Blockade of mineralocorticoid receptor has been shown to improve the clinical outcomes of proteinuric kidney diseases. However, little is known about the regulation of mineralocorticoid receptor-dependent transcriptional activity in renal disease. Here we identify a new role for Rac1, a member of the Rho family GTPases, as a potent activator of mineralocorticoid receptor signal transduction both in vitro and in vivo. Transient transfection assays in HEK 293 cells revealed that constitutively active Rac1 (CA-Rac1) enhanced mineralocorticoid receptor-dependent reporter activity, which was accompanied by increased nuclear translocation of mineralocorticoid receptor. CA-Rac1 facilitated mineralocorticoid receptor nuclear accumulation also in podocytes via p21-activated kinase phosphorylation. In mice lacking Rho GDP-dissociation inhibitor-alpha (Arhgdia(-/-) mice), renal abnormalities, including heavy albuminuria and podocyte damage, were associated with increased Rac1 (but not RhoA) and mineralocorticoid receptor signaling in the kidney, without alteration in systemic aldosterone status. Pharmacological intervention with a Rac-specific small-molecule inhibitor diminished mineralocorticoid receptor overactivity and renal damage in this model. Furthermore, albuminuria and histological changes in Arhgdia(-/-) mice were suppressed by mineralocorticoid receptor blockade, confirming the pathological role of Rac1-mineralocorticoid receptor interaction. Our results provide evidence that signaling cross-talk between Rac1 and mineralocorticoid receptor modulates mineralocorticoid receptor activity and identify Rac1 as a therapeutic target for chronic kidney disease.

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.

Nephrin is localized in slit diaphragm in the matured glomeruli and is identical with mAb 5-1-6 antigen. Nephrin is involved in the development of proteinuria not only in mAb 5-1-6 nephropathy, but also in puromycin aminonucleoside nephropathy.

CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1) is a unique homeostatic chemokine that signals through its cognate receptor, CXCR4. CXCL12/CXCR4 signaling is essential for the formation of blood vessels in the gastrointestinal tract during development, but its contribution to renal development remains unclear. Here, we found that CXCL12-secreting stromal cells surround CXCR4-positive epithelial components of early nephrons and blood vessels in the embryonic kidney. In glomeruli, we observed CXCL12-secreting podocytes in close proximity to CXCR4-positive endothelial cells. Both CXCL12-and CXCR4-deficient kidneys exhibited identical phenotypes; there were no apparent abnormalities in early nephrogenesis or in differentiation of podocytes and tubules, but there was defective formation of blood vessels, including ballooning of the developing glomerular tuft and disorganized patterning of the renal vasculature. To clarify the relative importance of different cellular defects resulting from ablation of CXCL12 and CXCR4, we established endothelial cell-specific CXCR4-deficient mice, which recapitulated the renal phenotypes of conventional CXCR4-deficient mice. We conclude that CXCL12 secreted from stromal cells or podocytes acts on endothelial cells to regulate vascular development in the kidney. These findings suggest new potential therapeutic targets for remodeling the injured kidney. 20: 171420: -172320: , 200920: . doi: 10.1681 Nephrogenesis requires a coordinated process during development and has two distinct embryologic aspects. One is the development of epithelial components. They originate from interactions between the metanephric blastema, a group of mesenchymal cells in the genital ridge, and the ureteric bud (UB), an epithelial outgrowth of the nephric duct. When the tips of the UB invade the metanephric blastema, mutual inductive signals initiate a cascade of events, including UB branching and mesenchymal aggregation, which is followed by formation of nephrons. The other essential aspect is assembly of renal microcirculation, a multistep process including differentiation of endothelial progenitor cells, recruitment of endothelial cells into the glomerular J Am Soc Nephrol

The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in the early stage of diabetic nephropathy. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (50 mg/kg, i.v.). After 2 weeks, albumin delivery in the proximal tubule was measured using micropuncture and the endocytosis process of FITC-labeled albumin was evaluated with immunoelectron microscopy. Albumin was significantly reabsorbed in the proximal convoluted tubule (PCT) of controls (0.39+/-0.05 ng/min at early PCT to 0.17+/-0.08 at late PCT, P<0.05), whereas albumin reabsorption was inhibited in diabetic rats (0.27+/-0.05 to 0.21+/-0.08). Immunogold study revealed that FITC-albumin was significantly less reabsorbed in endosomes and lysosomes of S1 segments in diabetic rats than in controls (endosome: 1.20+/-0.10 vs 2.16+/-0.15 microm-1, P<0.0001; lysosome: 0.26+/-0.03 vs 0.83+/-0.07, P<0.0001). The expression of megalin, an endocytosis receptor, was decreased at the apical membrane of PCT in diabetic rats. The lipid peroxidation production in the proximal tubule was significantly increased in diabetic rats. In conclusion, albuminuria in early-stage diabetic rats can be partly explained by a decreased albumin endocytosis with reduced megalin expression and with increased lipid peroxidation in the proximal tubule.

Abstract. Podocin is identified as a product of the gene mutated in a patient with autosomal recessive steroid-resistant nephrotic syndrome. Although podocin is reported to be located at the slit diaphragm area, the precise role of podocin for maintaining the barrier function of the slit diaphragm has not been clearly elucidated. A rat homologue of podocin was cloned, and the expression of podocin was investigated and then compared with the nephrin and the ZO-1 expressions in rat experimental proteinuric models and in developing glomeruli. Amino acid sequences of rat and human podocin are highly homologous (84.3% identity). The domain structure of podocin is also highly conserved between rat and human. The mRNA expression for podocin was detected in glomeruli and the nerve tissues. The localization of podocin has close proximity to that of nephrin in normal adult rat glomeruli. Podocin staining was restricted to the basal side of the podocyte of the early developing stage, whereas nephrin staining was detected on the basolateral surface of podocyte. The redistribution of podocin was observed in the anti-nephrin antibody (ANA)-induced nephropathy and puromycin aminonucleoside (PAN) nephropathy. The redistribution of podocin paralleled with nephrin in ANA nephropathy but not in PAN nephropathy. Podocin is observed at the site of tight junction newly formed in proteinuric state in PAN nephropathy. It is postulated that podocin is one of the critical components of a slit diaphragm for maintaining the barrier function of the glomerular capillary wall. kawachi@med.niigata-u.ac.jpThe primary barrier for ultrafiltration of plasma in a glomerular capillary wall comprises three layers: a fenestrated endothelial cell, a glomerular basement membrane (GBM), and a glomerular epithelial cell (podocyte). Although the role of the GBM in restricting the presence of macromolecules has been emphasized for nearly two decades (1,2), several recent studies have shown that slit diaphragms located between the foot processes play the critical role of a barrier to retain macromolecules (3-7). In the past several years, some molecules were reported to be associated with the slit diaphragm (8 -11). Nephrin is considered to be a critical component of the slit diaphragm for maintaining the barrier function (9,(12)(13)(14)(15). CD2AP is reported to be associated with nephrin (16). Boute et al. (17) cloned a novel gene, NPHS2, mutated in patients with autosomal recessive steroid-resistant nephrotic syndrome and reported that this gene product, podocin, is located at the slit diaphragm area (18). Podocin is an integral membrane protein of 383 amino acids, with a single membrane domain forming a hairpin-like structure and with both N-and C-terminal domains in the cytosol. Recent reports indicate that podocin interacts with CD2AP and nephrin (19,20); however, the precise role of podocin for maintaining the barrier function of the slit diaphragm is not yet clarified. To address this, more studies using experimental models would be necessary. We cl...

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.