMA1)-this was supported by reverse transcription-PCR experiments that showed the presence of the BK encoding mRNA, along with expression of KCNMB subunit types 3 and 4. In sections of human glomeruli, immunocytochemistry revealed that BK co-localizes with the podocyte-specific protein nephrin, indicating that these channels are present in native human podocytes. In whole-cell experiments, penitrem A inhibited outward currents to the same extent as tetra-ethyl ammonium (TEA) but did not affect the membrane potential. Channel activity was also increased by applying suction to the patch pipette or by dilution of the bathing medium, indicating that these channels are stretch sensitive. Thus, these channels do not contribute to the resting membrane potential but are activated by a rise in intracellular Ca(2+), membrane depolarization, cell swelling, or membrane stretch. By implication, these results suggest that podocytes may be able to respond to changes in the glomerular capillary pressure and modulate the GFR.

Calcium↗
PubMed2003

Coxsackievirus and adenovirus receptor, a tight junction membrane protein, is expressed in glomerular podocytes in the kidney.

In nephrosis, filtration slits of podocytes are greatly narrowed, and slit diaphragms are displaced by junctions with close contact. Freeze-fracture studies have shown that the newly formed junctions consist of tight junctions and gap junctions. Several tight-junction proteins are known as integral membrane components, including occludin and claudins; but none of them have been found in podocytes. Coxsackievirus and adenovirus receptor (CAR) has recently been identified as a virus receptor that is a 46-kDa integral membrane protein with two Ig-like domains in the extracellular region. In polarized epithelial cells, CAR is expressed at the tight junction, where it associates with ZO-1 and plays a role in the barrier to the movement of macromolecules and ions. In the present study, we investigated the expression and localization of CAR in rat kidneys treated with puromycin aminonucleoside (PAN) and in rat kidneys perfused for 15 minutes with protamine sulfate (PS). Both the experimental models have been used to induce tight junctions in podocytes. Ribonuclease protection assay and Western blot analysis revealed a distinct increase of CAR transcript and protein in glomeruli during PAN nephrosis but no increase in glomeruli by PS perfusion. Immunohistochemistry revealed a significant increase in CAR staining intensity along the glomerular capillary wall in PAN nephrosis and after PS perfusion. Immunoelectron microscopy demonstrated in both the models that the immunogold particles for CAR along the capillary wall were found predominantly at close cell-cell contact sites of podocytes but were rarely found at slit diaphragms. In cultured podocytes, CAR was localized at cell-cell contact sites. CAR distribution was identical to that of ZO-1 and different from that of a gap junction protein, connexin43. These findings indicate that CAR is an integral membrane component of tight junction in podocytes and that CAR expression in podocytes is regulated at the transcriptional level and in the redistribution of protein.

Amino Acid Sequence↗
PubMed2004

Transgene of MIF induces podocyte injury and progressive mesangial sclerosis in the mouse kidney.

BACKGROUND: Recent evidence suggests that macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pathogenic role in glomerulonephritis. Renal expression of MIF is up-regulated in infiltrating and intrinsic renal cells, which include glomerular epithelial cells. The aim of the current study was to further clarify the role of MIF produced by podocytes in the process of renal disease. METHODS: We generated transgenic mice carrying a murine MIF cDNA driven by cytomegalovirus enhancer and beta-actin/beta-globin promoter, a hybrid promoter transactivated in podocytes in vivo. RESULTS: MIF expression was markedly up-regulated in podocytes in neonatal and adult transgenic kidneys. A longitudinal study of the MIF transgenic mice demonstrated a progressive matrix increase in mesangium accompanied by collagen IV accumulation, representing no significant glomerular cell hypercellularity. The glomeruli in transgenic kidney were not accompanied by influx of macrophages and T cells at the early stage of disease progression. Although a significant number of the mice showing higher expression of MIF died from renal failure at 8 weeks, most of them survived with significant proteinuria and progressive renal failure. Podocytes of transgenic mice frequently underwent characteristic ultrastructural changes, such as cell flattening, contracted foot processes, and villous transformation. In addition, immunohistochemical expression of synaptopodin, an actin-associated protein distributed in differentiated podocyte foot process, was significantly attenuated in transgenic kidney. CONCLUSION: Our results indicate that podocyte-expressed MIF could induce an injury of podocytes themselves, thereby accelerating the progression of glomerulosclerosis and leading to end-stage renal failure.

Animals↗
PubMed2004

In vivo expression of podocyte slit diaphragm-associated proteins in nephrotic patients with NPHS2 mutation.

BACKGROUND: Mutations in NPHS2, encoding podocin, are a prevalent cause of autosomal-recessive steroid-resistant nephrotic syndrome (SRNS). Podocin is a protein associated with the slit diaphragm that interacts with nephrin and CD2-associated protein (CD2AP) within lipid rafts. METHODS: Using renal biopsies of six patients, we analyzed the in vivo consequences of different types of NPHS2 mutations on (1) the podocyte expression and distribution of podocin using in situ hybridization and immunohistology and (2) the distribution of related podocyte proteins and glomerular extracellular matrix components. RESULTS: In two patients with homozygous 855_856delAA or 419delG mutation, absence of podocyte labeling with the antibodies against the C-terminal domain contrasted with the normal expression of the N-terminal domain of the protein along the glomerular basement membrane (GBM). In patients carrying compound heterozygous mutations or variants (R168S/467_468insT, R138Q/V180M, and R291W/R229Q), or single heterozygous 976_977insA, podocin transcription appeared unchanged but the distribution of the protein was modified. Podocin was restricted to the podocyte body in the patient carrying the R168S/467_468insT mutation whereas strong immunolabeling of the podocyte body was associated with discrete labeling along the GBM in the three others. In all cases, podocin defect was associated with changes in the distribution of nephrin, CD2AP, and alpha-actinin: the proteins were mainly detected in the podocyte body, with mild expression along the GBM. There were no detectable changes in the distribution of other podocyte proteins or glomerular extracellular matrix components. CONCLUSION: NPHS2 mutations result in profound alteration of podocin expression and/or distribution. Secondary changes in the distribution of nephrin, CD2AP, and alpha-actinin are additional evidences for the scaffolding role of podocin in the organization of the slit diaphragm.

Actinin↗
PubMed2005

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

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

Animals↗