Abstract
Abstract Background and Aims Diabetic kidney disease (DKD) is a major cause of chronic kidney disease and end stage renal disease, therefore identification of novel therapeutic strategies that reduce the risk of DKD is a research priority. Recent large clinical trials suggest that improved renal outcomes by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects. Enhanced glucose reabsorption in diabetes leads to tubular hypoxia triggering fibrotic response. Hyperglycemia is in strong association with increased protein O-GlcNAcylation, a post-translational modification contributing to renal fibrosis. Considering the proximal tubular involvement in DKD pathogenesis and the key role of SGLT2 in glucose metabolism, here we investigated the effects of SGLT2i on tubular hypoxia and O-GlcNAcylation. Method Diabetes (D) was induced by streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day, po.). Metabolic parameters and renal function were evaluated. Novel urinary biomarkers of extracellular matrix remodeling (Pro-C3, uC3M, tumstatin) and profibrotic growth factors (TGF-β, CTGF, PDGF) were determined. Histological evaluation of glomerular damage (PAS), tubulointerstitial fibrosis (Masson’s trichrome, Picrosirius red) and fibronectin accumulation were performed. The effect of hyperglycemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions for 24 hours. HG cells were treated with 10 µM DAPA. O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber (1% O2) for 2 hours. Hypoxic injury was investigated using three different methods (qRT-PCR, Western blot, immunofluorescence analysis). HIF-1α, EPO, VEGFA and profibrotic factors were measured. Results DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p<0.05) and improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p<0.01). In parallel, novel urinary biomarkers of extracellular matrix remodeling, profibrotic growth factor expressions and extensive fibrotic tissue accumulation were reduced in the kidney. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Moreover, DAPA treatment prevented HIF-1α translocation to the nucleus, thereby confirming abolished HIF-1α activation. EPO, VEGFA and profibrotic factor levels were also increased in hypoxia and DAPA prevented EPO, TGFB and PDGF elevation. Conclusion 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, which seem to be targeted by SGLT2i. All these mechanisms are important parts in the puzzle of the complex system behind the protective effect of SGLT2i. OTKA-FK124491-K135398, 2017-1.3.1-VKE-2017-00006, 2020-4.1.1.-TKP2020-6183169273, 2020-4.1.1.-TKP2020-6183069269
Published Version
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