Mutant Podocytes Research Articles

Engineered human iPS cell models reveal altered podocytogenesis and glomerular capillary wall in CHD-associated SMAD2 mutations.

Early developmental programming involves extensive cell lineage diversification through shared molecular signaling networks. Clinical observations of congenital heart disease (CHD) patients carrying SMAD2 genetic variants revealed correlations with multi-organ impairments at the developmental and functional levels. For example, many CHD patients present with glomerulosclerosis, periglomerular fibrosis, and albuminuria. Still, it remains largely unknown whether SMAD2 variants associated with CHD can directly alter kidney cell fate, tissue patterning, and organ-level function. To address this question, we engineered human iPS cells (iPSCs) and organ-on-a-chip systems to uncover the role of pathogenic SMAD2 variants in kidney podocytogenesis. Our results show that abrogation of SMAD2 causes altered patterning of the mesoderm and intermediate mesoderm (IM) cell lineages, which give rise to nearly all kidney cell types. Upon further differentiation of IM cells, the mutant podocytes failed to develop arborizations and interdigitations. A reconstituted glomerulus-on-a-chip platform exhibited significant proteinuria as clinically observed in glomerulopathies. This study implicates CHD-associated SMAD2 mutations in kidney tissue malformation and provides opportunities for therapeutic discovery in the future.

#718 N-Ethylmaleimide Sensitive Factor (NSF) is essential for maintaining podocyte focal adhesion

Abstract Background and Aims Cell-matrix adhesion molecules in podocytes play a critical role in attaching these cells to the glomerular basement membrane, ensuring the proper functioning of the glomerular filtration barrier. Previous studies have shown that patients with proteinuric glomerular diseases often have higher levels of podocyturia. This increase is notably linked to a reduction in focal adhesion proteins, especially in cases of focal segmental glomerulosclerosis. Our study aims to explore the regulation of these focal adhesion molecules in podocytes. Method Doxycycline (Dox) inducible podocyte-specific Tln1 knockout mice (Tln1fl/fl Nphs2-rtTA TetO-Cre (Tln1 KO)) were used as the model for podocyte injury. These mice are genetically modified to lack Talin 1, a key protein in focal adhesion. The absence of Talin 1 in these mice leads to the development of pathological changes akin to focal segmental glomerulosclerosis, characterized by extensive proteinuria, thereby providing a valuable model for understanding this kidney disease (J Clin Invest. 2019 1;129(3):1295-13). As Tln1 KO mice 10 days after Dox induction (Tln1 KO 10 d) showed proteinuria without elevated serum creatinine, these mice were considered as the early phase of podocyte injury. Single-cell RNA sequencing (scRNAseq) was performed using these mice kidney samples, followed by differentially expressed genes (DEG) analyses. Podocyte-specific Nsf KO (Nphs2-Cre Rosa-DTRflox Nsf fl/fl (Nsf cKO)) mice were generated based on the result of the DEG analyses and the effect of NSF on focal adhesion was examined using primary podocyte culture. Results Nsf was identified as one of the most significantly down-regulated DEGs in the podocytes ofTln1 KO 10d mice. Further analysis of public scRNAseq data revealed a decrease in Nsf mRNA expression levels also in lipopolysaccharide-treated mouse podocytes. Notably, proteinuria was observed in Nsf conditional knockout (cKO) mice as early as 2 weeks of age, with all Nsf cKO mice succumbing to end-stage kidney disease by 6 weeks post-birth. Examination of isolated mutant podocytes showed a marked reduction in cell spreading. Additionally, a significant decrease in Integrin β1 expression was observed in primary podocytes of Nsf cKO mice, as evidenced by immunoblot analysis. In vivo studies using transmission electron microscopy revealed endoplasmic reticulum (ER) expansion in the podocytes of Nsf cKO mice. Immunofluorescence staining indicated a substantial increase in the ER and ER-Golgi intermediate compartment (ERGIC) in isolated Nsf cKO podocytes. These findings suggest that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi protein transport, subsequently impairing the trafficking of focal adhesion proteins. Conclusion This study provides new insights into the molecular mechanisms underlying podocyte injury and the progression of kidney diseases. NSF plays a pivotal role in maintaining podocyte focal adhesion.

A bigenic mouse model of FSGS reveals perturbed pathways in podocytes, mesangial cells and endothelial cells

Focal segmental glomerulosclerosis is a major cause of end stage renal disease. Many patients prove unresponsive to available therapies. An improved understanding of the molecular basis of the disease process could provide insights leading to novel therapeutic approaches. In this study we carried out an RNA-seq analysis of the altered gene expression patterns of podocytes, mesangial cells and glomerular endothelial cells of the bigenic Cd2ap+/-, Fyn-/- mutant mouse model of FSGS. In the podocytes we observed upregulation of many genes related to the Tgfβ family/pathway, including Gdnf, Tgfβ1, Tgfβ2, Snai2, Vegfb, Bmp4, and Tnc. The mutant podocytes also showed upregulation of Acta2, a marker of smooth muscle and associated with myofibroblasts, which are implicated in driving fibrosis. GO analysis of the podocyte upregulated genes identified elevated protein kinase activity, increased expression of growth factors, and negative regulation of cell adhesion, perhaps related to the observed podocyte loss. Both podocytes and mesangial cells showed strong upregulation of aldehyde dehydrogenase genes involved in the synthesis of retinoic acid. Similarly, the Cd2ap+/-, Fyn-/- mesangial cells, as well as podocytes in other genetic models, and the glomeruli of human FSGS patients, all show upregulation of the serine protease Prss23, with the common thread suggesting important functionality. Another gene with strong upregulation in the Cd2ap+/-, Fyn-/- mutant mesangial cells as well as multiple other mutant mouse models of FSGS was thrombospondin, which activates the secreted inactive form of Tgfβ. The Cd2ap+/-, Fyn-/- mutant endothelial cells showed elevated expression of genes involved in cell proliferation, angioblast migration, angiogenesis, and neovasculature, all consistent with the formation of new blood vessels in the diseased glomerulus. The resulting global definition of the perturbed molecular pathways in the three major cell types of the mutant glomerulus provide deeper understanding of the molecular pathogenic pathways.

402 Variable effects of the ITGA3R274Q on epithelial keratinocytes and kidney epithelial cells, insights into genotype-phenotype correlations in ILNEB

Interstitial lung disease, nephrotic syndrome and epidermolysis bullosa (ILNEB) is a rare autosomal recessive disorder, which is caused by mutations in ITGA3, encoding the integrin a3 subunit. ILNEB keratinocytes lacking integrin a3 (A3-) demonstrate reduced adhesion to own extracellular matrix and disturbed homeostasis of the cellular milieu. Here, we studied the molecular consequences of the ITGA3 mutation c.821G>A, p.R274Q, which has been reported in patients with pulmonary and cutaneous involvement, but without renal disease. Using site-directed mutagenesis and the retroviral system we cloned the missense mutation ITGA3R274Q or the full-length normal ITGA3 cDNA in A3- keratinocytes and in the human podocyte cell line AB8/13 (Po) in which ITGA3 was stably knocked out using CRISPR-Cas9 (Po-KO). Functional assays were performed to address why ITGA3R274Q affects keratinocytes but has no apparent effect on podocytes. We found that in keratinocytes, substitution of arginine 274 with a glutamine residue affects the furin cleavage site (aa745R and aa746Q) leading to about 10.5 % reduction in formation of ITGA3 heavy and light chains. The mutant protein ITGA3R274Q accumulated in the endoplasm reticulum, and only 50 % of the mature ITGA3R274Q reached the cell surface. Interestingly, no abnormal ITGA3R274Q cleavage was observed in mutant podocytes. Moreover, the cell morphology and ITGA3R274Q cell surface expression were not altered in Po-KO-A3R274Q. With these novel findings we conclude that ITGA3R274Q has a variable effect on epidermal keratinocytes and podocytes. The results show that 50 % of mutant ITGA3R274Q on surface of keratinocytes is not sufficient for normal cell function and leads to a skin phenotype. ITGA3 processing seems to differ in keratinocytes and podocytes. These genotype-phenotype correlations extend the spectrum of non-lethal ITGA3-associated disorders.

Organoids from Nephrotic Disease-Derived iPSCs Identify Impaired NEPHRIN Localization and Slit Diaphragm Formation in Kidney Podocytes

SummaryMutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. However, methods for SD reconstitution have been unavailable, thereby limiting studies in the field. In the present study, we established human induced pluripotent stem cells (iPSCs) from a patient with an NPHS1 missense mutation, and reproduced the SD formation process using iPSC-derived kidney organoids. The mutant NEPHRIN failed to become localized on the cell surface for pre-SD domain formation in the induced podocytes. Upon transplantation, the mutant podocytes developed foot processes, but exhibited impaired SD formation. Genetic correction of the single amino acid mutation restored NEPHRIN localization and phosphorylation, colocalization of other SD-associated proteins, and SD formation. Thus, these kidney organoids from patient-derived iPSCs identified SD abnormalities in the podocytes at the initial phase of congenital nephrotic disease.

Mutant Actn4 alters the distribution of intracellular stress within podocytes and contributes to their mechanical failure in response to stretch

Alpha‐actinin‐4 (ACTN4 in humans, Actn4 in mice) crosslinks actin filaments in podocytes, providing them mechanical resilience as they help maintain the glomerular filtration barrier. Although human disease‐causing mutant ACTN4 leads to brittle reconstituted actin networks in vitro, how this affects the ability of podocytes to withstand the external mechanical challenges within the glomerulus remains unknown. We studied primary podocytes isolated from wild‐type (WT) and disease‐causing mutant Actn4 knock‐in mice. We previously reported that, after transient stretch, WT cells recovered their baseline contraction and actin cytoskeleton breakages, whereas mutant podocytes developed irrecoverable reductions in their contraction and irreparable disruptions in their cytoskeletons. We now report that after stretch, when challenged in a centrifugal detachment assay, mutant podocytes (11/23) had a higher probability of detachment than WT (2/19) (odds ratio =7.42, 95% CI 1.27, 80.75; p=0.017). Through an analysis of baseline biophysical properties, we found that the actin filaments of mutant Actn4 podocytes displayed a more uniform distribution of orientation (aligned more parallel to each other) compared with the filaments of WT podocytes (which displayed more random orientation). We then computed baseline intracellular stress in terms of two constituent components: the magnitude of mean principal stress as tension (σ), and the orientation of first principal stress as principal orientation (θ). The spatial distribution of intracellular stress within the podocytes was measured using the autocorrelation function C(r). The distance over which the C(r) decays to a value of 0.5 was extrapolated as a singular value from the autocorrelation curves and is termed correlation length, or r1/2. The greater the r1/2 value, the greater the distance over which intracellular stress maintains uniformity. We found that mutant podocytes displayed a more uniform distribution of intracellular stress across the cell, both in terms of magnitude (r1/2 σ mean ± SE: mutant 18.0 ± 0.8 μm vs. WT 13.4 ± 0.8 μm) and orientation (r1/2 θ mean ± SE: mutant 34.7 ± 2.2 μm vs. WT 25.9 ± 2.3 μm). Moreover, the more uniform the intracellular stress was distributed at baseline, the less likely mutant podocytes recovered their baseline contraction after periodic stretch. Taken together, our findings provide novel insight into the mechanism by which ACTN4 mutation alters the alignment of the cytoskeleton and impairs the resiliency of the podocyte, leaving it vulnerable to mechanical stretch and more prone to detachment in its glomerular environment.Support or Funding InformationR37DK059588 and T32DK007199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury

Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics invitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.

A novel mechanism for enhanced neutrophil recruitment, retention, and podocyte damage in immune complex-mediated glomerular disease.

Abstract Glomerulonephritis (GN) is characterized by immune complex deposition and ensuing inflammation within the glomerulus of the kidney. Frank J. Dixon, a pioneer in immunologic kidney disease research, reported in 1965 that injection of nephrotoxic antibodies in rats and rabbits resulted in proteinuria and glomerular accumulation of neutrophils, and that depletion of circulating neutrophils reduced this immune-mediated glomerular damage and proteinuria. In the decades since, studies have confirmed a role for glomerular neutrophil accumulation in human GN, but the mechanisms for neutrophil recruitment and neutrophil-directed injury have not been resolved. We previously reported genetic variants for a NF-κB inhibitor ABIN1 as risks for GN. Others also report that glomerular ABIN1 gene expression is altered in GN in mice and humans. Here we present that transgenic disruption of ABIN1 function results in exacerbated podocyte injury, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in a mouse model of acute antibody-mediated GN. Moreover, a novel inhibitor of neutrophil granule release (SNAP23 peptide) also attenuates podocyte injury in this model. These in vivo findings are supported by in vitro experiments showing that secretome from homologous ABIN1 mutant podocytes activates neutrophil chemotaxis and granule release and neutrophil granule contents specifically disrupts cytoskeletal organization of ABIN1 mutant podocytes. These studies unfold a role for ABIN1 dysfunction in a novel neutrophil-mediated mechanism of podocyte injury in GN and presents inhibition of neutrophil granule release as a promising novel therapeutic direction for kidney inflammation.

Disease-causing mutations of RhoGDIα induce Rac1 hyperactivation in podocytes

ABSTRACTNephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidney's glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.

Pathogenic pathways are activated in each major cell type of the glomerulus in the Cd2ap mutant mouse model of focal segmental glomerulosclerosis.

BackgroundMutations in several genes expressed in podocytes, including Cd2ap, have been associated with focal segmental glomerulosclerosis in humans. Mutant mouse models provide an opportunity to better understand the molecular pathology that drives these diseases.MethodsIn this report we use a battery of transgenic-GFP mice to facilitate the purification of all three major cell types of the glomerulus from Cd2ap mutant mice. Both microarrays and RNA-seq were used to characterize the gene expression profiles of the podocytes, mesangial cells and endothelial cells, providing a global dual platform cross-validating dataset.ResultsThe mesangial cells showed increased expression of profibrotic factors, including thrombospondin, Tgfb2 and Tgfb3, as well as the angiogenesis factor Vegf. They also showed upregulation of protective genes, including Aldh1a2, involved in retinoic acid synthesis and Decorin, a Tgfb antagonist. Of interest, the mesangial cells also showed significant expression of Wt1, which has generally been considered podocyte specific. The Cd2ap mutant podocytes showed upregulation of proteases as well as genes involved in muscle and vasculature development and showed a very strong gene expression signature indicating programmed cell death. Endothelial cells showed increased expression of the leukocyte adhesion associated factors Vcam1 and Sele, as well as Midkine (promoting angiogenesis), endothelin and many genes responsive to cytokines and interferons.ConclusionsThis study provides a comprehensive analysis of the changing properties of the three cell types of the glomerulus in Cd2ap mutants, identifying activated and repressed pathways and responsible genes, thereby delivering a deeper molecular understanding of this genetic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-015-0063-z) contains supplementary material, which is available to authorized users.

N‐sulfation of Cell Surface Heparan Sulfate Glycosaminoglycans is Critical for Podocyte‐Matrix Interactions

Podocytes adhere to the glomerular basement membrane via cell surface receptors which include heparan sulfate (HS) proteoglycans (PG). Podocytes unable to assemble HS glycosaminoglycan chains have defective cell attachment and spreading, and cytoskeletal disruption in vitro and in vivo. The current report explores the importance of heparan sulfation in regulating podocyte cell‐matrix interactions, using an immortalized podocyte cell line having the genomic deletion of N‐deacetylase/ N‐sulfotransferase 1 (NDST1), the enzyme responsible for N‐sulfation of HS. Compared to wild type podocytes, mutant podocytes attached, spread and migrated less efficiently in assays than control cells. In the process of cell‐matrix interaction, the mutant cells showed decreased clustering of syndecan 4, the cell surface HSPG involved in focal adhesion formation, and decreased recruitment of PKCα, α‐actinin 4, vinculin, and pFAK to focal adhesions. Cell surface integrin expression and integrin activation/signaling was significantly diminished in the mutant cells. These results serve to highlight the critical role of HS N‐sulfation in regulating the ability of podocytes to interact with the extracellular matrix.Grant Funding Source: NIDDK RO1‐DK07786

Podocyte culture: Tricks of the trade

Podocytes (glomerular epithelial cells) lie on the urinary aspect of the glomerular capillary and play a key role in the selective filter that underlies kidney function. They are injured in various forms of renal disease: the extents of this injury and its reversibility have major implications for treatment and prognosis. Until recently, podocytes were difficult to study in vitro because of a previous lack of techniques for obtaining differentiated cells in quantities adequate for research. In recent years, this problem has been solved for rodent and human podocytes and there has been an explosion of research using cultured cells. These authors have led the development and characterization of human podocyte cell lines and in this article describe the methods that have allowed them to do this.

355 A Novel Role for Cd2Ap in Normal Podocyte Differentiation

Background: CD2AP is a multifunctional adaptor molecule, binding other key podocyte proteins within the slit-diaphragm complex. Methods: The cellular phenotype of conditionally immortalized wild-type (WT) and CD2AP mutant podocytes was compared by light microscopy. Immunoflourescence and western blot was then used to examine the expression of characteristic podocyte markers nephrin, podocin, CD2AP, PAX2 WT1 as well as WTIP and mesenchymal markers fibronectin and α-SMA in CD2AP mutant podocytes. To investigate differences in localisation of WTIP further, nuclear and cytoplasmic fractions were isolated from each cell line and nuclear/cytoplasmic expression ratios compared. Results: In contrast to WT podocytes, CD2AP mutant demonstrated spindle shaped fibroblast like morphology similar to WT1 mutatant podocytes and were unable to form recognizable cell-cell contacts. Although nephrin, CD2AP podocin and WT1 were expressed at comparable levels in both cell lines, mesenchymal markers fibronectin and α-SMA were significantly overexpressed in CD2AP mutants compared with WT. Western blot and immunoflourescence data showed that PAX-2 was also upregulated in CD2AP mutant cells in keeping with a WT1-related defect. Immunofluorescence staining confirmed that WTIP co-localized at the cell membrane in WT podocytes, whereas in mutant cells WTIP expression was primarily nuclear. This observation was confirmed by a significant increase. (p=0.03) in the nuclear/cytoplasmic ratio of WTIP expression in CD2AP mutants. Conclusion: a role for CD2AP in maintainance of the normal differentiated podocyte phenotype. This may be partly mediated through WT1. Our findings have clear implications for a novel role for CD2AP in development and progression of glomerular disease.

Actin-depolymerizing Factor Cofilin-1 Is Necessary in Maintaining Mature Podocyte Architecture

Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.

Deletion of Von Hippel-Lindau in Glomerular Podocytes Results in Glomerular Basement Membrane Thickening, Ectopic Subepithelial Deposition of Collagen α1α2α1(IV), Expression of Neuroglobin, and Proteinuria

Vascular endothelial growth factor, which is critical for blood vessel formation, is regulated by hypoxia inducible transcription factors (HIFs). A component of the E3 ubiquitin ligase complex, von Hippel-Lindau (VHL) facilitates oxygen-dependent polyubiquitination and proteasomal degradation of HIFalpha subunits. Hypothesizing that deletion of podocyte VHL would result in HIFalpha hyperstabilization, we crossed podocin promoter-Cre transgenic mice, which express Cre recombinase in podocytes beginning at the capillary loop stage of glomerular development, with floxed VHL mice. Vascular patterning and glomerular development appeared unaltered in progeny lacking podocyte VHL. However, urinalysis showed increased albumin excretion by 4 weeks when compared with wild-type littermates with several sever cases (>1000 microg/ml). Many glomerular ultrastructural changes were seen in mutants, including focal subendothelial delamination and widespread podocyte foot process broadening, and glomerular basement membranes (GBMs) were significantly thicker in 16-week-old mutants compared with controls. Moreover, immunoelectron microscopy showed ectopic deposition of collagen alpha1alpha2alpha1(IV) in GBM humps beneath podocytes. Significant increases in the number of Ki-67-positive mesangial cells were also found, but glomerular WT1 expression was significantly decreased, signifying podocyte death and/or de-differentiation. Indeed, expression profiling of mutant glomeruli suggested a negative regulatory feedback loop involving the HIFalpha prolyl hydroxylase, Egln3. In addition, the brain oxygen-binding protein, Neuroglobin, was induced in mutant podocytes. We conclude that podocyte VHL is required for normal maintenance of podocytes, GBM composition and ultrastructure, and glomerular barrier properties.

A murine model of Denys–Drash syndrome reveals novel transcriptional targets of WT1 in podocytes

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Podocyte-Specific Deletion of Dicer Alters Cytoskeletal Dynamics and Causes Glomerular Disease

MicroRNAs (miRNAs) regulate gene expression by binding the 3' untranslated region of mRNAs. To define their role in glomerular function, miRNA biogenesis was disrupted in mouse podocytes using a conditional Dicer allele. Mutant mice developed proteinuria by 3 wk after birth and progressed rapidly to end-stage kidney disease. Podocyte pathology included effacement, vacuolization, and hypertrophy with crescent formation. Despite normal expression of WT1, podocytes underwent dedifferentiation, exemplified by cytoskeletal disruption with early transcriptional downregulation of synaptopodin. These abnormalities differed from Cd2ap(-/-) mice, indicating they were not a general consequence of glomerular disease. Glomerular labeling of ezrin, moesin, and gelsolin was altered at 3 wk, but expression of nestin and alpha-actinin was unchanged. Abnormal cell proliferation or apoptosis was not responsible for the glomerular injury. Mutant podocytes were incapable of synthesizing mature miRNA, as revealed by their loss of miR-30a. In contrast, expression of glomerular endothelial and mesangial cell miRNAs (miR-126 and miR-145, respectively) was unchanged. These findings demonstrate a critical role for miRNA in glomerular function and suggest a pathway that may participate in the pathogenesis of kidney diseases of podocyte origin. The unique architecture of podocytes may make them especially susceptible to cytoskeletal alterations initiated by aberrant miRNA dynamics.

Partial Rescue of Glomerular Laminin α5 Mutations by Wild-Type Endothelia Produce Hybrid Glomeruli

Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5.

Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis.

Denys-Drash syndrome (DDS) is caused by dominant mutations of the Wilms' tumour suppressor gene, WT1, and characterized by a nephropathy involving diffuse mesangial sclerosis, male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that heterozygosity for the Wt1tmT396 mutation induces DDS in heterozygous and chimeric (Wt1tmT396/+<-->+/+) mice. In the present study, the fate of Wt1 mutant cells in chimeric kidneys was assessed by in situ marker analysis, and immunocytochemistry was used to re-examine the claim that glomerulosclerosis (GS) is caused by loss of WT1 and persistent Pax-2 expression by podocytes. Wt1 mutant cells colonized glomeruli efficiently, including podocytes, but some sclerotic glomeruli contained no detectable Wt1 mutant cells. The development of GS was preceded by widespread loss of ZO-1 signal in podocytes (even in kidneys where <5% of glomeruli contained Wt1 mutant podocytes), increased intra-renal renin expression, and de novo podocyte TGF-beta1 expression, but not podocyte Pax-2 expression or loss of WT1, synaptopodin, alpha-actinin-4 or nephrin expression. However, podocytes in partially sclerotic glomeruli that still expressed WT1 at high levels showed reduced vimentin expression, cell cycle re-entry, and re-expressed desmin, cytokeratin and Pax-2. The results suggest that: (i) GS is not due to loss of WT1 expression by podocytes; (ii) podocyte Pax-2 expression reflects re-expression rather than persistent expression, and is the consequence of GS; (iii) GS is mediated systemically and the mechanism involves activation of the renin-angiotensin system; and (iv) podocytes undergo typical maturational changes but subsequently de-differentiate and revert to an immature phenotype during disease progression.

High mortality in lower salaried Norwegian men: the healthy worker effect?

This study aimed to examine whether the high mortality in lower salaried men in Norway was related to an inflow into this group of unskilled workers with high mortality. Individual information on occupation was derived from the 1970 and the 1980 censuses and linked to mortality data for the period 1980-5 by the official, individual identification numbers. The study population included Norwegian men aged 20-64 years in 1980. All men enumerated in the 1980 census (and who were registered in the 1970 census) within the two occupational groups, unskilled workers and lower salaried employees, were included. The subjects were allocated to the two occupational groups according to the official Norwegian classification of socioeconomic status. Mortality was measured by standardised mortality ratios (SMR). The findings suggest that a fairly large number of unskilled workers with high mortality move into the lower salaried employee group. This transition produces a higher SMR in lower salaried employees and, simultaneously, an artificially but modestly lower SMR in unskilled workers. The difference in SMR between the two groups is thereby exaggerated by 44%. The difference among the elderly was smaller. Occupational mobility consistent with "the healthy worker effect" increases the SMR of lower salaried men and, at the same time, reduces slightly the SMR of unskilled workers.