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Transforming growth factor-β (TGF-β) plays crucial roles in the development of focal segmental glomerulosclerosis, but key molecular pathways remain unknown. Here, we identified the regulation of mammalian target of rapamycin complex1 (mTORC1) by TGF-β via ERK1/2 in the Adriamycin-induced murine model of focal segmental glomerulosclerosis. Adriamycin administration elicited early activation of TGF-β-ERK1/2-mTORC1 in podocytes, which persisted at later stages of albuminuria and glomerulosclerosis. Phosphorylation of the TGF-β receptor-I (TGF-βRI), Smad3, ERK1/2 and ribosomal protein S6 were evident in the glomeruli of adriamycin-treated mice. Targeting TGFβ-RI and mTORC1 with pharmacological inhibitors suppressed TGF-β signaling in glomeruli and significantly reduced albuminuria, glomerulosclerosis, protein levels of collagen 4α3, plasminogen activator inhibitor-1, and vimentin and restored mRNA levels of podocyte markers. Low dose US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor trametinib/GSK1120212 blunted TGF-β1-induced mTORC1 activation in podocytes, ameliorated up-regulation of TGF-β, plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, fibronectin and α-smooth muscle actin and prevented albuminuria and glomerulosclerosis with improved serum albumin. In cultured podocytes, this pathway was found to be associated with translation of fibrogenic collagen 4α3 and plasminogen activator inhibitor-1, without influencing their transcription. Notably, rapamycin suppressed upstream p-TGF-βRI, p-Smad3 and p-ERK1/2, and trametinib down-regulated upstream p-Smad3 in ex vivo and in vivo studies, indicating that harmful paracrine signaling among glomerular cells amplified the TGF-β-ERK1/2-mTORC1 axis by forming a positive feedback loop. Thus, an accentuated TGF-β-ERK1/2-mTORC1 pathway is suggested as a central upstream mediator to develop proteinuria and glomerulosclerosis. Hence, preventing activation of this vicious loop by trametinib may offer a new therapeutic strategy for glomerular disease treatment.

The beneficial effects of simvastatin on fibrosis in various organs have been reported. In addition, bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been suggested as an effective therapy for hepatic fibrosis and cirrhosis. Recent evidence suggests that pharmacological treatment devoted to regulating stem cell function is a potential new therapeutic strategy that is drawing nearer to clinical practice. The aim of this study was to determine whether the combination treatment of simvastatin plus MSCs (Sim-MSCs) could have a synergistic effect on hepatic fibrosis in a thioacetamide (TAA)-induced cirrhotic rat model and hepatic stellate cells (HSCs). Cirrhotic livers from rats treated with Sim-MSCs exhibited histological improvement compared to those treated with simvastatin alone. Sim-MSCs combination treatment decreased hepatic collagen distribution, lowered the hydroxyproline content, and rescued liver function impairment in rats with TAA-induced cirrhosis. These protective effects were more potent with Sim-MSCs than with simvastatin alone. The upregulation of collagen-1, α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, and phospho-Smad3 in cirrhotic livers was prevented by the administration of Sim-MSCs. Intriguingly, Sim-MSCs inhibited both TGF-β/Smad3 signaling and α-SMA in HSCs. The Sim-MSCs combination treatment exerted strong protective effects against hepatic fibrosis by suppressing TGF-β/Smad signaling. Simvastatin could act synergistically with MSCs as an efficient therapeutic approach for intractable cirrhosis.

Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic β-cells. Released insulin from β-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal β-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3-kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca(2+) ([Ca(2+)]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca(2+)]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion.

TGF-β is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-β1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-β receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-β1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-β1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTORactivation. Our data suggest that TGF-β1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases.

Major pelvic ganglia (MPG) are relay centers for autonomic reflexes such as micturition and penile erection. MPG innervate the urogenital system, including bladder. γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and may also play an important role in some peripheral autonomic ganglia, including MPG. However, the electrophysiological properties and function of GABAA receptor in MPG neurons innervating bladder remain unknown. This study examined the electrophysiological properties and functional roles of GABAA receptors in bladder-innervating neurons identified by retrograde Dil tracing. Neurons innervating bladder showed previously established parasympathetic properties, including small membrane capacitance, lack of T-type Ca(2+) channel expression, and tyrosine-hydroxylase immunoreactivity. GABAA receptors were functionally expressed in bladder innervating neurons, but GABAC receptors were not. GABA elicited strong depolarization followed by increase of intracellular Ca(2+) in neurons innervating bladder, supporting the hypothesis GABA may play an important role in bladder function. These results provide useful information about the autonomic function of bladder in physiological and pathological conditions.

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca 2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP 2 , protons, and divalent ions Ca 2 + and Mg 2 + . Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca 2 + entry and increased intracellular Ca 2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1 -encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La 3 + . In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K + secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.

Summary Background PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca 2+ . Although availability of the phosphatidylinositol-4,5-biphosphate (PIP 2 ) substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Results Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca 2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP 2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gα q are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by G q -coupled receptors, and basal input from G q is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. Conclusions WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP 2 is itself a signaling molecule, regulation of PIP 2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP 2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk, and a means to independently control PLC signaling and PIP 2 availability.

Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 μM), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca²+](i) transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 μM), a tyrosine phosphatase inhibitor, significantly augmented the ACh-induced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion.

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.