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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]

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]

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]

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]

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSCs administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies char by dysregulated Akt. [ABSTRACT FROM AUTHOR]

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.

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1∗±0.8 vs. 1.4∗±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2. [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 LMX1B R246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1B WT or myc-LMX1B R246Q , 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 LMX1B R246Q -mediated downregulation of WT1(-KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1B R246Q 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.

TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-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-alpha production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha 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-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappaB signaling in TNF-alpha 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.

Although tumor necrosis factor (TNF)-alpha is implicated in numerous cardiac pathologies, the intracellular events leading to its production by heart cells are largely unknown. The goal of the present study was to identify the role of the transcription factor nuclear factor (NF)-kappaB in this process. Among the many inducers of TNF-alpha expression in myeloid cells, only lipopolysaccharide (LPS) led to its induction in cultured neonatal myocytes. LPS also activated the NF-kappaB pathway, as evidenced by the degradation of the inhibitory protein IkappaB and the appearance of NF-kappaB-binding complexes in nuclear extracts. Furthermore, inhibitors of NF-kappaB activation, such as lactacystin, MG132, and pyrrolidine dithiocarbamate, were found to completely block the production of TNF-alpha in response to LPS stimulation, indicating a requirement of NF-kappaB for TNF-alpha expression. However, interleukin-1beta and phorbol 12-myristate 13-acetate also activated NF-kappaB but did not evoke TNF-alpha expression, revealing that this factor is not sufficient for cytokine production. Detailed examination of the NF-kappaB cascade revealed that cardiac cells displayed a unique pattern of IkappaB degradation in response to LPS, with IkappaBbeta but not IkappaBalpha being degraded upon stimulation. Additionally, two specific p65-containing DNA-binding complexes were observed in the nuclear extracts of neonatal cardiomyocytes: an inducible complex that is necessary for TNF-alpha expression and a constitutive species. Taken together, these results reveal that NF-kappaB is not only involved in cytokine production but also may be linked to other pathways that subserve a constitutive, protective mechanism for the heart cell.