Desmin-positive epithelial cells outgrowing from rat encapsulated glomeruli

Decapsulated glomeruli, encapsulated glomeruli and tubular fragments were each selected and cultured in order to identify the origin of polygonal epithelial cells in glomerular outgrowths and to characterize them by double-label immunofluorescence microscopy using antibodies against cytoskeletal proteins. Polygonal cells outgrew from less than 1% of decapsulated glomeruli, 34.8 to 65.1% of encapsulated glomeruli and 62.4 to 79.7% of tubular fragments in culture. These data support the idea that polygonal cells in glomerular culture are derived mainly from parietal epithelial cells of Bowman's capsule but not visceral cells, and indicate that polygonal cells of tubular epithelial origin are also present. Polygonal cells from encapsulated glomeruli consisted of intensely vimentin-positive (IV) cells and weakly vimentin-positive (WV) ones. Most of the IV cells showed various degrees of staining with anti-desmin antibody, and some of them also expressed cytokeratins and alpha-smooth muscle actin. By contrast, all of the WV cells were stained with anti-cytokeratin antibody but not with anti-desmin antibody. Polygonal cells in cultures of tubular fragments were negative for desmin. These findings suggest that parietal cells express desmin in culture, even though they show no desmin staining in kidney sections.     

Characterization of a simian virus 40-transformed human podocyte cell line producing type IV collagen and exhibiting polarized response to atrial natriuretic peptide.

Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.     

Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.     

De novo expression of podocyte proteins in parietal epithelial cells in experimental aging nephropathy

A progressive decrease in podocyte number underlies the development of glomerulosclerosis and reduced kidney function in aging nephropathy. Recent data suggest that under certain disease states, parietal epithelial cells (PECs) begin to express proteins considered specific to podocytes. To determine whether this phenomenon increases in aging kidneys, 4-, 12-, and 20-mo ad libitum-fed and 20-mo calorie-restricted (CR) rats were studied. Single and double immunostaining were performed with antibodies to the PEC protein paired box gene 2 (PAX2) and tight junction protein claudin-1, the podocyte-specific protein Wilms' tumor 1 (WT-1), and the proliferating cell protein (Ki-67). ImageJ software measured Bowman's basement membrane (BBM) length and glomerular tuft area in individual glomeruli from each animal to assess glomerular size. The results showed that in aged ad libitum rats, the decrease in number of podocytes/glomerular tuft area was accompanied by an increase in the number of PECs/BBM length at 12 and 20 mo (P < 0.01 vs. 4 mo). The increase in PEC number was due to proliferation (increase in PAX2/Ki-67 double-positive cells). Aging was accompanied by a progressive increase in the number of glomerular cells double staining for PAX2 and WT-1. In contrast, the control 20-mo-old CR rats had no increase in glomerular size, and podocyte and PEC number were not altered. These results suggest that although the number of PECs and PECs expressing podocyte proteins increase in aging nephropathy, they are likely not sufficient to compensate for the decrease in podocyte number.     

Cellular aspects of rabbit Masugi nephritis V. Ultrastructural cytochemistry of monocytic cells

Ultrastructural cytochemistry for nonspecific esterase (NSE) was performed on normal and nephritic rabbit kidneys. In normal glomeruli distinct reaction deposits appearing as electron-dense granules were present in visceral epithelial cells (podocytes) and epithelial cells of Bowman's capsule, but only a few small or minute deposits were seen in some endothelial and mesangial cells. In acute proliferative glomerulonephritis (Masugi nephritis), many reaction deposits occurred in mononuclear cells accumulating in glomerular tufts which presented the characteristic features of monocytic cells. Macrophages which had migrated into subendothelial space as well as epithelioid cells and multinucleated giant cells, all of which are known to be derived from monocytes, also exhibited the reaction product. The NSE granules in mesangial and endothelial cells were much smaller and fewer in number than those in monocytic cells. The present method may contribute to the more precise differentiation of monocytic cells from mesangial and endothelial cells in proliferative glomerulonephritis.     

NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli

Using a novel approach for analysis of TRPC channel activity, we report here that NSAIDs are involved into regulation of TRPC channels in the podocytes of the freshly isolated decapsulated glomeruli. Fluorescence and electron microscopy techniques confirmed the integrity of podocytes in the glomeruli. Western blotting showed that TRPC1, 3 and 6 are highly expressed in the glomeruli. Single-channel patch clamp analysis revealed cation currents with distinct TRPC properties. This is the first report describing single TRPC-like currents in glomerular podocytes. Furthermore, our data provide a novel mechanism of NSAIDs regulation of TRPC channels, which might be implicated in maintaining the glomerular filtration barrier.     

Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D₃ through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25‐dihydroxyvitamin D₃ analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)‐diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose‐mediated down‐regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose‐induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co‐localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down‐regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ‐diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.     

Podocytes in metanephric organ culture express characteristic in vivo phenotypes

 Podocytes outgrown from isolated glomeruli in vitro have failed to express fully differentiated in vivo phenotypes. In an attempt to determine whether podocytes in metanephric culture accomplish terminal differentiation, as observed in vivo, we investigated expression of their characteristic phenotypic features in rat metanephric organ cultures using immunohistochemistry and electron microscopy. Rat metanephroi were harvested on embryonic day 12.5 and cultured on transmembrane filters for 9 days. Morphological examination revealed two maturation stages when the podocytes resembled those of the S-shaped body stage and maturational stages of glomeruli in vivo. Electron microscopy revealed that, firstly podocytes lost their intercellular contacts and, simultaneously, the tight junctions shifted into close proximity to cell bases, followed by foot process development. Immunohistochemistry demonstrated that the tight junction protein, ZO-1, and specific podocytic markers, pp44, 5-1–6, podocalyxin and vimentin were expressed in a cell maturity-dependent manner, as observed in newborn rat kidneys. Furthermore, glomerular basement membrane components, collagen type IV and laminin, were expressed in the glomerular center. Our findings that cell maturity-dependent expression of structural and functional phenotypes in podocytes in metanephric culture was the same as that observed in developing kidneys in vivo indicate that podocyte differentiation during glomerulogenesis may be operated by an intrinsic property, such as programmed cell fate. Furthermore, these highly differentiated podocytes in vitro may provide clues that will help to establish a podocyte culture system. Accepted: 26 February 1997     

Dynamics of glial fibrillary acidic protein distribution in cultured glomerular podocytes and mesangial cells of the rat kidney

Glial fibrillary acidic protein (GFAP) has recently been shown to be expressed in the glomerular podocytes and mesangial cells (MC) of kidney (Buniatian et al (1998) Biol Cell 90, 53-61). The different localization of GFAP in podocytes and MC has raised the question whether this might reflect specific cellular functions. To address this question, in the present study podocytes and MC in early (2, 3 day-old), prolonged (5, 7 day-old) and late (14, 21 day-old) primary cultures from out-growths of glomerular explants were used. Double-immunolabeling studies demonstrated that podocytes transiently acquire myofibroblastic features, characterized by the expression of smooth muscle alpha-actin (SMAA) and increased perinuclear reaction of GFAP in prolonged cultures. The morphological differentiation of cobblestone-like podocytes into process-bearing cells was followed by loss of the myofibroblastic marker, SMAA, de novo expression of desmin, and distribution of GFAP, vimentin and desmin into the processes. In late culture, GFAP and SMAA were nearly absent from the podocytes which maintained the cobblestone-like morphology. By contrast, the myofibroblastic features gained by MC during prolonged culturing increased with time. A myofibroblast-like cytoskeleton of podocytes and MC similar to that of healthy astrocytes suggest an increased spectrum of functional activities of these cells during the acquisition of myofibroblastic features. In addition, the present study provides a new combination of biochemical and biological features by which podocytes and MC can be distinguished in culture.     

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ～ JR > KE, and of doxorubicin was JR ～ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.     

Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model

Unilateral ureteral obstruction (UUO), employed extensively as a model of progressive renal interstitial fibrosis, results in rapid parenchymal deterioration. Atubular glomeruli are formed in many renal disorders, but their identification has been limited by labor-intensive available techniques. The formation of atubular glomeruli was therefore investigated in adult male mice subjected to complete UUO under general anesthesia. In this species, the urinary pole of Bowman's capsule is normally lined by tall parietal epithelial cells similar to those of the proximal tubule, and both avidly bind Lotus tetragonolobus lectin. Following UUO, these cells became flattened, lost their affinity for Lotus lectin, and no longer generated superoxide (revealed by nitroblue tetrazolium infusion). Based on Lotus lectin staining, stereological measurements, and serial section analysis, over 80% of glomeruli underwent marked transformation after 14 days of UUO. The glomerulotubular junction became stenotic and atrophic due to cell death by apoptosis and autophagy, with concomitant remodeling of Bowman's capsule to form atubular glomeruli. In this degenerative process, transformed epithelial cells sealing the urinary pole expressed α-smooth muscle actin, vimentin, and nestin. Although atubular glomeruli remained perfused, renin immunostaining was markedly increased along afferent arterioles, and associated maculae densae disappeared. Numerous progressive kidney disorders, including diabetic nephropathy, are characterized by the formation of atubular glomeruli. The rapidity with which glomerulotubular junctions degenerate, coupled with Lotus lectin as a marker of glomerular integrity, points to new investigative uses for the model of murine UUO focusing on mechanisms of epithelial cell injury and remodeling in addition to fibrogenesis.     

A scanning electron-microscopic study of the peripolar cell of the rat renal glomerulus

Summary The interior of Bowman's capsules of rat kidneys has been examined by scanning electron microscopy, and a distinctive population of cells around the exposed vascular poles of glomerular tufts were identified. The cells were situated in the annular groove at the root of the glomerulus, between the parietal epithelial cells and the podocytes. These peripolar cells were dendritic cells with long processes embracing the glomerular arterioles. Up to three peripolar cells were present at each vascular pole and they were mainly distributed in the glomeruli of the outer third of the renal cortex. This first detailed study of the surface morphology of the glomerular peripolar cell supports the suggestion that changes in the diameter of the polar region of the glomerular tuft may cause variations in stretching of the cuff of peripolar cells, and hence modulation of their secretory activity.     

The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron

We studied the distribution of the alpha 1-alpha 6 subunits of beta 1 integrins in developing and adult human kidney using a panel of mAbs in indirect immunofluorescence microscopy. Uninduced mesenchyme displayed a diffuse immunoreactivity for only the alpha 1 integrin subunit. At the S-shaped body stage of nephron development, several of the alpha subunits were characteristically expressed in distinct fetal nephron segments, and the pattern was retained also in the adult nephron. Thus, the alpha 1 subunit was characteristically expressed in mesangial and endothelial cells, the alpha 2 in glomerular endothelium and distal tubules, the alpha 3 in podocytes, Bowman's capsule, and distal tubules, and the alpha 6 subunit basally in all tubules, and only transiently in podocytes during development. Unlike the alpha 3 and alpha 6 subunits, the alpha 2 subunit displayed an overall cell surface distribution in distal tubules. It was also distinctly expressed in glomerular endothelia during glomerulogenesis. The beta 4 subunit was expressed only in fetal collecting ducts, and hence the alpha 6 subunit seems to be complexed with the beta 1 rather than beta 4 subunit in human kidney. Of the two fibronectin receptor alpha subunits, alpha 4 and alpha 5, only the latter was expressed, confined to endothelia of developing and adult blood vessels, suggesting that these receptor complexes play a minor role during nephrogenesis. The present results suggest that distinct integrins play a role during differentiation of specific nephron segments. They also indicate that alpha 3 beta 1 and alpha 6 beta 1 integrin complexes may function as basement membrane receptors in podocytes and tubular epithelial cells.     

Nestin expression in adult and developing human kidney.

Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and alpha-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5.     

The ultrastructure of the renal corpuscle of a lizard

The few and small renal corpuscles of the lizard Podarcis (= Lacerta) taurica are composed of a tuft of three to four capillaries (glomerulus), Bowman's capsule and mesangium. The thin interdigitated capillary endothelial cells are, in most regions, in contact with the mesangium. In some regions, however, they rest on a bilaminate basement membrane with an electron-dense lamina densa and a less dense lamina rara. Bowman's capsule is composed of visceral and parietal layers. The epithelial cells (podocytes) of the visceral layer bear trabeculae connected to pedicels with microvilli. The pedicels rest on a bilaminate basement membrane which in some regions has a double-layered densa with connecting bands. Generally, this basement membrane is thicker than that of the capillary endothelial cells. The mesangium is composed mostly of irregular satellite cells with large nuclei and cytoplasmic processes, but also has smaller cells with kidney-shaped nuclei and cytoplasmic processes containing microfilaments. The mesangium cells are embedded in a collagenous matrix which extends to invade the area between the epithelial basement membrane and the capillary endothelium. These observations are discussed in relation to the structure and function of vertebrate renal corpuscles with special reference to the mesangium.     

Claudin-6 localized in tight junctions of rat podocytes

Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.