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Thyroid hormones, including 3,5,3′-triiodothyronine (T3), cause a wide spectrum of genomic effects on cellular metabolism and bioenergetic regulation in various tissues. The non-genomic actions of T3 have been reported but are not yet completely understood. Acute T3 treatment significantly enhanced basal, maximal, ATP-linked, and proton-leak oxygen consumption rates (OCRs) of primary differentiated mouse brown adipocytes accompanied with increased protein abundances of uncoupling protein 1 (UCP1) and mitochondrial Ca2+ uniporter (MCU). T3 treatment depolarized the resting mitochondrial membrane potential (Ψm) but augmented oligomycin-induced hyperpolarization in brown adipocytes. Protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were activated by T3, leading to the inhibition of autophagic degradation. Rapamycin, as an mTOR inhibitor, blocked T3-induced autophagic suppression and UCP1 upregulation. T3 increases intracellular Ca2+ concentration ([Ca2+]i) in brown adipocytes. Most of the T3 effects, including mTOR activation, UCP1 upregulation, and OCR increase, were abrogated by intracellular Ca2+ chelation with BAPTA-AM. Calmodulin inhibition with W7 or knockdown of MCU dampened T3-induced mitochondrial activation. Furthermore, edelfosine, a phospholipase C (PLC) inhibitor, prevented T3 from acting on [Ca2+]i, UCP1 abundance, Ψm, and OCR. We suggest that short-term exposure of T3 induces UCP1 upregulation and mitochondrial activation due to PLC-mediated [Ca2+]i elevation in brown adipocytes.

With‐no‐lysine 1 (WNK1) kinase is a substrate of the insulin receptor/Akt pathway. Impaired insulin signaling in skeletal muscle disturbs glucose transporter 4 (GLUT4) translocation associated with the onset of type 2 diabetes (T2D). WNK1 is highly expressed in skeletal muscle. However, it is currently unknown how insulin signaling targeting WNK1 regulates GLUT4 trafficking in skeletal muscle, and whether this regulation is perturbed in T2D. Hereby, we show that insulin phosphorylates WNK1 at its activating site via a phosphatidylinositol 3‐kinase‐dependent mechanism. WNK1 promotes the cell surface abundance of GLUT4 via regulating TBC1D4. Of note, we observed insulin resistance and decreased WNK1 phosphorylation in T2D db/db mice as compared to the control mice. These results provide a new perspective on WNK1 function in the pathogenesis of hyperglycemia in T2D.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are known to have an antifibrotic effect and could be used as vehicles for targeted gene delivery. Decorin plays a protective role against fibrogenesis by modulating the degradation of the extracellular matrix. The aim of this study was to determine whether the antifibrotic effect of a combination treatment consisting of BM-MSCs and decorin on hepatic fibrosis is superior to BM-MSCs alone. The effects of BM-MSCs infected with decorin-expressing adenovirus (DCN-MSCs) on hepatic fibrosis were examined in a rat model of thioacetamide (TAA)-induced cirrhosis. The effects of infection with decorin-expressing adenovirus and of incubation with the conditioned medium of DCN-MSCs on transforming growth factor-β (TGF-β) signaling were analyzed in immortalized human hepatic stellate cells (HSCs). According to the Laennec fibrosis scoring system, cirrhotic livers from rats treated with DCN-MSCs exhibited histological improvement compared with cirrhotic livers from rats treated with control adenovirus-infected MSCs (CA-MSCs). DCN-MSC treatment reduced 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 DCN-MSCs than with CA-MSCs. The upregulation of collagen-1, α-smooth muscle actin (α-SMA), TGF-β1, and Smad3 phosphorylation in cirrhotic livers was prevented by DCN-MSC administration. Intriguingly, medium from cultured DCN-MSCs blocked both Smad3 phosphorylation and exogenous TGF-β1 stimulated α-SMA synthesis in HSCs. DCN-MSCs exert strong protective effects against hepatic fibrosis by suppressing TGF-β/Smad signaling. Thus, treatment with DCN-MSCs is a potentially novel and efficient therapeutic approach for patients with intractable cirrhosis. Significance A combination treatment consisting of bone marrow-derived mesenchymal stem cells (BM-MSCs) and decorin strongly inhibited the progression of thioacetamide-induced hepatic fibrosis in rats, compared with BM-MSCs alone. Furthermore, the significant inhibitory effect of BM-MSCs infected with decorin-expressing adenovirus was attributed to suppressing transforming growth factor-β (TGF-β)/Smad signaling pathway, supported by attenuation of TGF-β1 expression and inhibition of Smad3 phosphorylation. Therefore, treatment with BM-MSCs infected with decorin-expressing adenovirus could constitute a novel and efficient therapeutic approach for patients with intractable cirrhosis.

Store-operated calcium (Ca2+) entry (SOCE) is the principal Ca2+ entry route in non-excitable cells, including cancer cells. We previously demonstrated that Orai1 and STIM1, the molecular components of SOCE, are involved in tumorigenesis of clear cell renal cell carcinoma (CCRCC). However, a clinical relevance of Orai1 and STIM1 expression in CCRCC has been ill-defined. Here, we investigated the expression of Orai1 and STIM1 in CCRCC, and compared their expression with clinico-pathological parameters of CCRCC and the patients’ outcome. Immunohistochemical staining for Orai1 and STIM1 was performed on 126 formalin fixed paraffin embedded tissue of CCRCC and western blot analysis for Orai1 was performed on the available fresh tissue. The results were compared with generally well-established clinicopathologic prognostic factors in CCRCC and patient survival. Membrane protein Orai1 is expressed in the nuclei in CCRCC, whereas STIM1 shows the cytosolic expression pattern in immunohistochemical staining. Orai1 expression level is inversely correlated with CCRCC tumor grade, whereas STIM1 expression level is not associated with tumor grade. The higher Orai1 expression is significantly associated with lower Fuhrman nuclear grade, pathologic T stage, and TNM stage and with favorable prognosis. The expression level of STIM1 is not correlated with CCRCC grade and clinical outcomes. Orai1 expression in CCRCC is associated with tumor progression and with favorable prognostic factors. These results suggest that Orai1 is an attractive prognostic marker and therapeutic target for CCRCC., Graphical Abstract

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.

Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.

Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca2+ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca2+ depletion mediate persistent Ca2+ influx, further impairing cytosolic and mitochondrial Ca2+ homeostasis. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

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.

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

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.

Diabetes & Metabolism - In Press.Proof corrected by the author Available online since mercredi 2 novembre 2016

Klotho is a type-1 membrane protein predominantly produced in the kidney, the extracellular domain of which is secreted into the systemic circulation. Membranous and secreted Klotho protect organs, including the kidney, but whether and how Klotho directly protects the glomerular filter is unknown. Here, we report that secreted Klotho suppressed transient receptor potential channel 6 (TRPC6)-mediated Ca2+ influx in cultured mouse podocytes by inhibiting phosphoinositide 3-kinase-dependent exocytosis of the channel. Furthermore, soluble Klotho reduced ATP-stimulated actin cytoskeletal remodeling and transepithelial albumin leakage in these cells. Overexpression of TRPC6 by gene delivery in mice induced albuminuria, and exogenous administration of Klotho ameliorated the albuminuria. Notably, immunofluorescence and in situ hybridization revealed Klotho expression in podocytes of mouse and human kidney. Heterozygous Klotho-deficient CKD mice had aggravated albuminuria compared with that in wild-type CKD mice with a similar degree of hypertension and reduced clearance function. Finally, disrupting the integrity of glomerular filter by saline infusion-mediated extracellular fluid volume expansion increased urinary Klotho excretion. These results reveal a potential novel function of Klotho in protecting the glomerular filter, and may offer a new therapeutic strategy for treatment of proteinuria.

Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal β cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPis) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of sodium-dependent phosphate transporter 1 and 2 (PiT-1 and -2), isotypes of type III NaPi, were up-regulated by high-Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na+-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition (mPT), and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.

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.

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca2+reabsorption in kidney. Many hormones alter renal Ca2+reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca2+reabsorption by PTH.

WNKs (with-no-lysine [K]) are a family of serine-threonine protein kinases with an atypical placement of the catalytic lysine relative to all other protein kinases. The roles of WNK kinases in regulating ion transport were first revealed by the findings that mutations of two members cause a genetic hypertension and hyperkalemia syndrome. More recent studies suggest that WNKs are pleiotropic protein kinases with important roles in many cell processes in addition to ion transport. Here, we review roles of WNK kinases in the regulation of ion balance, cell signaling, survival, and proliferation, and embryonic organ development.

WNK (with-no-lysine [K]) kinases (WNK1-4) are serine/threonine protein kinases with an atypical placement of the catalytic lysine and tissue-specific expression. WNK1, ubiquitously expressed including skeletal muscle, is known as a downstream effector of insulin signaling and WNKs have been implicated in protein trafficking highly correlated with insulin resistance or diabetes. However, expression pattern and functional role of WNKs in skeletal muscle and linkage between WNKs and insulin signaling effectors such as PKB/Akt and glucose transports in skeletal muscle with type II diabetes are ill-defined. In the present study, WNK1 and WNK2, not WNK4 are expressed in C2C12. The expression levels of WNK1 and WNK2 significantly decreased in db/db mice, a type II diabetic model, compared to that of wild type mice. Phosphorylated Akt in skeletal muscle decreased in db/db mice supporting insulin resistance in skeletal muscle. Insulin stimulates Akt followed by WNK1 phosphorylation. Insulin stimulation of WNK1 is ...

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pitransport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Piconcentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pion secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi(1∼5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨmabolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨmhyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pialso promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pidiminished ATP synthesis, cytosolic Ca2+oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Picauses mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.

The intracellular Ca(2+) regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca(2+) entry (SOCE) is a major Ca(2+) entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.

Klotho functions as a tumor suppressor predominantly expressed in renal tubular cells, the origin of clear cell renal cell carcinoma (ccRCC). Altered expression and/or activity of growth factor receptor have been implicated in ccRCC development. Although Klotho suppresses a tumor progression through growth factor receptor signaling including insulin-like growth factor-1 receptor (IGF-1R), the role of Klotho acting on IGF-1R in ccRCC and its clinical relevance remains obscure. Here, we show that Klotho is favorable prognostic factor for ccRCC and exerts tumor suppressive role for ccRCC through inhibiting IGF-1R signaling. Our data shows the following key findings. First, in tumor tissues, the level of Klotho and IGF-1R expression are low or high, respectively, compared to that of adjacent non-neoplastic parenchyma. Second, the Klotho expression is clearly low in higher grade of ccRCC and is closely associated with clinical outcomes in tumor progression. Third, Klotho suppresses IGF-1-stimulated cell proliferation and migration by inhibiting PI3K/Akt pathway. These results provide compelling evidence supporting that Klotho acting on IGF-1R signaling functions as tumor suppressor in ccRCC and suggest that Klotho is a potential carcinostatis substance for ccRCC.

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.

WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca(2+) channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca(2+) reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (approximately 215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca(2+) reabsorption by parathyroid hormones or the tissue kallikrein in vivo.

Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca 2+ channel TRPV5 by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose- N -acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of β-galactoside α2,6-sialyltransferase (ST6Gal-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6Gal-1, but is restored by forced expression of recombinant ST6Gal-1. Thus, Klotho participates in specific removal of α2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho.

The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside-out patches of Chinese hamster ovary cells. The slope conductance values (Na(+) as a charge carrier, between -25 and -75 mV) for full and subconductance opening at intracellular pH 7.4 were 59 +/- 6 and 29 +/- 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open-subconductance-closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance.

Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca2+ increase due to depletion of luminal ER Ca2+. Palmitate-induced ER Ca2+ depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca2+ pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca2+ depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca2+ loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca2+ depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca2+ depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.

Purpose Both insulin and insulin-like growth factor (IGF)-1 signaling are key regulators of energy metabolism, cellular growth, proliferation, and survival. The IGF-1 receptor (IGF-1R) is overexpressed in most types of human cancers including renal cell carcinoma (RCC) with poor prognosis. Insulin receptor (IR) shares downstream effectors with IGF-1R; however, the expression and function of IR in the tumorigenesis of renal cancer remains elusive. Therefore, we examined the expression of IR and its prognostic significance in clear cell RCC (CCRCC). Materials and Methods Immunohistochemical staining for IR was performed on 126 formalin-fixed paraffin-embedded CCRCC tissue samples. Eight of these cases were utilized for western blot analysis. The results were compared with various clinico-pathologic parameters of CCRCC and patient survival. Results IR was expressed in the nuclei of CCRCC tumor cells in 109 cases (87.9%). Higher IR expression was significantly correlated with the presence of cystic change, lower Fuhrman nuclear grade, lower pathologic T stage, and lower TNM stage, although it wasn't significantly related to diabetes status and patient survival. Western blot analyses supported the results of the immunohistochemistry studies. Conclusion IR expression in CCRCC may be associated with favorable prognostic factors.

AIM: To investigate serotonergic Ca2+ signaling and the expression of 5-hydroxytryptamine (5-HT) receptors, as well as Ca2+ transporting proteins, in hepatic stellate cells (HSCs). METHODS: The intracellular Ca2+ concentration ([Ca2+]i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system. Quantitative PCR was performed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum (ER) proteins involved in Ca2+ storage and release in cultured rat HSCs. RESULTS: Distinct from quiescent cells, activated HSCs exhibited [Ca2+]i transients following treatment with 5-HT, which was abolished by U-73122, a phospholipase C inhibitor. Upregulation of 5-HT2A and 5-HT2B receptors, but not 5-HT3, was prominent during trans-differentiation of HSCs. Pretreatment with ritanserin, a 5-HT2 antagonist, inhibited [Ca2+]i changes upon application of 5-HT. Expression of type 1 inositol-5’-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca2+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca2+ storage and release in activated HSCs. Ca2+ binding chaperone proteins of the ER, including calreticulin, calnexin and calsequestrin, were up-regulated following activation of HSCs. CONCLUSION: The appearance of 5-HT-induced [Ca2+]i response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca2+ transporting/chaperone ER proteins may participate in the activating process of HSCs.