Edited by Lukas Huber Keywords:Connective tissue growth factor Wnt Glycogen synthase kinase 3b b-Catenin LRP6 Diabetic nephropathy a b s t r a c tWe describe the activation of Wnt signalling in mesangial cells by CCN2. CCN2 stimulates phosphorylation of LRP6 and GSK-3b resulting in accumulation and nuclear localisation of b-catenin, TCF/LEF activity and expression of Wnt targets. This is coincident with decreased phosphorylation of b-catenin on Ser 33/37 and increased phosphorylation on Tyr142. DKK-1 and LRP6 siRNA reversed CCN2's effects. Microarray analyses of diabetic patients identified differentially expressed Wnt components. b-Catenin is increased in type 1 diabetic and UUO mice and in in vitro models of hyperglycaemia and hypertension. These findings suggest that Wnt/CCN2 signalling plays a role in the pathogenesis of diabetic nephropathy.

SummarySignalling interplay between transforming growth factor-b (TGFb) and CCN2 [also called connective tissue growth factor (CTGF)] plays a crucial role in the progression of diabetic nephropathy and has been implicated in cellular differentiation. To investigate the potential role of microRNAs (miRNAs) in the mediation of this signalling network, we performed miRNA screening in mesangial cells treated with recombinant human CCN2. Analysis revealed a cohort of 22 miRNAs differentially expressed by twofold or more, including members of the miR-302 family. Target analysis of miRNA to 39-untranslated regions (39-UTRs) identified TGFb receptor II (TbRII) as a potential miR-302 target. In mesangial cells, decreased TbRII expression was confirmed in response to CCN2 together with increased expression of miR-302d. TbRII was confirmed as an miR-302 target, and inhibition of miR-302d was sufficient to attenuate the effect of CCN2 on TbRII. Data from the European Renal cDNA Biopsy Bank revealed decreased TbRII in diabetic patients, suggesting pathophysiological significance. In a mouse model of fibrosis (UUO), miR-302d was increased, with decreased TbRII expression and aberrant signalling, suggesting relevance in chronic fibrosis. miR-302d decreased TGFb-induced epithelial mesenchymal transition (EMT) in renal HKC8 epithelial cells and attenuated TGFb-induced mesangial production of fibronectin and thrombospondin. In summary, we demonstrate a new mode of regulation of TGFb by CCN2, and conclude that the miR-302 family has a role in regulating growth factor signalling pathways, with implications for nephropathic cell fate transitions.

The critical involvement of TGF-β1 (transforming growth factor-β1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-β1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-β1 and its physiological significance. CTGF was determined to bind directly to the TβRIII (TGF-β type III receptor) and antagonize TGF-β1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-β1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-β1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-β1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF. Knockdown of TβRIII restored TGF-β1-mediated Smad signalling and cell contractility, suggesting that TβRIII is key for CTGF-mediated regulation of TGF-β1. Comparison of gene expression profiles from CTGF/TGF-β1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-β1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

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Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-β1 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.

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BackgroundCCN2/CTGF is an established effector of TGFβ driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFβ leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function.ResultsFrom a panel of genes regulated by TGFβ and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFβ and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy.ConclusionsTGFβ and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response.

We present a case of D-lactic acidosis presenting as a metabolic encephalopathy secondary to small intestinal bacterial overgrowth. This patient had a known history of short bowel syndrome. Of note, this case required the alteration of treatment to promote a sustained clinical and biochemical improvement. We discuss the pathophysiological mechanisms thought to be involved. We also review the current therapies as well as potential future strategies. This case highlights the importance of the prompt clinical recognition of signs and symptoms as well as the rapid initiation of management strategies to ameliorate this condition.