P0967ENHANCING PODOCYTE REGENERATION WITH BROMOINDIRUBIN-3´-OXIME (BIO) AS A POTENTIAL TERAPEUTIC TARGET IN DIABETIC KIDNEY DISEASE
Authors: Mesas Irene , Swiederska Monika , Anders Hans-Joachim , Anguiano Lidia 
Published: June 2020

Abstract

Background and Aims
Diabetic nephropathy (DN) is a leading cause of end-stage kidney disease. RAS and SGLT2 inhibitors are well established in reducing the progression of DN in early stages but true tissue regeneration is not achieved. BIO, a glucose synthase kinase 3 inhibitor (GSK3β), improves outcomes in non-diabetic kidney injury by enhancing podocyte regeneration from local podocyte progenitors. We hypothesized that treating podocytes in hyperglycemic conditions with BIO could revert the oxidative stress damage typically seen in DN.

Method
To study the effect of BIO on podocytes, we isolated glomeruli from wildtype mice by perfusing them with magnetic beads and stimulated the purified glomeruli for 24 hours with or without 10µM BIO in presence or absence of 30mM glucose. The response to the stimuli was assessed by characterizing the overall gene expression via RT-qPCR. Additionally, differentiated murine podocytes (K5P5) and murine glomerular endothelial cells (GEnCs) were stimulated under the same conditions with the same subsequent readouts to differentiate the response of podocytes from that of the endothelial compartment.
To understand, if BIO could represent a potential new therapeutic option under the aspect of pharmacological safety, we uninephrectomized male BKS-Lepr-/- mice, fed them with a salt-free chow to induce DN. To mimic the human condition more closely we treated the mice with and without BIO, but in presence of the human standard of care therapy (SOC; metformin, ramipril, empagliflozin), which could potentially identify adverse pharmacological interactions. After 16 weeks kidney, heart, pancreas, sciatic nerve, liver, blood, skeletal muscle and adipose tissue were collected for histopathological and gene expression analysis.

Results
While glucose exposure induced a strong reduction of nephrin gene expression in whole glomeruli, BIO reverted the effect, indicating a protective effect on podocytes (30 mM glucose: 0.447, ci 0.224 – 0.724; 30 mM glucose+BIO: 1.837, ci 1.205 – 2.505, p-value=0.029, Fig.A). Similar results were found in differentiated murine podocytes, in which BIO treatment significantly increased podocin gene expression after 7 days of culture (0.920, ci 0.820 – 1.34, 3.561 ci 1.756 – 7.909, p-value=0.029), also here suggesting a protective effect of BIO on glucose-induced podocyte (Fig.B). In contrast, BIO showed no effect on endothelial cell-specific markers in murine GEnCs, indicating a podocyte-specific effect of BIO.
A comparable effect of BIO on podocytes was observed in vivo, as well. Diabetic mice treated with SOC and BIO showed borderline increased renal podocin gene expression levels (2.177 ci 0.830 – 2.739) compared to SOC alone (1.188 ci 0.378 – 2.194 ,p-value = 0.064), but did not modify markers of inflammation, such as IL-6, or fibrosis, such as fibronectin (Fig.C). The extended safety analysis of non-renal organs did not reveal any safety concern of the SOC/BIO combination in comparison to SOC alone in any of the analyzed tissues.

Conclusion
The current study revealed that BIO has protective effects on podocytes in vitro and might as well have renoprotective effects in DN in vivo by enhancing podocyte regeneration without showing any safety-related issues.