Key points: Increased activation of the renin‐angiotensin‐aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes‐induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia‐induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis. In diabetic kidney disease (DKD) increased activation of renin‐angiotensin‐aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin‐induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet‐derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)‐induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha‐smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK‐2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia‐induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non‐antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.

Diabetic kidney disease is a worldwide epidemic, and therapies are incomplete. Clinical data suggest that improved renal outcomes by Na⁺-glucose cotransporter 2 inhibitor (SGLT2i) are partly beyond their antihyperglycemic effects; however, the mechanisms are still elusive. Here, we investigated the effect of the SGLT2i dapagliflozin (DAPA) in the prevention of elevated O-GlcNAcylation and tubular hypoxia as contributors of renal fibrosis. Type 1 diabetes was induced by streptozotocin in adult male Wistar rats. After the onset of diabetes, rats were treated for 6 wk with DAPA or DAPA combined with losartan (LOS). The effect of hyperglycemia was tested in HK-2 cells kept under normal or high glucose conditions. To test the effect of hypoxia, cells were kept in 1% O₂ for 2 h. Cells were treated with DAPA or DAPA combined with LOS. DAPA slowed the loss of renal function, mitigated renal tubular injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), and reduced tubulointerstitial fibrosis. DAPA diminished high glucose-induced protein O-GlcNAcylation and moderated the tubular response to hypoxia through the hypoxia-inducible factor pathway. DAPA alone was as effective as combined treatment with LOS in all outcome parameters. These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia, and fibrosis, a vicious trio that could be targeted by SGLT2i in kidney diseases of other origins as well.

Background: Renal fibrosis is the hallmark of chronic kidney disease (CKD) and is characterized by an imbalanced extracellular matrix remodelling. Endotrophin (ETP) is a signalling molecule released from collagen type VI (COL VI). ETP can be measured by the PRO-C6 assay, which quantifies the levels of COL VI formation. ETP levels were previously associated with mortality and disease progression in patients with CKD. We hypothesized that serum and urinary ETP levels correlate with the degree of interstitial fibrosis in kidney biopsies from patients with immunoglobulin A nephropathy (IgAN) and patients with anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV).Methods: We examined a cohort of 49 IgAN and 47 AAV patients. A validation cohort of 85 IgAN patients was included. ETP was measured in serum (S-ETP) and urine (U-ETP/Cr) samples, taken on the same day before renal biopsy was performed, using the enzyme-linked immunosorbent assay PRO-C6. The biopsies were evaluated for interstitial fibrosis and tubular atrophy according to the Banff and MEST-C scores.Results: S-ETP and U-ETP/Cr levels correlated with kidney function, increased CKD severity, correlated with the extent of interstitial fibrosis and gradually increased with increasing degree of interstitial fibrosis and tubular atrophy. ETP outperformed the known fibrosis biomarker Dickkopf-3 for discrimination of patients with high fibrotic burden. The association of S-ETP and U-ETP/Cr with the level of kidney fibrosis was confirmed in the validation cohort.Conclusions: We demonstrated that high levels of circulating and excreted ETP are not only indicative of lower kidney function, but also reflect the burden of fibrosis in the kidneys.

We evaluated biomarkers related to kidney fibrosis for the outcome of patients with IgA nephropathy (IgAN). Clinical parameters (estimated glomerular filtration rate, hypertension, proteinuria) and histological findings were assessed in 134 patients with IgAN at the time of diagnosis and followed up prospectively (mean follow-up time, 56.5 months). We measured biomarkers of collagen and laminin turnover in serum and urine collected at the time of kidney biopsy using a novel enzyme-linked immunosorbent assay. Linear discriminant analysis and logistic regression models were used to predict the patient’s kidney outcome. Five serum and urine biomarkers of laminin and collagen turnover (sLG1M, sPRO-C3, sPRO-C6, uPRO-C6/Cr, uC3M/Cr) could significantly differentiae IgAN patients with a worse prognosis. Clinical parameters (glomerular filtration rate (GFR), proteinuria) distinguished patients at risk of IgAN progression with a specificity of 87.3% and a sensitivity of 45.2% (area under the curve-AUC 0.751). The addition of the biomarkers significantly increased the prognostic ability with a specificity of 85.1% and a sensitivity of 73.3% (AUC 0.905). We have identified three serum (sLG1M, sPRO-C3, sPRO-C6) and two urinary markers (uPRO-C6/Cr, u-C3M /Cr) that significantly improve the prognostic ability of markers of kidney function to identify an IgAN patient’s risk of progressing to ESKD.

Key Points: In patients with AKI, endotrophin can predict long-term mortality and has a stronger association with mortality than eGFR. In patients with AKI, endotrophin can predict kidney disease progression at later time points and is superior to eGFR. Background: AKI involves a rapid decrease in kidney function that may be associated with structural damage. Early markers predicting AKI are emerging, but tools to assess patients’ long-term health risks after AKI are still lacking. Endotrophin (ETP) is a bioactive molecule released during the formation of collagen type VI. We evaluated the potential of circulating ETP as a prognostic biomarker of adverse outcomes after AKI. Methods: We measured ETP in plasma samples collected 1 year after an episode of AKI, using the PRO-C6 ELISA in 801 patients (393 patients with AKI and 408 controls) from the prospective AKI Risk in Derby (ARID) study (ISRCTN25405995), who were then followed until year 3. Kidney disease progression was defined as ≥25% decline in eGFR combined with a decline in CKD stage. Results: ETP levels were significantly higher in the AKI group compared with controls (P<0.001). In the AKI group, ETP could discriminate patients with kidney disease progression at year 3 (AUC=0.67, P<0.01), whereas eGFR could not (AUC=0.51, P=0.57). In logistic regression including common risk factors, ETP was independently associated with kidney disease progression in patients with AKI (OR=1.10, P<0.01). ETP could discriminate survivors from nonsurvivors at year 3 (AUC=0.64, P<0.01). In a Cox proportional hazards regression for mortality after AKI that included common risk factors, only ETP (HR=1.05; P<0.001) and age (HR=1.06, P<0.01) were retained in the final model. Conclusions: Patients in the AKI group had higher levels of plasma ETP at year 1 as compared with those who had not had AKI. In the AKI group, ETP levels predict kidney disease progression and mortality. Because ETP is a profibrotic molecule, our findings may indicate that ETP identifies patients with active fibrogenesis after AKI, suggestive of long-term renal remodeling, which is associated with patient outcome.

Kidney fibrosis is the hallmark of chronic kidney disease (CKD) and is characterized by an imbalanced extracellular matrix (ECM) remodeling. Collagen type III is one of the main ECM components of the interstitial matrix of the kidney. We hypothesized that measuring three biomarkers of collagen type III reflecting different aspects of this protein turnover (C3M, C3C, and PRO-C3) may provide different information about the fibrotic burden in patients with IgA nephropathy (IgAN). We examined a cohort of 134 patients with IgAN. The three collagen type III biomarkers were measured in serum (S) and in urine (U) samples taken on the same day before kidney biopsy was performed. Biopsies were evaluated for interstitial fibrosis and tubular atrophy, according to the Banff and MEST-C scores. S-PRO-C3 and S-C3C correlated with the degree of fibrosis in the biopsy, whereas U-C3M/Cr had an inverse correlation with fibrosis. U-C3M/Cr had the highest discrimination ability for advanced fibrosis, which was maintained after adjustment for the other collagen type III biomarkers, proteinuria, and serum creatinine. The data presented in this study indicate that measuring the different fragments of the same ECM protein and in different matrices provides a variety of information regarding pathological kidney tissue alterations in patients with IgAN.