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Glomerular visceral epithelial cells (i.e., podocytes) are an essential component of the tripartite glomerular filtration barrier. Healthy podocytes are terminally differentiated cells with limited replicative capacity; however, inappropriate cell cycle reentry can be induced in podocytes by various injurious stimuli. In this issue of the JCI, Yamaguchi et al. report on a somatic mosaic gain-of-function mutation in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic α subunit (p110α, encoded by PIK3CA). The study reveals that activating mutations of p110α can drive podocyte proliferation in PIK3CA-related overgrowth syndrome (PROS). They also showed that selective, small-molecule inhibitors of p110 may be useful for the treatment of proliferative glomerulonephritis. [ABSTRACT FROM AUTHOR]

Over a decade ago, mutations in the gene encoding TRPC6 (transient receptor potential cation channel, subfamily C, member 6) were linked to development of familial forms of nephrosis. Since this discovery, TRPC6 has been implicated in the pathophysiology of non-genetic forms of kidney disease including focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, immune-mediated kidney diseases, and renal fibrosis. On the basis of these findings, TRPC6 has become an important target for the development of therapeutic agents to treat diverse kidney diseases. Although TRPC6 has been a major focus for drug discovery, more recent studies suggest that other TRPC family members play a role in the pathogenesis of glomerular disease processes and chronic kidney disease (CKD). This review highlights the data implicating TRPC6 and other TRPC family members in both genetic and non-genetic forms of kidney disease, focusing on TRPC3, TRPC5, and TRPC6 in a cell type (glomerular podocytes) that plays a key role in proteinuric kidney diseases. [ABSTRACT FROM AUTHOR]

Background: Mutations in podocin (NPHS2) are the most common cause of childhood onset autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The disease is characterized by early-onset proteinuria, resistance to immunosuppressive therapy and rapid progression to end-stage renal disease. Compound heterozygous changes involving the podocin variant R229Q combined with another pathogenic mutation have been associated with a mild phenotype with disease onset often in adulthood. Methods: We screened 19 families with early-onset SRNS for mutations in NPHS2 and WT1 and identified four disease-causing mutations (three in NPHS2 and one in WT1) prior to planned whole-exome sequencing. Results: We describe two families with three individuals presenting in childhood who are compound heterozygous for R229Q and one other pathogenic NPHS2 mutation, either L327F or A297V. One child presented at age 4 years (A297V plus R229Q) and the other two at age 13 (L327F plus R229Q), one with steadily deteriorating renal function. Conclusions: These cases highlight the phenotypic variability associated with the NPHS2 R229Q variant plus pathogenic mutation. Individuals may present with early aggressive disease. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome (NS) is a clinicopathological entity characterized by proteinuria, hypoalbuminemia, peripheral edema, and hyperlipidemia. It is the most common cause of glomerular disease in children and adults. Although the molecular pathogenesis of NS is not completely understood, data from the study of familial NS suggest that it is a "podocytopathy." Virtually all of the genes mutated in hereditary NS localize to the podocyte or its secreted products and the slit diaphragm. Since the completion of human genome sequence and the advent of next generation sequencing, at least 29 causes of single-gene NS have been identified. However, these findings have not been matched by therapeutic advances owing to suboptimal in vitro and in vivo models for the study of human glomerular disease and podocyte injury phenotypes. Multidisciplinary collaboration between clinicians, geneticists, cell biologists, and molecular physiologists has the potential to overcome this barrier and thereby speed up the translation of genetic findings into improved patient care. [ABSTRACT FROM AUTHOR]

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr69/Thr70 (Thr69 in mice and Thr70 in humans) and Ser321/Ser322 (Ser321 in mice and Ser322 in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr69 in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-DL-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3=,4=-(methylenedioxy) benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr69 dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (−/−) mice. Hypoglycemia during fasting (plasma glucose <100 mg/dl) was prevented for >6 months by the dsAAV2/7, dsAAV2/8, and dsAAV2/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2/7 and dsAAV2/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (−/−) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2/9 vector. Hepatorenal correction in G6pase (−/−) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism. [ABSTRACT FROM AUTHOR]

Abstract: The Nuclear Factor-κB (NF-κB) signaling pathway has been linked to several pathologic processes in the myocardium including cardiomyocyte proinflammatory cytokine release, ischemia/reperfusion injury, hypertrophy and apoptosis. However, very little is known about the intracellular mechanisms that govern NF-κB activity in the myocardial cells. Recent advances in our understanding of the regulation of NF-κB signaling in non-myocyte systems suggest that the activity of the NF-κB pathway is tightly regulated by a diversity of stress-activated signaling intermediates through direct post-translational modification of various components of the NF-κB pathway. In this review, we will focus on these recent revelations and their implications not only in cardiac pathologies, but in the development of new therapeutic strategies to manage heart disease. [Copyright &y& Elsevier]

TNF-α is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-κB family of transcription factors are essential regulators of myocardial TNF-α gene expression, recent developments in our understanding of the modulation of NF-κB activity through posttranslational modification of NF-κB subunits suggest that the present view of NF-κB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-α production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-α production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-β reduced LPS-induced TNF-α production 38-fold, TNF-α mRNA levels 6-fold, and IκB-α phosphorylation 5-fold and degraded IκB-α 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-α production 3.5-fold, whereas overexpression of dominant-negative IKK-β reduced LPS-induced TNF-α production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-α had no effect on p65 phosphorylation or TNF-α production, revealing that IKK-β, not IKK-α, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-α production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-α gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-κB signaling in TNF-α gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction. [ABSTRACT FROM AUTHOR]

Elaborates the mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner. Effect of pregnancy on vascular guanosine triphosphatase (GTPase) activity; Measurement of guinea pig vascular GTPase activity during pregnancy and after estradiol treatment in aortas and uterine (UA) arteries, messenteric arteries and aortas; Investigates the result of the decreased GTPase activity at the level of the UA endothelial cell.

Interleukin (IL)-1 receptor type 1 (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated podocyte IL-1R1 in mice (PKO mice). We then subjected PKO mice and wild-type controls to two glomerular injury models: nephrotoxic serum (NTS)- and adriamycin-induced nephropathy. Surprisingly, we found that IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and adriamycin-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between wild-type and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease. [ABSTRACT FROM AUTHOR]

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the β-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated β-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine β-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated β-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the β-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the β-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated β-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage. [ABSTRACT FROM AUTHOR]

Mutations in the LIM homeobox transcription factor 1-beta (LMX1B) are a cause of nail patellar syndrome, a condition characterized by skeletal changes, glaucoma and focal segmental glomerulosclerosis. Recently, a missense mutation (R246Q) in LMX1B was reported as a cause of glomerular pathologies without extra-renal manifestations, otherwise known as nail patella-like renal disease (NPLRD). We have identified two additional NPLRD families with the R246Q mutation, though the mechanisms by which LMX1BR246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1BWT or myc-LMX1BR246Q, we observed dominant negative and haploinsufficiency effects of the mutation on the expression of podocyte genes such as NPHS1, GLEPP1, and WT1. Specifically, we observed a novel LMX1BR246Q-mediated downregulation of WT1(−KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1BR246Q mutation may be due to a dominant negative effect on WT1(−KTS) isoforms that may cause a disruption of the WT1 (−KTS):(+KTS) isoform ratio and a decrease in the expression of podocyte genes. Full delineation of the LMX1B gene regulon is needed to define its role in maintenance of glomerular filtration barrier integrity. [ABSTRACT FROM AUTHOR]

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes. [ABSTRACT FROM AUTHOR]

Focal and segmental glomerulosclerosis (FSGS) is a major cause of end-stage kidney disease. Recent advances in molecular genetics show that defects in the podocyte play a major role in its pathogenesis and mutations in inverted formin 2 (INF2) cause autosomal dominant FSGS. In order to delineate the role of INF2 mutations in familial and sporadic FSGS, we sought to identify variants in a large cohort of patients with FSGS. A secondary objective was to define an approach for genetic screening in families with autosomal dominant disease. A total of 248 individuals were identified with FSGS, of whom 31 had idiopathic disease. The remaining patients clustered into 64 families encompassing 15 from autosomal recessive and 49 from autosomal dominant kindreds. There were missense mutations in 8 of the 49 families with autosomal dominant disease. Three of the detected variants were novel and all mutations were confined to exon 4 of INF2, a regulatory region responsible for 90% of all changes reported in FSGS due to INF2 mutations. Thus, in our series, INF2 mutations were responsible for 16% of all cases of autosomal dominant FSGS, with these mutations clustered in exon 4. Hence, screening for these mutations may represent a rapid, non-invasive and cost-effective method for the diagnosis of autosomal dominant FSGS. [ABSTRACT FROM AUTHOR]

Background: Primary vesicoureteral reflux (PVUR) is the most common malformation of the kidney and urinary tract, and reflux nephropathy is a major cause of chronic kidney disease in children. Recently, we reported mutations in the tenascin XB gene ( TNXB) as a cause of PVUR with joint hypermobility. Methods: To define the role of rare variants in tenascin genes in the etiology of PVUR, we screened a cohort of patients with familial PVUR (FPVUR) and non-familial PVUR (NFPVUR) for rare missense variants in TNXB and the tenascin C gene ( TNC) after excluding mutations in ROBO2 and SOX17. Results: The screening procedure identified 134 individuals from 112 families with PVUR; two families with mutations in ROBO2 were excluded from further analysis. Rare missense variants in TNXB were found in the remaining 110 families, of which 5/55 (9 %) families had FPVUR and 2/55 (4 %) had NFPVUR. There were no differences in high-grade reflux or renal parenchymal scarring between patients with and without TNXB variants. All patients with TNXB rare variants who were tested exhibited joint hypermobility. Overall we were able to identify causes of FPVUR in 7/57 (12 %) families (9 % in TNXB and 3 % in ROBO2). Conclusions: In conclusion, the identification of a rare missense variant in TNXB in combination with a positive family history of VUR and joint hypermobility may represent a non-invasive method to diagnose PVUR and warrants further evaluation in other cohorts. [ABSTRACT FROM AUTHOR]

Abstract: Since massive irreversible loss of cardiac myocytes occurs following myocardial injury, injection of human mesenchymal stem cells (hMSCs) has emerged as a promising therapeutic intervention. Despite the growing enthusiasm for this approach, the understanding of how hMSCs evoke cardiac improvement is ever more controversial. The present study critically tests hypothesis that hMSCs provide specific benefit directly to damaged ventricular myocytes. Cultures of neonatal mouse ventricular cardiac myocytes (nMCM) were subjected to two distinct acute stress protocols; incubations with either endotoxin, lipopolysaccharide (LPS) or toxic cytokine, IL-1β. Myocyte injury was assessed in intracellular Ca2+ signaling assays in fluo-3-loaded nMCMs that were imaged with high temporal resolution by fluorescent microscopy. Following LPS or IL-1β treatment there was profound myocyte injury, manifest by chaotic [Ca2+]i handling, quantified as a 3- to 5-fold increase in spontaneous [Ca2+]i transients. Antibody neutralization experiments reveal such damage is mediated in part by interleukin-18 and not by tumor necrosis factor-α (TNF-α). Importantly, normal [Ca2+]i signaling was preserved when cardiomyocytes were co-cultured with hMSCs. Since normal [Ca2+]i handling was maintained in transwell cultures, where nMCMs and hMSCs were separated by a permeable membrane, a protective paracrine signaling cascade is operable. hMSCs provoke a genetic reprogramming of cardiomyocytes. LPS provokes release of TNFα from nMCMs which is blocked by hMSCs grown in co- or transwell cultures. Consistent with cytokine release, flow cytometry analyses reveal that hMSCs also block the LPS- and IL-1β-dependent activation of cardiac transcription factor, NF-κB. Importantly, hMSC-conditioned medium restores normal Ca2+ signaling in LPS- and IL-1β-damaged nMCMs. These results reveal new evidence that hMSCs elicit protective and reparative effects on cardiac tissue through molecular reprogramming of the cardiac myocytes themselves. Thus these studies provide novel new insight into the cellular and molecular mechanisms that underlie the therapeutic benefit of hMSCs in the setting of heart failure. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited". [Copyright &y& Elsevier]

Discusses a study that examined the expression of tumor necrosis factor (TNF)-α in mouse neonatal cardiomyocytes. Materials and methods used; Effect of nuclear factor (NF)-κ B inhibitors and activators on TNF-α secretion by lipopolysaccharide-stimulated cardiomyocytes; Importance of identifying the intracellular signaling cascades that underlie the production of TNF-α.