Balakuntalam S. Kasinath

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.

MicroRNA-21 Orchestrates High Glucose-induced Signals to TOR Complex 1, Resulting in Renal Cell Pathology in Diabetes

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3′-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.

Knockout of Toll-Like Receptor-2 Attenuates Both the Proinflammatory State of Diabetes and Incipient Diabetic Nephropathy

Objective—Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. Methods and Results—We induced T1DM in TLR2 knockout mice (TLR2−/−) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2−/− macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-&bgr; and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-&bgr; and laminin levels were decreased in TLR2−/−+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2−/−+STZ mice. Conclusion—These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3′UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.

Genome-Wide Scan for Estimated Glomerular Filtration Rate in Multi-Ethnic Diabetic Populations The Family Investigation of Nephropathy and Diabetes (FIND)

OBJECTIVE— Diabetic nephropathy, the most common cause of end-stage renal disease, aggregates in families and specific ethnic groups. Deconstructing diabetic nephropathy into intermediate, quantitative phenotypes may increase feasibility of detecting susceptibility loci by genetic screens. Glomerular filtration rate (GFR), which characterizes diabetic nephropathy, was employed as a quantitative trait in a preliminary whole-genome scan. RESEARCH DESIGN AND METHODS— Estimated GFR (eGFR) was calculated for 882 diabetic sibpairs (mean age 57 years) of African-American (25.6% of total), American Indian (8.6%), European-American (14.2%), and Mexican-American (51.6%) descent enrolled in the initial phase of the Family Investigation of Nephropathy and Diabetes (FIND). A whole-genome scan was performed using 404 microsatellite markers (average spacing 9 cM) and model-free linkage analysis. RESULTS— For all ethnicities combined, strong evidence for linkage was observed on chromosomes 1q43 (P = 3.6 × 10−3), 7q36.1 (P = 2.1 × 10−4), 8q13.3 (P = 4.6 × 10−4), and 18q23.3 (P = 2.7 × 10−3). Mexican-American families, who comprised the major ethnic subpopulation in FIND, contributed to linkage on chromosomes 1q43, 2p13.3, 7q36.1, 8q13.3, and 18q23.3, whereas African-American and American-Indian families displayed linkage peaks on chromosomes 11p15.1 and 15q22.3, respectively. CONCLUSIONS— We have demonstrated multiple chromosomal regions linked to eGFR in a multi-ethnic collection of families ascertained by a proband with diabetic nephropathy. Identification of genetic variants within these loci that are responsible for the linkage signals could lead to predictive tests or novel therapies for subsets of patients at risk for diabetic nephropathy.

Hydrogen Sulfide Inhibits High Glucose-induced Matrix Protein Synthesis by Activating AMP-activated Protein Kinase in Renal Epithelial Cells

Background: Whether hydrogen sulfide regulates protein synthesis is not known. Results: In kidney cells, hydrogen sulfide inhibited high glucose-induced synthesis of proteins including matrix proteins by activating AMP-activated protein kinase and inhibiting events in mRNA translation. Conclusion: Hydrogen sulfide reduces high glucose stimulation of matrix protein synthesis in renal cells. Significance: Hydrogen sulfide induction may inhibit kidney matrix protein accumulation in diabetes. Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H2S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

High Glucose, High Insulin, and Their Combination Rapidly Induce Laminin-β1 Synthesis by Regulation of mRNA Translation in Renal Epithelial Cells

Laminin is a glycoprotein that contributes to renal extracellular matrix expansion in diabetes. We investigated regulation of laminin-β1 synthesis in murine renal proximal tubular epithelial cells by 30 mmol/l glucose (high glucose), 1 nmol/l insulin (high insulin), and their combination (high glucose+high insulin), simulating conditions observed during progression of type 2 diabetes. Compared with 5 mmol/l glucose and no insulin (control), high glucose alone, high insulin alone, or high glucose+high insulin together increased laminin-β1 chain protein synthesis within 5 min, lasting for up to 60 min with no change in laminin-β1 mRNA levels. Cycloheximide, but not actinomycin-D, abrogated increased laminin-β1 synthesis. High glucose, high insulin, and high glucose+high insulin stimulated phosphorylation of 4E-BP1, a repressor binding protein for eukaryotic initiation factor 4E (eIF4E), that was dependent on activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin. High glucose, high insulin, and high glucose+high insulin also promoted release of eIF4E from 4E-BP1, phosphorylation of eIF4E, and increase in eIF4E association with eIF4G, critical events in the initiation phase of mRNA translation. High glucose, high insulin, and high glucose+high insulin increased Erk phosphorylation, which is an upstream regulator of eIF4E phosphorylation, and PD098059, which is a MEK inhibitor that blocks Erk activation, abolished laminin-β1 synthesis. This is the first demonstration of rapid increment in laminin-β1 synthesis by regulation of its mRNA translation by cells exposed to high glucose, high insulin, or high glucose+high insulin.

Genome-Wide Scan for Estimated GFR in Multi-Ethnic Diabetic Populations: The Family Investigation of Nephropathy and Diabetes

OBJECTIVE— Diabetic nephropathy, the most common cause of end-stage renal disease, aggregates in families and specific ethnic groups. Deconstructing diabetic nephropathy into intermediate, quantitative phenotypes may increase feasibility of detecting susceptibility loci by genetic screens. Glomerular filtration rate (GFR), which characterizes diabetic nephropathy, was employed as a quantitative trait in a preliminary whole-genome scan. RESEARCH DESIGN AND METHODS— Estimated GFR (eGFR) was calculated for 882 diabetic sibpairs (mean age 57 years) of African-American (25.6% of total), American Indian (8.6%), European-American (14.2%), and Mexican-American (51.6%) descent enrolled in the initial phase of the Family Investigation of Nephropathy and Diabetes (FIND). A whole-genome scan was performed using 404 microsatellite markers (average spacing 9 cM) and model-free linkage analysis. RESULTS— For all ethnicities combined, strong evidence for linkage was observed on chromosomes 1q43 (P = 3.6 × 10−3), 7q36.1 (P = 2.1 × 10−4), 8q13.3 (P = 4.6 × 10−4), and 18q23.3 (P = 2.7 × 10−3). Mexican-American families, who comprised the major ethnic subpopulation in FIND, contributed to linkage on chromosomes 1q43, 2p13.3, 7q36.1, 8q13.3, and 18q23.3, whereas African-American and American-Indian families displayed linkage peaks on chromosomes 11p15.1 and 15q22.3, respectively. CONCLUSIONS— We have demonstrated multiple chromosomal regions linked to eGFR in a multi-ethnic collection of families ascertained by a proband with diabetic nephropathy. Identification of genetic variants within these loci that are responsible for the linkage signals could lead to predictive tests or novel therapies for subsets of patients at risk for diabetic nephropathy.

mRNA Translation: Unexplored Territory in Renal Science

Ambient protein levels are under coordinated control of transcription, mRNA translation, and degradation. Whereas transcription and degradation mechanisms have been studied in depth in renal science, the role of mRNA translation, the process by which peptide synthesis occurs according to the genetic code that is present in the mRNA, has not received much attention. mRNA translation occurs in three phases: Initiation, elongation, and termination. Each phase is controlled by unique eukaryotic factors. In the initiation phase, mRNA and ribosomal subunits are brought together. During the elongation phase, amino acids are added to the nascent peptide chain in accordance with codon sequences in the mRNA. During the termination phase, the fully synthesized peptide is released from the ribosome for posttranslational processing. Signaling pathways figure prominently in regulation of mRNA translation, particularly the phosphatidylinositol 3 kinase-Akt-mammalian target of rapamycin pathway, the AMP-activated protein kinase-tuberous sclerosis complex protein 1/tuberous sclerosis complex protein 2-Rheb pathway, and the extracellular signal-regulated kinase 1/2 type mitogen-activated protein kinase signaling pathway; there is significant cross-talk among these pathways. Regulation by mRNA translation is suggested when changes in mRNA and protein levels do not correlate and in the setting of rapid protein synthesis. Ongoing work suggests an important role for mRNA translation in compensatory renal growth, hypertrophy and extracellular matrix synthesis in diabetic nephropathy, growth factor synthesis by kidney cells, and glomerulonephritis. Considering that mRNA translation plays an important role in cell growth, development, malignancy, apoptosis, and response to stress, its study should provide novel insights in renal physiology and pathology.

Glycogen Synthase Kinase 3β Is a Novel Regulator of High Glucose- and High Insulin-induced Extracellular Matrix Protein Synthesis in Renal Proximal Tubular Epithelial Cells

High glucose (30 mm) and high insulin (1 nm), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininβ1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3β (GSK3β) by high glucose and high insulin induces increase in synthesis of laminin β1 via activation of eIF2Bϵ. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3β at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bϵ and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3β or constitutively active kinase led to increased and diminished laminin β1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin β1 synthesis and phosphorylation of GSK3β were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3β phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3β. Status of GSK3β was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3β and decreased phosphorylation of eIF2Bϵ, which correlated with renal hypertrophy at 2 weeks, and increased laminin β1 and fibronectin protein content at 2 months. GSK3β and eIF2Bϵ play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.