ABSTRACT
Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.
Subject(s)
Diabetic Nephropathies/metabolism , Gene Expression , Insulin Receptor Substrate Proteins/metabolism , Kidney Tubules/metabolism , Adolescent , Adult , Animals , Base Sequence , Binding Sites , Bone Morphogenetic Protein 7/physiology , Case-Control Studies , Cell Line , Child , Epithelium/metabolism , Female , Humans , Insulin Receptor Substrate Proteins/genetics , Kidney Tubules/pathology , Male , Mice , Middle Aged , Phosphorylation , Protein Processing, Post-Translational , Signal Transduction , Smad4 Protein/genetics , Transcriptional Activation , Young AdultABSTRACT
It is clear that the well-described phenomenon of epithelial-mesenchymal transition (EMT) plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. EMTs have been classified into three subtypes based on the functional consequences and biomarker context in which they are encountered. This review will highlight findings on type II EMT as a direct contributor to the kidney myofibroblast population in the development of renal fibrosis, specifically in diabetic nephropathy, the signalling molecules and the pathways involved in type II EMT and changes in the expression of specific miRNA with the EMT process. These findings have provided new insights into the activation and development of EMT during disease processes and may lead to possible therapeutic interventions to suppress EMTs and potentially reverse organ fibrosis.
Subject(s)
Epithelial-Mesenchymal Transition/physiology , Fibrosis/pathology , Kidney Diseases/pathology , Kidney/pathology , Animals , Biomarkers/metabolism , Diabetic Nephropathies/pathology , Humans , MicroRNAs/metabolism , Signal Transduction/physiologyABSTRACT
Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3-kinase and mitogen-activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)-ß1 is the primary cytokine shown to induce epithelial-mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF-ß1-induced epithelial-mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF-ß1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)-mediated targeting of IRS2 increased E-cadherin expression, although it did not alter TGF-ß1-mediated E-cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24 h greatly reduced FoxO3a phosphorylation on Ser253 but over-expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF-ß1-mediated E-cadherin repression at 24 h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E-cadherin.
Subject(s)
Cadherins/metabolism , Forkhead Transcription Factors/metabolism , Insulin Receptor Substrate Proteins/metabolism , Kidney Tubules, Proximal/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Antigens, CD , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Cell Line, Transformed , Cells, Cultured , Epithelial-Mesenchymal Transition , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Gene Silencing , HEK293 Cells , Humans , Insulin Receptor Substrate Proteins/antagonists & inhibitors , Insulin Receptor Substrate Proteins/genetics , Kidney Glomerulus/cytology , Kidney Glomerulus/metabolism , Membrane Proteins/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Phosphorylation , Protein Processing, Post-Translational , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering , Superoxide Dismutase/metabolismSubject(s)
Diabetes Complications/physiopathology , MicroRNAs/physiology , Animals , Cardiomyopathies/etiology , Diabetes Complications/genetics , Diabetes Mellitus, Type 2/genetics , Diabetic Nephropathies/etiology , Diabetic Nephropathies/genetics , Diabetic Retinopathy/etiology , Humans , Myocardium/metabolism , Receptor for Advanced Glycation End Products , Receptors, Immunologic/physiologyABSTRACT
BACKGROUND: Male Irs2-/- mice develop fatal type 2 diabetes at 13-14 weeks. Defects in neuronal proliferation, pituitary development and photoreceptor cell survival manifest in Irs2-/- mice. We identify retarded renal growth in male and female Irs2-/- mice, independent of diabetes. RESULTS: Kidney size and kidney:body weight ratio were reduced by approximately 20% in Irs2-/- mice at postnatal day 5 and was maintained in maturity. Reduced glomerular number but similar glomerular density was detected in Irs2-/- kidney compared to wild-type, suggesting intact global kidney structure. Analysis of insulin signalling revealed renal-specific upregulation of PKBbeta/Akt2, hyperphosphorylation of GSK3beta and concomitant accumulation of beta-catenin in Irs2-/- kidney. Despite this, no significant upregulation of beta-catenin targets was detected. Kidney-specific increases in Yes-associated protein (YAP), a key driver of organ size were also detected in the absence of Irs2. YAP phosphorylation on its inhibitory site Ser127 was also increased, with no change in the levels of YAP-regulated genes, suggesting that overall YAP activity was not increased in Irs2-/- kidney. CONCLUSIONS: In summary, deletion of Irs2 causes reduced kidney size early in mouse development. Compensatory mechanisms such as increased beta-catenin and YAP levels failed to overcome this developmental defect. These data point to Irs2 as an important novel mediator of kidney size.
Subject(s)
Glycogen Synthase Kinase 3/metabolism , Insulin Receptor Substrate Proteins/metabolism , Kidney/growth & development , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Female , Glycogen Synthase Kinase 3 beta , Insulin/metabolism , Insulin Receptor Substrate Proteins/genetics , Male , Mice , Organ Size , Phosphoproteins/genetics , Phosphorylation , Signal Transduction , YAP-Signaling ProteinsABSTRACT
The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.
