nephrin and Renal-Insufficiency--Chronic

Diabetes is the leading cause of microalbuminuria and end-stage renal failure in industrial countries. Disruption of the filtration barrier, seen in almost all nephrotic diseases and diabetes, is the result of the loss or effacement of the podocyte foot process, notably damage of proteins within the slit diaphragm such as nephrin. For many years, nephrin has been viewed as a structural component of the slit diaphragm. It is now well recognized that nephrin contains several tyrosine residues in its cytoplasmic domain, which influences the development of glomerular injury. In this review, we propose an overview of nephrin signaling pathways in kidney injury.

Topics: Animals; Diabetic Nephropathies; Humans; Kidney; Membrane Proteins; Phosphorylation; Protein Processing, Post-Translational; Renal Insufficiency, Chronic; Signal Transduction

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Differentiation; Cytoskeletal Proteins; Glomerular Filtration Rate; Humans; Induced Pluripotent Stem Cells; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Mechanoreceptors; Membrane Proteins; Multiprotein Complexes; Podocytes; Proteinuria; rac1 GTP-Binding Protein; Renal Insufficiency, Chronic; Signal Transduction; TRPC Cation Channels; TRPC6 Cation Channel

We established a novel mouse model of chronic kidney disease (CKD) using acetic acid and compared it with the 5/6-nephrectomized mouse model. In our novel model, significant increases were observed in blood biochemical values and urinary parameters. Moreover, a decrease in creatinine clearance (Ccr) was observed. This model also demonstrated a higher survival rate than the 5/6-nephrectomized model. Observed histological changes in our model included cell infiltration in the renal interstitium, tubular dilation, regenerated tubules, and glomerulosclerosis. Inflammation of the renal interstitium was particularly remarkable. TNF-α, IL-1β, and ICAM-1 mRNA expression were up-regulated prior to elevation of mean blood pressure and prior to changes in blood biochemical values and urinary parameters. Up-regulation of TGF-β mRNA and down-regulation of nephrin mRNA were also observed at 12 weeks after acetic acid treatment. However, no correlation between the progression of CKD and the decrease in renal blood flow was observed. Finally, repeated losartan administration attenuated the effects of acetic acid-induced renal injury. Our findings suggest that chronic kidney conditions associated with this model may be triggered by interstitial inflammation. Moreover, we suggest that this model is useful for understanding the pathophysiological mechanisms of CKD, and for evaluating the effects of therapeutic agents.

Topics: Acetic Acid; Animals; Creatine; Disease Models, Animal; Gene Expression; Interleukin-1beta; Kidney; Losartan; Male; Membrane Proteins; Metabolic Clearance Rate; Mice, Inbred Strains; Nephrectomy; Renal Insufficiency, Chronic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Numerous disease-causing gene mutations have been identified in proteinuric diseases, such as nephrotic syndrome and glomerulosclerosis. This report describes the results of comprehensive genetic diagnosis of Japanese patients with severe proteinuria. In addition, the report describes the clinical characteristics of patients with monogenic disease-causing mutations. We conducted comprehensive gene screening of patients who had either congenital nephrotic syndrome, infantile nephrotic syndrome, steroid-resistant nephrotic syndrome, or focal segmental glomerular sclerosis. Using targeted next-generation sequencing, 60 podocyte-related genes were screened in 230 unrelated patients with proteinuria. A retrospective review of clinical data was conducted for these patients. We detected monogenic disease-causing mutations in 30% (69 of 230) of patients among 19 of the screened genes. Common genes with disease-causing mutations were WT1 (25%), NPHS1 (12%), INF2 (12%), TRPC6 (10%), and LAMB2 (9%). With various immunosuppressive or renoprotective therapies, remission of proteinuria in patients with unknown causative mutations was observed in 26% of patients, whereas only 5% of patients with monogenic disease-causing mutations exhibited complete remission. We assessed the genetic backgrounds of Japanese patients with severe proteinuria. The proportion of patients with gene defects was similar to that of other reports, but the disease-causing gene mutation frequency was considerably different.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Genetic Testing; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Humans; Infant; Infant, Newborn; Japan; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Odds Ratio; Proteinuria; Renal Insufficiency, Chronic; Retrospective Studies; Severity of Illness Index; WT1 Proteins; Young Adult

Chronic kidney disease is a public health burden and it remains unknown which genetic loci are associated with kidney function in the Japanese population, our genome-wide association study using the Biobank Japan dataset (excluding secondary kidney diseases, such as diabetes mellitus) clearly revealed that almost half of the top 50 single nucleotide polymorphisms associated with estimated glomerular filtration rate are located in the SHROOM3 gene, suggesting that SHROOM3 will be responsible for kidney function. Thus, to confirm this finding, supportive functional analyses were performed on Shroom3 in mice using fullerene-based siRNA delivery, which demonstrated that Shroom3 knockdown led to albuminuria and podocyte foot process effacement. The in vitro experiment shows that knockdown of Shroom3 caused defective formation of lamellipodia in podocyte, which would lead to the disruption of slit diaphragm. These results from the GWAS, in vivo and in vitro experiment were consistent with recent studies reporting that albuminuria leads to impairment of kidney function.

Topics: Albuminuria; Animals; Base Pairing; Female; Gene Knockdown Techniques; Genetic Association Studies; Genetic Loci; Genetic Predisposition to Disease; Genome-Wide Association Study; Glomerular Filtration Rate; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Podocytes; Pseudopodia; Rats; Renal Insufficiency, Chronic

Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.

Topics: Adolescent; Age of Onset; Cell Line; Child; Child, Preschool; Consanguinity; Disease Progression; DNA Mutational Analysis; Drug Resistance; Exome Sequencing; Female; Follow-Up Studies; Gene Frequency; Glomerular Basement Membrane; Glucocorticoids; Homozygote; Humans; Male; Membrane Proteins; Microscopy, Electron, Transmission; Mutation; Nephrotic Syndrome; Pedigree; Podocytes; Renal Insufficiency, Chronic

Proteinuric chronic kidney disease (CKD) remains a major health problem worldwide. While it is well established that the progression of primary glomerular disease induces tubulointerstitial lesions, how tubular injury triggers glomerular damage is poorly understood. We hypothesized that injured tubules secrete mediators that adversely affect glomerular health. To test this, we used conditional knockout mice with tubule-specific ablation of β-catenin (Ksp-β-cat-/-) and subjected them to chronic angiotensin II (Ang II) infusion or Adriamycin. Compared with control mice, Ksp-β-cat-/- mice were dramatically protected from proteinuria and glomerular damage. MMP-7, a downstream target of β-catenin, was upregulated in treated control mice, but this induction was blunted in the Ksp-β-cat-/- littermates. Incubation of isolated glomeruli with MMP-7 ex vivo led to nephrin depletion and impaired glomerular permeability. Furthermore, MMP-7 specifically and directly degraded nephrin in cultured glomeruli or cell-free systems, and this effect was dependent on its proteolytic activity. In vivo, expression or infusion of exogenous MMP-7 caused proteinuria, and genetic ablation of MMP-7 protected mice from Ang II-induced proteinuria and glomerular injury. Collectively, these results demonstrate that β-catenin-driven MMP-7 release from renal tubules promotes glomerular injury via direct degradation of the key slit diaphragm protein nephrin.

Topics: Angiotensin II; Animals; beta Catenin; Cells, Cultured; Disease Models, Animal; Doxorubicin; Humans; Kidney Tubules; Male; Matrix Metalloproteinase 7; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Podocytes; Primary Cell Culture; Proteolysis; Rats; Renal Insufficiency, Chronic

Podocyte injury is a key event for progressive renal failure. We have previously established a mouse model of inducible podocyte injury (NEP25) that progressively develops glomerulosclerosis after immunotoxin injection. We performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice, in which a hemagglutinin tag is attached to ribosomal protein L22 selectively in podocytes. Podocyte-specific polysomes were successfully obtained by immunoprecipitation with an antihemagglutinin antibody from glomerular homogenate and analyzed using a microarray. Compared with glomerular cells, 353 genes were highly expressed and enriched in podocytes; these included important podocyte genes and also heretofore uncharacterized genes, such as Dach1 and Foxd2. Podocyte injury by immunotoxin induced many genes to be upregulated, including inflammation-related genes despite no infiltration of inflammatory cells in the glomeruli. MafF and Egr-1, which structurally have the potential to antagonize MafB and WT1, respectively, were rapidly and markedly increased in injured podocytes before MafB and WT1 were decreased. We demonstrated that Maff and Egr1 knockdown increased the MafB targets Nphs2 and Ptpro and the WT1 targets Ptpro, Nxph3, and Sulf1, respectively. This indicates that upregulated MafF and Egr-1 may promote deterioration of podocytes by antagonizing MafB and WT1. Our systematic microarray study of the heretofore undescribed behavior of podocyte genes may open new insights into the understanding of podocyte pathophysiology.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Gene Expression Profiling; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Interleukin-2 Receptor alpha Subunit; Membrane Proteins; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Phenotype; Podocytes; Polyribosomes; Renal Insufficiency, Chronic; Transcriptome

Topics: Adolescent; Biopsy; Cardiomyopathy, Dilated; Child; Child, Preschool; Deafness; Genetic Testing; Humans; Infant; Infant, Newborn; Kidney; Kidney Transplantation; Longitudinal Studies; Male; Membrane Proteins; Mitochondrial Diseases; Nephrectomy; Nephrotic Syndrome; Renal Insufficiency, Chronic; Young Adult

Topics: Adolescent; Biopsy; Cardiomyopathy, Dilated; Child; Child, Preschool; Deafness; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Introns; Kidney; Longitudinal Studies; Male; Membrane Proteins; Mitochondrial Diseases; Mutation; Nephrotic Syndrome; Renal Insufficiency, Chronic; Young Adult

Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix.. To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model.. Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified. Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin

Topics: Animals; Cells, Cultured; Drosophila; Flow Cytometry; Gene Expression Regulation; Humans; Integrin beta1; Membrane Proteins; Microscopy, Electron, Transmission; Phosphorylation; Podocytes; Renal Insufficiency, Chronic; Signal Transduction; Statistics, Nonparametric

We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development.. We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli.. Expression data from the Nephroseq database showed glomerular and nonglomerular. We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

Topics: Actin Cytoskeleton; Adolescent; Adult; Aged; Albuminuria; Allografts; Animals; Child; Child, Preschool; Enhancer Elements, Genetic; Female; Gene Knockdown Techniques; Glomerular Filtration Rate; Homozygote; Humans; Kidney; Kidney Transplantation; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Middle Aged; Phosphorylation; Podocytes; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins c-fyn; Renal Insufficiency, Chronic; RNA, Small Interfering; Signal Transduction; src Homology Domains; Young Adult

Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression.

Topics: Albuminuria; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Cell Line; Disease Models, Animal; DNA Methylation; Dose-Response Relationship, Drug; Doxorubicin; Epigenesis, Genetic; Irbesartan; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Promoter Regions, Genetic; Renal Insufficiency, Chronic; Renin-Angiotensin System; Signal Transduction; Tetrazoles; Time Factors; Transfection

The protective effects of Rho kinase inhibitor fasudil against renal diseases have recently been reported. We compared the therapeutic effects of fasudil on the spontaneously hypercholesterolemic (SHC) rat, a model of chronic kidney disease (CKD) with proteinuria, with those of the angiotensin receptor blocker olmesartan (OL) by paying attention to the proteinuria and the macrophage phenotype. SHC rats were allocated to six treatment groups: a vehicle (Ve) group, a low-dose fasudil (FL) group, a high-dose fasudil (FH) group, an OL group, a combination of low-dose fasudil and OL (CL) group, and a combination of high-dose fasudil and OL (CH) group. Sprague-Dawley rats treated with vehicle served as a control (n = 7/each). The rats were treated for 24 wk. Compared with the Ve group, proteinuria was significantly decreased in the FH, OL, and CL groups, and it completely disappeared in the CH group. Glomerular stainings of nephrin and F-actin were focally impaired in the Ve group but were restored in the CH group. Western blotting showed that the CH group had significantly increased renal nephrin expression compared with the Ve group. Interstitial infiltration of macrophages was significantly increased in the Ve group, which was significantly attenuated in all treatment groups. The ratio of CD206 (M2 macrophage marker) to CD68 mRNA was significantly greater in the CH group than in the Ve group. These results indicate that fasudil with OL reduces proteinuria by protecting podocyte integrity and alters the interstitial macrophage density/phenotype, thereby exerting renoprotective effects against CKD.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Angiotensin II Type 1 Receptor Blockers; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Enzyme Inhibitors; Hypercholesterolemia; Imidazoles; Kidney Glomerulus; Lectins, C-Type; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Membrane Proteins; Microscopy, Electron; Phenotype; Proteinuria; Rats; Receptors, Cell Surface; Renal Insufficiency, Chronic; rho-Associated Kinases; RNA, Messenger; Tetrazoles

Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.

Topics: Adaptor Proteins, Signal Transducing; Albuminuria; Animals; Cell Line, Transformed; Cytoskeletal Proteins; Gene Expression Regulation; Glomerular Filtration Rate; Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Proteins; Mice; Morphine; Narcotics; p38 Mitogen-Activated Protein Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Receptors, Opioid, kappa; Receptors, Opioid, mu; Renal Insufficiency, Chronic