Meenalakshmi M. Mariappan

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.

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.

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.

Regulation of mRNA translation in renal physiology and disease

Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

Signaling mechanisms in the regulation of renal matrix metabolism in diabetes

Renal hypertrophy and accumulation of extracellular matrix proteins are among cardinal manifestations of diabetic nephropathy. TGF beta system has been implicated in the pathogenesis of these manifestations. Among signaling pathways activated in the kidney in diabetes, mTOR- (mammalian target of rapamycin-)regulated pathways are pivotal in orchestrating high glucose-induced production of ECM proteins leading to functional and structural changes in the kidney culminating in adverse outcomes. Understanding signaling pathways that influence individual matrix protein expression could lead to the development of new interventional strategies. This paper will highlight some of the diverse components of the signaling network stimulated by hyperglycemia with an emphasis on extracellular matrix protein metabolism in the kidney in diabetes.

Molecular events in matrix protein metabolism in the aging kidney

We explored molecular events associated with aging‐induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle‐aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age‐phase‐specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle‐aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ‐regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR‐21 and miR‐200c, but not miR‐192, miR‐200a, or miR‐200b, was increased with aging. Increased miR‐21 and miR‐200c contents were associated with reduced expression of their targets, Sprouty‐1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age‐phase‐specific regulation of matrix protein synthesis occurs and involves matrix protein–specific transcriptional and post‐transcriptional mechanisms.

Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells

Diabetes promotes protein synthesis to induce kidney hypertrophy and increase renal matrix proteins. Increased capacity for mRNA translation by way of ribosomal biogenesis facilitates sustained stimulation of protein synthesis. We tested the hypothesis that high glucose induces ribosomal biogenesis as indicated by an increase in rRNA synthesis in the setting of augmented protein synthesis. High glucose (30 mM) increased global protein synthesis, expression of matrix proteins, laminin γ1 and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388 phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, along with increased phosphorylation of Erk and p70S6 kinase. Inactivation of Erk and p70S6 kinase either by their respective chemical inhibitors or by expression of their inactive mutant constructs blocked high-glucose-induced UBF phosphorylation. High glucose reduced nuclear content of p19ARF and promoted dissolution of inactive UBF-p19ARF complex. High glucose also promoted association of UBF with RPA194, a subunit of RNA polymerase I. Inhibition of Erk, p70S6 kinase, and UBF1 by transfecting GECs with their respective inactive mutants abolished laminin γ1 synthesis, protein synthesis, and rDNA transcription. Renal cortex from type 1 diabetic rats and type 2 diabetic db/db mice showed increased phosphorylation of UBF, Erk, and p70S6 kinase coinciding with renal hypertrophy and onset of matrix accumulation. Our data suggest that augmented ribosome biogenesis occurs in an UBF-dependent manner during increased protein synthesis induced by high glucose in the GECs that correlates with UBF activation and renal hypertrophy in rodents with type 1 and type 2 diabetes.