Female protection against diabetic kidney disease is regulated by kidney-specific AMPK activity.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes were evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2KO) male and female mice. In WT males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2KO. Whereas WT females had protection against diabetes induced kidney injury, KTAMPKγ2KO led to loss of female protection against kidney disease. 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA-seq and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.

Oncoprotein DJ-1 interacts with mTOR complexes to effect transcription factor Hif1α-dependent expression of collagen I (α2) during renal fibrosis.

Proximal tubular epithelial cells respond to transforming growth factor ß (TGFß) to synthesize collagen I (α2) during renal fibrosis. The oncoprotein DJ-1 has previously been shown to promote tumorigenesis and prevent apoptosis of dopaminergic neurons; however, its role in fibrosis signaling is unclear. Here, we show TGFß-stimulation increased expression of DJ-1, which promoted noncanonical mTORC1 and mTORC2 activities. We show DJ-1 augmented the phosphorylation/activation of PKCßII, a direct substrate of mTORC2. In addition, coimmunoprecipitation experiments revealed association of DJ-1 with Raptor and Rictor, exclusive subunits of mTORC1 and mTORC2, respectively, as well as with mTOR kinase. Interestingly, siRNAs against DJ-1 blocked TGFß-stimulated expression of collagen I (α2), while expression of DJ-1 increased expression of this protein. In addition, expression of dominant negative PKCßII and siRNAs against PKCßII significantly inhibited TGFß-induced collagen I (α2) expression. In fact, constitutively active PKCßII abrogated the effect of siRNAs against DJ-1, suggesting a role of PKCßII downstream of this oncoprotein. Moreover, we demonstrate expression of collagen I (α2) stimulated by DJ-1 and its target PKCßII is dependent on the transcription factor hypoxia-inducible factor 1α (Hif1α). Finally, we show in the renal cortex of diabetic rats that increased TGFß was associated with enhanced expression of DJ-1 and activation of mTOR and PKCßII, concomitant with increased Hif1α and collagen I (α2). Overall, we identified that DJ-1 affects TGFß-induced expression of collagen I (α2) via an mTOR-, PKCßII-, and Hif1α-dependent mechanism to regulate renal fibrosis.

High glucose-stimulated enhancer of zeste homolog-2 (EZH2) forces suppression of deptor to cause glomerular mesangial cell pathology.

Function of mTORC1 and mTORC2 has emerged as a driver of mesangial cell pathologies in diabetic nephropathy. The mechanism of mTOR activation is poorly understood in this disease. Deptor is a constitutive subunit and a negative regulator of both mTOR complexes. Mechanistic investigation in mesangial cells revealed that high glucose decreased the expression of deptor concomitant with increased mTORC1 and mTORC2 activities, induction of hypertrophy and, expression of fibronectin and PAI-1. shRNAs against deptor mimicked these pathologic outcomes of high glucose. Conversely, overexpression of deptor significantly inhibited all effects of high glucose. To determine the mechanism of deptor suppression, we found that high glucose significantly increased the expression of EZH2, resulting in lysine-27 tri-methylation of histone H3 (H3K27Me3). Employing approaches including pharmacological inhibition, shRNA-mediated downregulation and overexpression of EZH2, we found that EZH2 regulates high glucose-induced deptor suppression along with activation of mTOR, mesangial cell hypertrophy and fibronectin/PAI-1 expression. Moreover, expression of hyperactive mTORC1 reversed shEZH2-mediated inhibition of hypertrophy and expression of fibronectin and PAI-1 by high glucose. Finally, in renal cortex of diabetic mice, we found that enhanced expression of EZH2 is associated with decreased deptor levels and increased mTOR activity and, expression of fibronectin and PAI-1. Together, our findings provide a novel mechanism for mTOR activation via EZH2 to induce mesangial cell hypertrophy and matrix expansion during early progression of diabetic nephropathy. These results suggest a strategy for leveraging the intrinsic effect of deptor to suppress mTOR activity via reducing EZH2 as a novel therapy for diabetic nephropathy.

Chloride channel accessory 1 integrates chloride channel activity and mTORC1 in aging-related kidney injury.

The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging-related kidney injury, we performed RNA-Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H2 S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H2 S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl- current via the Ca++ -dependent Cl- channel TMEM16A (anoctamin-1) by patch-clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence-associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1-TMEM16A-Cl- current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H2 S.

Marmoset as a Model to Study Kidney Changes Associated With Aging.

We evaluated whether the marmoset, a nonhuman primate, can serve as a good model to study aging-related changes in the kidney by employing healthy young and aged marmosets of both sexes. Aging was associated with glomerulosclerosis, interstitial fibrosis, and arteriolosclerosis in both sexes; correspondingly, the content of matrix proteins was increased. Functionally, aging resulted in an increase in urinary albumin and protein excretion. There was a robust correlation between markers of fibrosis and functional changes. We explored signaling pathways as potential mechanistic events. Aging in males, but not in females, was associated with reduced renal cortical activity of AMP-activated protein kinase (AMPK) and a trend toward activation of mechanistic target of rapamycin complex 1 (mTORC1); upstream of AMPK and mTORC1, Akt and IGF-1 receptor were activated. In both sexes, aging promoted kidney activation of transforming growth factor ß-1 signaling pathway. While the expression of cystathionine ß-synthase (CBS), an enzyme involved hydrogen sulfide (H2S) synthesis, was reduced in both aged males and females, decreased H2S generation was seen in only males. Our studies show that the marmoset is a valid model to study kidney aging; some of the signaling pathways involved in renal senescence differ between male and female marmosets.

Akt2 causes TGFß-induced deptor downregulation facilitating mTOR to drive podocyte hypertrophy and matrix protein expression.

TGFß promotes podocyte hypertrophy and expression of matrix proteins in fibrotic kidney diseases such as diabetic nephropathy. Both mTORC1 and mTORC2 are hyperactive in response to TGFß in various renal diseases. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. We identified that deptor downregulation by TGFß maintains hyperactive mTOR in podocytes. To unravel the mechanism, we found that TGFß -initiated noncanonical signaling controls deptor inhibition. Pharmacological inhibitor of PI 3 kinase, Ly 294002 and pan Akt kinase inhibitor MK 2206 prevented the TGFß induced downregulation of deptor, resulting in suppression of both mTORC1 and mTORC2 activities. However, specific isoform of Akt involved in this process is not known. We identified Akt2 as predominant isoform expressed in kidney cortex, glomeruli and podocytes. TGFß time-dependently increased the activating phosphorylation of Akt2. Expression of dominant negative PI 3 kinase and its signaling inhibitor PTEN blocked Akt2 phosphorylation by TGFß. Inhibition of Akt2 using a phospho-deficient mutant that inactivates its kinase activity, as well as siRNA against the kinase markedly diminished TGFß -mediated deptor suppression, its association with mTOR and activation of mTORC1 and mTORC2. Importantly, inhibition of Akt2 blocked TGFß -induced podocyte hypertrophy and expression of the matrix protein fibronectin. This inhibition was reversed by the downregulation of deptor. Interestingly, we detected increased phosphorylation of Akt2 concomitant with TGFß expression in the kidneys of diabetic rats. Thus, our data identify previously unrecognized Akt2 kinase as a driver of TGFß induced deptor downregulation and sustained mTORC1 and mTORC2 activation. Furthermore, we provide the first evidence that deptor downstream of Akt2 contributes to podocyte hypertrophy and matrix protein expression found in glomerulosclerosis in different renal diseases.

Hydrogen sulfide ameliorates aging-associated changes in the kidney.

Aging is associated with replacement of normal kidney parenchyma by fibrosis. Because hydrogen sulfide (H2S) ameliorates kidney fibrosis in disease models, we examined its status in the aging kidney. In the first study, we examined kidney cortical H2S metabolism and signaling pathways related to synthesis of proteins including matrix proteins in young and old male C57BL/6 mice. In old mice, increase in renal cortical content of matrix protein involved in fibrosis was associated with decreased H2S generation and AMPK activity, and activation of insulin receptor (IR)/IRS-2-Akt-mTORC1-mRNA translation signaling axis that can lead to increase in protein synthesis. In the second study, we randomized 18-19 month-old male C57BL/6 mice to receive 30 µmol/L sodium hydrosulfide (NaHS) in drinking water vs. water alone (control) for 5 months. Administration of NaHS increased plasma free sulfide levels. NaHS inhibited the increase in kidney cortical content of matrix proteins involved in fibrosis and ameliorated glomerulosclerosis. NaHS restored AMPK activity and inhibited activation of IR/IRS-2-Akt-mTORC1-mRNA translation axis. NaHS inhibited age-related increase in kidney cortical content of p21, IL-1ß, and IL-6, components of the senescence-associated secretory phenotype. NaHS abolished increase in urinary albumin excretion seen in control mice and reduced serum cystatin C levels suggesting improved glomerular clearance function. We conclude that aging-induced changes in the kidney are associated with H2S deficiency. Administration of H2S ameliorates aging-induced kidney changes probably by inhibiting signaling pathways leading to matrix protein synthesis.

Nox4 is a Target for Tuberin Deficiency Syndrome.

The mechanism by which TSC2 inactivation or deficiency contributes to the pathology of tuberous sclerosis complex (TSC) is not fully clear. We show that renal angiomyolipomas from TSC patients and kidney cortex from Tsc2+/- mice exhibit elevated levels of reactive oxygen species (ROS). Downregulation of tuberin (protein encoded by TSC2 gene) in renal proximal tubular epithelial cells significantly increased ROS concomitant with enhanced Nox4. Similarly, we found elevated levels of Nox4 in the renal cortex of Tsc2+/- mice and in the renal angiomyolipomas from TSC patients. Tuberin deficiency is associated with activation of mTORC1. Rapamycin, shRNAs targeting raptor, or inhibition of S6 kinase significantly inhibited the expression of Nox4, resulting in attenuation of production of ROS in tuberin-downregulated proximal tubular epithelial cells. In contrast, activation of mTORC1 increased Nox4 and ROS. These results indicate that Nox4 may be a potential target for tuberin-deficiency-derived diseases. Using a xenograft model from tuberin-null tubular cells in nude mice, both anti-sense Nox4 and GKT137831, a specific inhibitor of Nox1/4, significantly inhibited the tumor growth. Thus, our results demonstrate the presence of an antagonistic relationship between tuberin and Nox4 to drive oncogenesis in the tuberin deficiency syndrome and identify Nox4 as a target to develop a therapy for TSC.

microRNA-181a downregulates deptor for TGFß-induced glomerular mesangial cell hypertrophy and matrix protein expression.

TGFß contributes to mesangial cell hypertrophy and matrix protein increase in various kidney diseases including diabetic nephropathy. Deptor is an mTOR-interacting protein and suppresses mTORC1 and mTORC2 activities. We have recently shown that TGFß-induced inhibition of deptor increases the mTOR activity. The mechanism by which TGFß regulates deptor expression is not known. Here we identify deptor as a target of the microRNA-181a. We show that in mesangial cells, TGFß increases the expression of miR-181a to downregulate deptor. Decrease in deptor augments mTORC2 activity, resulting in phosphorylation/activation of Akt kinase. Akt promotes inactivating phosphorylation of PRAS40 and tuberin, leading to stimulation of mTORC1. miR-181a-mimic increased mTORC1 and C2 activities, while anti-miR-181a inhibited them. mTORC1 controls protein synthesis via phosphorylation of translation initiation and elongation suppressors 4EBP-1 and eEF2 kinase. TGFß-stimulated miR-181a increased the phosphorylation of 4EBP-1 and eEF2 kinase, resulting in their inactivation. miR-181a-dependent inactivation of eEF2 kinase caused dephosphorylation of eEF2. Consequently, miR-181a-mimic increased protein synthesis and hypertrophy of mesangial cells similar to TGFß. Anti-miR-181a blocked these events in a deptor-dependent manner. Finally, TGFß-miR-181a-driven deptor downregulation increased the expression of fibronectin. Our results identify a novel mechanism involving miR-181a-driven deptor downregulation, which contributes to mesangial cell pathologies in renal complications.

Tyrosines-740/751 of PDGFRß contribute to the activation of Akt/Hif1α/TGFß nexus to drive high glucose-induced glomerular mesangial cell hypertrophy.

Glomerular mesangial cell hypertrophy contributes to the complications of diabetic nephropathy. The mechanism by which high glucose induces mesangial cell hypertrophy is poorly understood. Here we explored the role of the platelet-derived growth factor receptor-ß (PDGFRß) tyrosine kinase in driving the high glucose-induced mesangial cell hypertrophy. We show that high glucose stimulates the association of the PDGFRß with PI 3 kinase leading to tyrosine phosphorylation of the latter. High glucose-induced Akt kinase activation was also dependent upon PDGFRß and its tyrosine phosphorylation at 740/751 residues. Inhibition of PDGFRß activity, its downregulation and expression of its phospho-deficient (Y740/751F) mutant inhibited mesangial cell hypertrophy by high glucose. Interestingly, expression of constitutively active Akt reversed this inhibition, indicating a role of Akt kinase downstream of PDGFRß phosphorylation in this process. The transcription factor Hif1α is a target of Akt kinase. siRNAs against Hif1α inhibited the high glucose-induced mesangial cell hypertrophy. In contrast, increased expression of Hif1α induced hypertrophy similar to high glucose. We found that inhibition of PDGFRß and expression of PDGFRß Y740/751F mutant significantly inhibited the high glucose-induced expression of Hif1α. Importantly, expression of Hif1α countered the inhibition of mesangial cell hypertrophy induced by siPDGFRß or PDGFRß Y740/751F mutant. Finally, we show that high glucose-stimulated PDGFRß tyrosine phosphorylation at 740/751 residues and the tyrosine kinase activity of the receptor regulate the transforming growth factor-ß (TGFß) expression by Hif1α. Thus we define the cell surface PDGFRß as a major link between high glucose and its effectors Hif1α and TGFß for induction of diabetic mesangial cell hypertrophy.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells.

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

Hydrophobic motif site-phosphorylated protein kinase CßII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

PKCßII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCßII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCßII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCßII, dominant negative PKCßII, and PKCßII hydrophobic motif phosphorylation-deficient mutant, we found that PKCßII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCßII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCßII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCßII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCßII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCßII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression.

High glucose milieu inhibits PTEN expression to activate Akt kinase and induces glomerular mesangial cell hypertrophy and matrix protein expression in diabetic nephropathy. Specific mechanism by which high glucose inhibits PTEN expression is not clear. We found that high glucose increased the expression of the microRNA-26a (miR-26a) in mesangial cells. Using a sensor plasmid with 3'UTR-driven luciferase, we showed PTEN as a target of miR-26a in response to high glucose. Overexpression of miR-26a reduced the PTEN protein levels resulting in increased Akt kinase activity similar to high glucose treatment. In contrast, anti-miR-26a reversed high glucose-induced suppression of PTEN with concomitant inhibition of Akt kinase activity. Akt-mediated phosphorylation of tuberin and PRAS40 regulates mTORC1, which is necessary for mesangial cell hypertrophy and matrix protein expression. Inhibition of high glucose-induced miR-26a blocked phosphorylation of tuberin and PRAS40, which lead to suppression of phosphorylation of S6 kinase and 4EBP-1, two substrates of mTORC1. Furthermore, we show that expression of miR-26a induced mesangial cell hypertrophy and increased fibronectin and collagen I (α2) expression similar to that observed with the cells incubated with high glucose. Anti-miR-26a inhibited these phenomena in response to high glucose. Together our results provide the first evidence for the involvement of miR-26a in high glucose-induced mesangial cell hypertrophy and matrix protein expression. These data indicate the potential therapeutic utility of anti-miR-26a for the complications of diabetic kidney disease.

Tadalafil Integrates Nitric Oxide-Hydrogen Sulfide Signaling to Inhibit High Glucose-induced Matrix Protein Synthesis in Podocytes.

Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase ß. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.

Activation of glycogen synthase kinase 3ß ameliorates diabetes-induced kidney injury.

Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3ß (GSK3ß) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3ß by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3ß and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3ß, indicating that NO may mediate SNP stimulation of GSK3ß. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3ß by activation of Src, Pyk2, Akt, and ERK; GSK3ß inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3ß by SNP ameliorates kidney injury induced by diabetes.

TGFß-induced deptor suppression recruits mTORC1 and not mTORC2 to enhance collagen I (α2) gene expression.

Enhanced TGFß activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFß rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFß treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFß reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFß treatment. Importantly, downregulation of deptor by TGFß increased the expression of Hif1α by increasing translation of its mRNA. TGFß-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFß-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFß-induced renal fibrosis.

microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKß-mTORC1 axis to regulate renal cancer cell invasion.

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKß, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKß blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKß. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4 and dominant negative IKKß. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor-PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells.