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PubMed1993

Lectin-binding sites during postnatal differentiation of normal and cystic rabbit renal corpuscles.

Fluorochrome-labeled lectins were used to study the expression of glycoconjugates during the postnatal differentiation of normal and cystic rabbit renal corpuscles. Glomerular cysts (GC) are induced in the rabbit by a single injection of corticoids. The Bowman's capsule of these cysts is exclusively formed of podocytes (parietal podocytes). During normal development, the cell coat of the podocytes is intensely positive for wheat germ agglutinin (WGA) and Maclura pomifera agglutinin (MPA). This reaction decreases considerably during maturation, in parallel with an increase in the number of binding sites masked by terminal sialylation. Throughout the stages studied, the podocyte coat is peanut agglutinin (PNA)-negative, but it becomes intensely positive after neuraminidase treatment. Visceral and parietal podocytes in the glomerular cysts show the same pattern of glycosylation as the normal podocytes. In contrast, normal parietal cells only transiently expressed a weak reactivity to WGA and MPA during the first stages of differentiation, and did not express cryptic binding sites at any stage. The glomerular basement membrane (GBM) is positive to WGA, to succinylated WGA, and to MPA, in all the stages studied. Maturation of the GBM is characterized by expression of cryptic MPA-binding sites, and by a considerable increase in the number of cryptic PNA-binding sites. The basement membrane of the parietal layer of the cystic Bowman's capsule shows the same pattern of glycosylation, despite the fact that this epithelial layer is solely formed of podocytes and lacks endothelial cells. In contrast, the normal parietal basement membrane does not express PNA or MPA cryptic sites at any stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture.

The origin of cobblestone-like polygonal cells (the most numerous in renal glomerular cell culture) remains controversial; they could be either dedifferentiated podocytes or parietal epithelial cells (PECs) of Bowman's capsule. Poor cellular outgrowth from glomeruli devoid of Bowman's capsule (decapsulated glomeruli) hinders podocytes being obtained without contamination of PECs in culture. Since podocytes are easily damaged during the isolation of glomeruli by the conventional sieving method, we devised a gentle isolation method without forced sieving, resulting in substantial numbers of arborized cells growing out from decapsulated glomeruli. The cells were distinctly different from cobblestone-like polygonal cells in their irregular and often arborized shape and extended long cytoplasmic processes that often crossed over adjacent cells. The arborized cells from decapsulated glomeruli showed intense staining for a podocyte-specific marker, podocalyxin, but no staining for markers specific to PECs (pan cadherin), mesangial cells (Thy-1) or endothelial cells (von Willebrand factor, RECA-1), indicating their podocyte origin. Polygonal cells growing out from encapsulated glomeruli were negative for podocalyxin and positive for pan cadherin at the peripheral cell-cell contact. Thus, the cell population from decapsulated glomeruli is distinctly different from that from encapsulated glomeruli, supporting the idea that polygonal cells originate from PECs, although immunocytochemical markers specific to podocytes in vivo such as WT1, synaptopodin, HSP27 and P-31 antigen were expressed significantly in the polygonal cells. Occasionally, large irregular-shaped cells appeared at the periphery of the outgrowths from encapsulated glomeruli. They were similar in shape to the arborized cells from decapsulated glomeruli but were identical in antigenic properties to cobblestone-like polygonal cells and thus may be named "pseudo-arborized cells". We conclude that PECs in culture modulate their phenotype to resemble podocytes.

Animals↗
PubMed2003

CD2-associated protein and glomerular disease.

CONTEXT: Proteinuria is a major cause of progression in renal disease. The glomerular ultrafiltration barrier, containing highly differentiated podocytes, normally restricts protein access to the urine. Patients with urinary protein in the nephrotic range (>3.5 g daily) often have effaced podocyte foot-processes. Slit diaphragms span the gaps between foot processes as a barrier to macromolecules. Nephrin and podocin are slit-diaphragm proteins identified in families with congenital nephrotic syndromes. CD2-associated protein (CD2AP) is an adapter protein originally identified as a novel ligand interacting with the T-cell-adhesion protein CD2. CD2AP knockout (-/-) mice develop a congenital nephrotic syndrome with podocyte foot-process effacements and die at 6 weeks of age from renal failure. CD2AP localises to the slit diaphragm and links nephrin and podocin to phosphoinositide 3-OH kinase; this complex has cell-signalling properties. STARTING POINT: The CD2AP +/- heterozygous mice developed by Jeong Kim and colleagues (Science 2003; 300: 1298-300) are haploinsufficient and develop glomerular changes at 9 months of age with a histological pattern similar to that in human focal segmental glomerulosclerosis. These researchers found that 2 of 30 African-American patients with idiopathic focal segmental glomerulosclerosis had a CD2AP mutation that ablated expression of one allele. WHERE NEXT? Further studies should address the normal distribution of the CD2AP heterozygous mutation in different ethnic populations, because the association with human idiopathic focal segmental glomerulosclerosis could be accidental. Decreased expression of CD2AP in podocytes of individuals with the CD2AP heterozygous mutation would help to understand how the haploinsufficiency translates into increased susceptibility to renal disease. Transfection of podocytes with mutated CD2AP or study of cultured podocytes from CD2AP +/- mice would provide further insight into whether the nephrin-podocin-CD2AP signal-transduction pathway is altered and leads to increased apoptosis of podocytes. Assuming that a decrease in CD2AP attenuates clearance of glomerular immune complexes, patients with other types of idiopathic glomerulonephritis should also have a CD2AP mutation. However, first studies looking at the most common form of glomerulonephritis, IgA nephropathy, have failed to show decreased renal CD2AP expression.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury.

BACKGROUND: Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. METHODS: The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. RESULTS: Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. CONCLUSIONS: Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.

Aging↗
PubMed2002

Proteinuria is preceded by decreased nitric oxide synthesis and prevented by a NO donor in cholesterol-fed rats.

BACKGROUND: Hypercholesterolemia decreases nitric oxide (NO) availability in the circulation and induces podocyte activation and renal injury in rats. It is unknown whether hypercholesterolemia decreases renal NO availability. To dissociate the injury-independent effect of hypercholesterolemia on renal NO availability from secondary effects of proteinuria, increasing concentrations of cholesterol were administered. To determine whether podocyte activation and renal injury were associated with NO deficiency, molsidomine, an exogenous NO donor, was administered to hypercholesterolemic rats. METHODS: Female rats were fed 0, 0.5, 1, or 2% cholesterol for 24 weeks. Rats fed 2% cholesterol were also studied for two weeks. In addition rats fed 0 or 1% cholesterol received 120 mg molsidomine/L drinking water. Renal NO availability was determined by measuring renal NO synthesis and superoxide activity. Podocyte activation was monitored by desmin staining. RESULTS: Hypercholesterolemia dose-dependently increased proteinuria. In the absence of proteinuria, hypercholesterolemia decreased renal NO synthesis (4.2 +/- 0.5 in 0.5% cholesterol vs. 6.8 +/- 0.6 pmol/min/mg protein in controls; P < 0.05). With the exception of neuronal nitric oxide synthase (nNOS), renal NOS protein mass remained unaffected. Renal superoxide activity was dose-dependently increased, thus further lowering renal NO availability. Podocyte injury was dose-dependently increased even in the absence of proteinuria (score, 40 +/- 4 in 0.5% cholesterol vs. 9 +/- 4 in controls; P < 0.05). After two weeks, hypercholesterolemia caused no proteinuria, but did cause some podocyte injury. Renal NOS activity was decreased, but glomerular endothelial NOS (eNOS) staining was unchanged. Molsidomine prevented proteinuria, podocyte activation, and all further renal injury. CONCLUSIONS: Hypercholesterolemia decreases renal NO synthesis, and induces podocyte activation before proteinuria appears. Renal superoxide activity is increased once rats are proteinuric, further lowering renal NO availability. All of these changes can be prevented by a NO donor.

Animals↗
PubMed2002

Inhibitory smads and tgf-Beta signaling in glomerular cells.

Smad6 and Smad7 are inhibitory SMADs with putative functional roles at the intersection of major intracellular signaling networks, including TGF-beta, receptor tyrosine kinase (RTK), JAK/STAT, and NF-kappaB pathways. This study reports differential functional roles and regulation of Smad6 and Smad7 in TGF-beta signaling in renal cells, in murine models of renal disease and in human glomerular diseases. Smad7 is upregulated in podocytes in all examined glomerular diseases (focal segmental glomerulosclerosis [FSGS], minimal-change disease [MCD], membranous nephropathy [MNP], lupus nephritis [LN], and diabetic nephropathy [DN]) with a statistically significant upregulation in "classical" podocyte-diseases such as FSGS and MCD. TGF-beta induces Smad7 synthesis in cultured podocytes and Smad6 synthesis in cultured mesangial cells. Although Smad7 expression inhibited both Smad2- and Smad3-mediated TGF-beta signaling in podocytes, it inhibited only Smad3 but not Smad2 signaling in mesangial cells. In contrast, Smad6 had no effect on TGF-beta/Smad signaling in podocytes and enhanced Smad3 signaling in mesangial cells. These data suggest that Smad7 is activated in injured podocytes in vitro and in human glomerular disease and participates in negative control of TGF-beta/Smad signaling in addition to its pro-apoptotic activity, whereas Smad6 has no role in TGF-beta response and injury in podocytes. In contrast, Smad6 is upregulated in the mesangium in human glomerular diseases and may be involved in functions independent of TGF-beta/Smad signaling. These data indicate an important role for Smad6 and Smad7 in glomerular cells in vivo that could be important for the cell homeostasis in physiologic and pathologic conditions.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Disruption of glomerular cell-cell and cell-matrix interactions in hydrocarbon nephropathy.

Environmental chemicals play an etiological role in greater than 50% of idiopathic glomerular diseases. The present studies were conducted to define mechanisms of renal cell-specific hydrocarbon injury. Female rats were given 10 mg/kg benzo(a)pyrene (BaP) once a week for 16 wk. Progressive elevations in total urinary protein, protein/creatinine ratios, and microalbuminuria were observed in rats treated with BaP for up to 16 wk. The nephropathic response involved early reductions in mesangial cell numbers and fibronectin levels by 8 wk, coupled to transient increases in podocyte cellularity. Changes in podocyte numbers subsided by 16 wk and correlated with rebound increases in mesangial cell numbers and fibronectin levels, along with increased alpha-smooth muscle actin and Cu/Zn superoxide dismutase and fusion of podocyte foot processes. In culture, mesangial cells were more sensitive than podocytes to hydrocarbon injury and expressed higher levels of inducible aryl hydrocarbon hydroxylase activity. Naïve mesangial cells exerted a strong inhibitory influence on podocyte proliferation under both direct and indirect coculture conditions, and this response involved a mesangial cell-derived matrix that selectively inhibited podocyte proliferation. These findings indicate that hydrocarbon nephropathy in rats involves disruption of glomerular cell-cell and cell-matrix interactions mediated by deposition of a mesangial cell-derived growth-inhibitory matrix that regulates podocyte proliferation.

Animals↗
PubMed2000

Identification and characterization of a glomerular-specific promoter from the human nephrin gene.

Podocytes are highly specialized cells that make up a major portion of the glomerular filtration barrier in the kidney. They are also believed to play a pivotal role in the progression of chronic renal disease due to diverse causes that include diabetes (3, 20, 24) and aging (1, 7). Despite the importance of podocytes for kidney function and disease, studies of this cell type have been hindered due to a lack of model systems. Recently, the gene responsible for congenital Finnish nephropathy was identified and named nephrin (13). Nephrin expression is restricted to slit diaphragms of podocytes (11, 30). Infants with congenital Finnish nephropathy develop massive proteinuria and subsequent kidney failure due to podocyte injury. We have identified a 1.25-kb DNA fragment from the human nephrin promoter and 5'-flanking region that is capable of directing podocyte-specific expression in transgenic mice; this represents the first glomerular-specific promoter to be identified. Use of this transgene will facilitate studies of the podocyte in vivo and allow the identification of transacting factors that are required for podocyte-specific expression.

Base Sequence↗
PubMed2003

Hemoperfusion with polymyxin B-immobilized fiber in septic patients with methicillin-resistant Staphylococcus aureus-associated glomerulonephritis.

BACKGROUND/AIMS: We investigated whether urinary podocytes are present in septic patients with methicillin-resistant Staphylococcus aureus (MRSA)-associated glomerulonephritis and whether polymyxin B-immobilized fiber (PMX-F) treatment affects proteinuria and urinary podocyte excretion in these patients. METHODS: Twenty septic patients with MRSA-associated glomerulonephritis (mean age: 63.7 years) and 80 septic patients whose MRSA infection was not followed by glomerulonephritis (mean age: 60.5 years) were included in this study. All septic patients were treated with fosfomycin sodium, beta-lactams, arbekacin sulfate, and teicoplanin, or a combination of these. Twenty septic patients with MRSA-associated glomerulonephritis were randomly assigned to one of two treatments: PMX-F treatment (group A, n = 10) and conventional treatment (group B, n = 10). PMX-F treatment was repeated twice. RESULTS: Urinary podocytes and urinary protein excretion were not detected in MRSA septic patients without glomerulonephritis. However, urinary podocytes (1.7 +/- 0.6 cells/ml) and proteinuria (2.6 +/- 0.6 g/d) were detected in the 20 septic patients with MRSA-associated glomerulonephritis. Plasma endotoxin levels were decreased from 13.6 +/- 4.6 pg/ml to 6.6 +/- 2.2 pg/ml (p < 0.05) in group A. Levels in group B, however, showed little difference after treatment. Urinary podocytes were reduced in group A (from 1.8 +/- 0.6 cells/ml to 0.4 +/- 0.2 cells/ml, p < 0.01) as was urinary protein excretion (from 3.0 +/- 0.5 g/d to 0.8 +/- 0.4 g/d, p < 0.01) but urinary podocytes and protein excretion levels showed little difference after treatment in group B. CONCLUSION: PMX-F treatment may be effective in reducing urinary protein and urinary podocyte excretion in septic patients with MRSA-associated glomerulonephritis.

Endotoxins↗
PubMed2002

Glomerular-specific gene excision in vivo.

Podocytes (glomerular visceral epithelial cells) are highly specialized cells that are found in the renal glomerulus and make up a major portion of the filtration barrier between the blood and urinary spaces. Recently, the identification of a number of genes responsible for both autosomal dominant and recessive forms of human nephrotic syndrome has provided insight into a number of molecules responsible for unique features of the podocyte such as the slit diaphragms. Despite these major advances in our understanding of podocyte biology, the function of many genes expressed in the podocyte remains unknown. Targeted gene disruption using homologous recombination in murine embryonic stem cells (ES cells) is a powerful tool to determine the biologic function of genes in vivo. However, resulting embryonic lethal or pleiotropic phenotypes often preclude the analysis of genes in specific renal cell types. To overcome this problem, a glomerular-specific Cre-recombinase transgenic murine line under the control of the Nphs1 (nephrin) promoter (Neph-Cre) was generated. This article reports successful Cre-mediated excision of a 'floxed' transgene specifically in podocytes in vivo. This murine founder line represents a powerful new tool for the manipulation of the expression of genes in podocytes and will provide valuable insight into podocyte biology in the whole animal.

Animals↗
PubMed1990

Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium and podocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappeared from glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomeruli for hours or days. The sites of binding of the MAbs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (anti-DPPIV) bound to both the luminal endothelial membrane and the apical and basal domains of podocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal domain, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and micro-domains of the endothelium and podocyte are accessible to circulating antibodies; and 3) the fate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others.

Animals↗
PubMed2006

An inducible mouse model for PAX2-dependent glomerular disease: insights into a complex pathogenesis.

Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2006

Large-scale identification of genes implicated in kidney glomerulus development and function.

To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large-scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non-glomerular kidney tissue, FACS-sorted podocytes and brain capillaries, which identified over 300 glomerular cell-enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus-enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

Animals↗
PubMed2006

Glomerular sclerosis in kidneys with congenital nephrotic syndrome (NPHS1).

Congenital nephrotic syndrome of the Finnish type (NPHS1) is a rare genetic disease caused by mutations in the NPHS1 gene encoding a major podocyte slit-diaphragm protein, nephrin. Patients with NPHS1 have severe nephrotic syndrome from birth and develop renal fibrosis in early childhood. In this work, we studied the development of glomerular sclerosis in kidneys removed from 4- to 44-month-old NPHS1 patients. The pathological lesions and expression of glomerular cell markers were studied in nephrectomized NPHS1 and control kidneys using light and electron microscopy and immunohistochemistry. An analysis of 1528 glomeruli from 20 patients revealed progressive mesangial sclerosis and capillary obliteration. Although few inflammatory cells were detected in the mesangial area, paraglomerular inflammation and fibrosis was common. The podocytes showed severe ultrastructural changes and hypertrophy with the upregulation of cyclins A and D1. Podocyte proliferation, however, was rare. Apoptosis was hardly detected and the expression of antiapoptotic B-cell lymphoma-2 and proapoptotic p53 were comparable to controls. Moderate amounts of podocytes were secreted into the urine of NPHS1 patients. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. The results indicate that, in NPHS1 kidneys, the damaged podocytes induce progressive mesangial expansion and capillary obliteration. Podocyte depletion, glomerular tuft adhesion, and misdirected filtration, however, seem to play a minor role in the nephron destruction.

Apoptosis↗
PubMed2006

Update on HIV-associated nephropathy.

PURPOSE OF REVIEW: HIV-associated nephropathy is characterized by a constellation of pathologic findings including a collapsing glomerulopathy, tubular dilatation, and interstitial infiltration with leukocytes. This review summarizes some of the recent advances in our understanding of the gene products and signaling pathways that contribute to the pathogenesis of HIV-associated nephropathy. RECENT FINDINGS: Podocytes infected with HIV-associated nephropathy exhibit podocyte proliferation and de-differentiation. Restriction of HIV-1 transgene expression to the podocyte in a murine model supports the belief that podocyte infection is pivotal to the development of the disease. Recent studies have provided compelling in-vitro and in-vivo evidence that expression of the HIV-1 accessory gene nef is critical in altering the phenotype of mature podocytes and causing injury to these cells. An in-vitro study suggests that nef's effects in the podocyte appear to be mediated through Src kinase-dependent activation of the signal transducer and activator of transcription 3 and mitogen-activated protein kinase 1,2 signaling pathways. SUMMARY: Recent evidence demonstrates that the viral protein nef plays a critical role in the development of HIV-associated nephropathy and provides a foundation for developing new therapeutic strategies for patients afflicted with this disease.

AIDS-Associated Nephropathy↗
PubMed2007

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.

Aborted Fetus↗
PubMed2005

NF-kappaB regulates Fas-mediated apoptosis in HIV-associated nephropathy.

Renal parenchymal injury in HIV-associated nephropathy (HIVAN) is characterized by epithelial proliferation, dedifferentiation, and apoptosis along the entire length of the nephron. Although apoptotic cell death in HIVAN has been well documented, the mechanism for HIV-induced apoptosis is poorly understood. Whether the epithelial apoptosis in HIVAN is mediated by NF-kappaB-activated Fas ligand expression was investigated here. In human HIVAN and HIV-1 transgenic mouse kidney specimens, the expression of Fas receptor and ligand proteins were markedly upregulated on epithelium in diseased glomerular and tubulointerstitial compartments when compared with normal. Podocyte cell lines that were derived from HIV-1 transgenic mice showed a similar upregulation of Fas receptor expression and de novo expression of Fas ligand by semiquantitative reverse transcription-PCR and Western blotting. In cultured podocytes, cross-linking of the Fas receptor to mimic ligand binding induced caspase 8 activity and apoptosis in both normal and HIVAN podocytes. Because constitutive NF-kappaB activity has been demonstrated in HIVAN epithelia, evidence for transcriptional control of the Fas ligand expression by NF-kappaB was sought. With the use of cultured podocytes, expression of a Fas ligand promoter reporter plasmid was higher in HIVAN podocytes, indicating increased transcriptional activity. In addition, chromatin immunoprecipitation assays were performed to demonstrate that p65-containing (RelA) complexes bound the Fas ligand promoter and that suppression of activated NF-kappaB with a peptide inhibitor could reduce the expression of Fas ligand mRNA in HIVAN podocytes. These results suggest that NF-kappaB may regulate Fas-mediated apoptosis in HIVAN by controlling the expression of Fas ligand in renal epithelium.

AIDS-Associated Nephropathy↗
PubMed2005

Glomerular permeability is altered by loss of P0, a myelin protein expressed in glomerular epithelial cells.

The myelin protein 0 (MPZ or P0) is a transmembrane glycoprotein that represents the most abundant myelin component. Mutations in the P0 gene are associated with one form of autosomal dominant demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1B (CMT1B). Because CMT1 may be associated with renal involvement, mostly focal segmental glomerulosclerosis, we hypothesized that P0 could be expressed in the kidney. P0 mRNA was detected by reverse transcriptase-PCR in the human and mouse renal cortex. P0 transcripts were identified by in situ hybridization at different stages of the mouse kidney development, especially in embryonic structures that give rise to the glomerulus. P0 protein was also detected by Western blot in human and rat glomerular extracts and in a human podocyte cell line using a monoclonal anti-P0 antibody. Immunofluorescence studies on human kidney sections showed that the podocytes were intensely labeled. Immunogold electron microscopy disclosed a predominant staining of the membranes of intracellular vesicles in podocytes. P0 was also detected in the podocyte cell membrane, including at the foot processes. P0(-/-) mice exhibited mild growth retardation and demyelinating neuropathy similar to the one observed in patients with CMT1B. They also presented mild albuminuria, without significant ultrastructural change of the glomerular basement membrane or the podocytes. These results demonstrate that P0, the major myelin protein, is also expressed during nephrogenesis and in mature kidney, mostly in podocytes. They suggest that P0 gene mutations might be involved in renal diseases.

Adult↗