MO625INHIBITION OF SGLT-2 CANNOT RESCUE NEPHRIN EXPRESSION IN DIABETIC NEPHROPATHY. (29th May 2021)

• Record Type: Journal Article
• Authors: Kotb, Ahmed; Klanke, Bernd; Kroll, Jens; Schiffer, Mario
• Is Part Of: Nephrology dialysis transplantation. Volume 36(2021)Supplement 1
• Publication Date: 2021-05-29
• Subjects: Nephrology -- Periodicals; Hemodialysis -- Periodicals; Kidneys -- Transplantation -- Periodicals; Hemodialysis; Kidneys -- Transplantation; Nephrology; Periodicals; 616.61
• DOI: 10.1093/ndt/gfab093.006
• Languages: English
• ISSNs: 0931-0509

Abstract

Abstract: Background and Aims: Early glomerular damage in diabetes is induced by high blood glucose level (hyperglycemia) which affects the glomerular filtration barrier and leads to proteinuria. Podocyte-specific proteins like the transmembrane protein nephrin form the slit diaphragm that is important for proper function of the glomerular filtration barrier. Sodium-glucose co-transporter 2 (SGLT2) specific proteins are involved in glucose reabsorption in the kidney and maintain the normal glucose level in the blood. Recent studies showed remarkable success of SGLT2-inhibition in patients with diabetic nephropathy. Therefore we wanted to study if hyperglycemia induced reduction of nephrin expression is affected bySGLT-2 inhibition. Therefore we induced hyperglycemia in zebrafish larvae by knockdown of Pancreatic duodenal homeobox 1 (Pdx1) transcription factor and treated zebrafish larvae with Empagliflozin. In parallel we treated Bl/6 mice with streptozotozin and treated them with Empagliflozin. We then analyzed nephrin expression in both model systems. Method: Zebrafish is an ideal model to study glomerular diseases, because the zebrafish larvae develops a pronephros with high homology to the human glomerulus. In order to inhibit SLGT-2 after Pdx1-knockdown, we treated both control and diabetic zebrafish larvae with 10µM Empagliflozin from 1dpf to 5dpf. We used a zebrafish line that expresses a fluorescent Vitamin D binding plasma protein Tg(l-fabp:DBP:eGFP) to measure proteinuria by measuring the GFP signal in both retinal and glomerular vessels of 120 hpf larvae. Immunohistochemistry against nephrin was performed using a specific zebrafish antibody. Results: Pdx1 knockdown induces a glomerular phenotype We found that Pdx1 knockdown larvae developed pericardial edema and proteinuria two hallmarks for kidney disease. To investigate the efficiency of the glomerular filtration barrier, we measured the clearance of the GFP-vitamin D-binding protein by measuring fluorescence activity of the retinal blood vessel plexus and in glomerular vessels. We found that GFP signal in the retinal (Eye assay) and glomerular (Cryosections) vessels was markedly decreased after Pdx1 knockdown indicating a leakage of the filtration barrier compared to control injected larvae. In contrast to control, Pdx1 knockdown resulted in significant changes of the morphology of the glomerulus. Pdx1 knockdown glomeruli display a dilated Bowman's space and capillaries. Immunostaining with a specific antibody for zebrafish nephrin, a podocyte-specific protein essential for blood filtration, revealed that the expression of nephrin was significantly decreased in podocytes of Pdx1 knockdown larvae in contrast to podocytes of the control larvae. SLGT-2 inhibitor could not rescue the diabetic glomerular phenotype In order to investigate the SLGT-2 inhibitor effect on the diabetic phenotype, we treated diabetic zebrafish larvae with Empagliflozin. 5 dpf Empagliflozin treated larvae still continuously develop pericardial edema. In addition, Empagliflozin treated larvae showed low GFP signal in the retinal vessels (Eye assay) compare to control, confirming that Empagliflozin treated larvae still have a leaky glomerular filtration barrier. A confirmatory experiment with SLGT-2 inhibitor treatment of mice after STZ induced diabetes showed similar results. Conclusion: Despite the promising effects of SLGT-2 inhibitor treatment in patients with diabetic nephropathy the early effects on nephrin expression are not addressed and remain an unchanged problem by this novel treatment option.