CCN2/CTGF increases expression of miR-302 microRNAs, which target the TGFß type II receptor with implications for nephropathic cell phenotypes.

Signalling interplay between transforming growth factor-ß (TGFß) 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 3'-untranslated regions (3'-UTRs) identified TGFß receptor II (TßRII) as a potential miR-302 target. In mesangial cells, decreased TßRII expression was confirmed in response to CCN2 together with increased expression of miR-302d. TßRII was confirmed as an miR-302 target, and inhibition of miR-302d was sufficient to attenuate the effect of CCN2 on TßRII. Data from the European Renal cDNA Biopsy Bank revealed decreased TßRII in diabetic patients, suggesting pathophysiological significance. In a mouse model of fibrosis (UUO), miR-302d was increased, with decreased TßRII expression and aberrant signalling, suggesting relevance in chronic fibrosis. miR-302d decreased TGFß-induced epithelial mesenchymal transition (EMT) in renal HKC8 epithelial cells and attenuated TGFß-induced mesangial production of fibronectin and thrombospondin. In summary, we demonstrate a new mode of regulation of TGFß 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.

TGFß and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation.

BACKGROUND: CCN2/CTGF is an established effector of TGFß driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFß 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. RESULTS: From a panel of genes regulated by TGFß 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 TGFß and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy. CONCLUSIONS: TGFß 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.

Connective tissue growth factor antagonizes transforming growth factor-ß1/Smad signalling in renal mesangial cells.

The critical involvement of TGF-ß1 (transforming growth factor-ß1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-ß1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-ß1 and its physiological significance. CTGF was determined to bind directly to the TßRIII (TGF-ß type III receptor) and antagonize TGF-ß1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-ß1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-ß1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-ß1-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 TßRIII restored TGF-ß1-mediated Smad signalling and cell contractility, suggesting that TßRIII is key for CTGF-mediated regulation of TGF-ß1. Comparison of gene expression profiles from CTGF/TGF-ß1-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-ß1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

The strategic use of biopsy in the diagnosis of coeliac disease in adults.

BACKGROUND: The paediatric guidelines support the use of the 'No Biopsy Approach' in the diagnosis of coeliac disease (CD). We aimed to determine the correlation between anti tissue transglutaminase (anti-TTG serology) ≥10 times the upper limit of normal (ULN), using the Celikey ® ELiA assay and histological findings. Our secondary aim was to determine the safety of this approach in our centre. METHODS: A retrospective analysis of adult patients referred to a tertiary referral centre with raised anti-TTG titres and/or histological changes of coeliac on D2 biopsies between 2014 - 2019. Excluded patients were those who did not have a biopsy performed, or whose biopsy was unavailable for review, selective IgA deficiency, and gluten elimination prior to biopsy. Biopsies were classified according to Marsh, by two independent pathologists, blinded to the anti-TTG titre. RESULTS: 164 patients had positive anti-TTG serology and duodenal biopsy in our centre prior to starting a gluten free diet (GFD) in the period 2014 - 2019. Of these 164 patients (median age 40yrs, 62% female), 68 (33%) had an anti-TTG titre ≥10 x ULN, 99% of which had a Marsh grading ≥ 3 and 1% had a Marsh of 2 on biopsy. 91% had either a normal index gastroscopy or findings of mild gastritis/oesophagitis. CONCLUSIONS: We found a 98.5% positive predictive value (PPV) of determining CD (i.e., Marsh ≥ 3) in those with an anti-TTG ≥10 x ULN. In those with moderate to high-risk clinical suspicion of CD we propose that duodenal biopsy is unnecessary for diagnosis.

D-lactic acidosis presenting as metabolic encephalopathy in a patient with short bowel syndrome.

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.

Is Endoscopic Band Ligation a Superior Treatment Modality for Gastric Antral Vascular Ectasia Compared to Argon Plasma Coagulation?

BACKGROUND/AIMS: Gastric antral vascular ectasia (GAVE) is a rare acquired vascular lesion of the gastric antrum. The most frequent presentation of GAVE is iron deficiency anemia. Endoscopic therapy is the mainstay of treatment. However, there is no consensus regarding the optimal treatment modality. METHODS: A retrospective cohort study was performed on patients with GAVE, including patients receiving endoscopic therapy. Treatment was with either argon plasma coagulation (APC) or endoscopic band ligation (EBL). Basic demographic data, indication for index procedure, number of sessions, and pre- and post-hemoglobin levels were collected. The aim of the study was to compare outcomes across the two treatment modalities. RESULTS: One hundred and seventeen diagnoses of GAVE were made. Sixty-two patients (53%) required endoscopic treatment for symptomatic GAVE (female, n=38, 61%; mean age of 74.4 years). Two hundred and eighteen procedures were performed during the study period. APC was performed (n=161, 74%) more frequently than EBL (n=57, 26%). Patients treated with APC at index required a median 5 subsequent therapeutic interventions (APC or EBL), while those treated with EBL at index required a further 2.9 treatments (EBL only) (p<0.05). CONCLUSION: APC was the most common treatment modality employed. We demonstrate an increasing incidence of EBL. Patients treated with EBL at index treatment required fewer subsequent treatment sessions and had a greater mean rise in hemoglobin. This suggests a more effective endoscopic response with EBL.

Coeliac disease enteropathy and symptoms may be aggravated by angiotensin receptor blockers in patients on a gluten-free diet.

BACKGROUND: Angiotensin receptor blocker-associated enteropathy (ARB-e) is an increasingly recognised clinical entity with symptoms and histological findings identical to coeliac disease (CD). There is evidence to suggest immune-mediated mucosal injury in ARB-e with a high prevalence of DQ2/DQ8; however, as IgA anti-tissue transglutaminase (anti-TTG) is usually negative, an insult other than TTG-mediated injury is suspected. The impact of ARBs on disease activity in patients with CD is not known. OBJECTIVE: To assess the effect of ARB exposure on patients with established CD. METHODS: A patient record search of 1142 individual patients attending a dedicated coeliac clinic from 2010 to the present identified 59 patients treated with ARB. Those with CD confirmed by serology (TTG + ve/EMA + ve) and histopathology (Marsh criteria) were included (n = 40, 0.52%). Data collected included disease duration, compliance with gluten-free diet (GFD), reported symptoms (diarrhoea, weight loss and abdominal pain), surrogate markers of absorption (Vitamin D, Iron, Calcium and Haemoglobin), in addition to anti-TTG titre and histological grade at last follow up. Patients were age and sex-matched in a 1:2 ratio with CD patients not taking ARBs (controls), with comparable rates of disease duration and compliance with GFD. RESULTS: The ARB and control groups were matched in terms of age (mean 66.2 years) and gender (female 63%). Strict compliance with GFD was reported in 55% and 56%, respectively. Persistent symptoms were reported in 10/40 (25%) of the ARB group compared with 7/82 (9%) of controls (p = 0.0181). There were lower rates of mucosal healing (Marsh grade 0) in the ARB group (36% n = 11) compared to controls (55%, n = 33). There was no significant difference in anti-TTG titres. Surrogate markers of absorption were comparable across the groups, except for Vitamin D which was lower in those taking olmesartan (p = 0.0015). CONCLUSIONS: ARBs may aggravate the enteropathy and lead to increased symptoms in patients with bone fide diagnosed CD following a GFD.

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR.

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKCalpha, and PKCepsilon to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed cells.

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy.

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.

CTGF/CCN2 activates canonical Wnt signalling in mesangial cells through LRP6: implications for the pathogenesis of diabetic nephropathy.

We describe the activation of Wnt signalling in mesangial cells by CCN2. CCN2 stimulates phosphorylation of LRP6 and GSK-3ß resulting in accumulation and nuclear localisation of ß-catenin, TCF/LEF activity and expression of Wnt targets. This is coincident with decreased phosphorylation of ß-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. ß-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.

DNA methylation profiling in cell models of diabetic nephropathy.

We have previously identified differentially expressed genes in cell models of diabetic nephropathy and renal biopsies. Here we have performed quantitative DNA methylation profiling in cell models of diabetic nephropathy. Over 3,000 CpG units in the promoter regions of 192 candidate genes were assessed in unstimulated human mesangial cells (HMCs) and proximal tubular epithelial cells (PTCs) compared to HMCs or PTCs exposed to appropriate stimuli. A total of 301 CpG units across 38 genes (~20%) were identified as differentially methylated in unstimulated HMCs versus PTCs. Analysis of amplicon methylation values in unstimulated versus stimulated cell models failed to demonstrate a >20% difference between amplicons. In conclusion, our results demonstrate that (1) specific DNA methylation signatures are present in HMCs and PTCs, and (2) standard protocols for exposure of renal cells to stimuli that alter gene expression may be insufficient to replicate possible alterations in DNA methylation profiles in diabetic nephropathy.