P0972INHIBITION OF LYSINE63 UBIQUITINATION PREVENTS THE PROGRESSION OF RENAL FIBROSIS IN DIABETIC NEPHROPATHY IN VITRO AND IN VIVO
Authors: Conserva Francesca , Pontrelli Paola , Menghini Rossella , Rossini Michele , Stasi Alessandra , Divella Chiara , Cinefra Claudia , Simone Simona , Pertosa Giovanni Battista , Gallone Anna , Federici Massimo , Gesualdo Loreto 
Published: June 2020

Abstract

Background and Aims
Diabetic Nephropathy (DN) is the primary cause of end stage renal disease (ESRD). Our group demonstrated that in DN an accumulation of lysine63 (K63)-ubiquitinated proteins at tubular level is involved in the progression of renal damage, in particular renal fibrosis. Current treatments do not provide complete renoprotection and targeted therapies that prevent fibrosis or delay its progression are still lacking.
Aim of the present study was to evaluate the renoprotective effect of specific drug and their combinations, including an inhibitor of K63 ubiquitination (K63Ub) and/or an anti-hypertensive agent, in vitro and in vivo in a murine model of DN.

Method
Renal Proximal Tubule Epithelial Cells (HK2) were pre-incubated with a specific inhibitor of K63Ub and/or with the ACE-inhibitor Ramipril. Accumulation of K63 ubiquitinated proteins along with α-sma expression, indicator of epithelial-to-mesenchymal transition (EMT), were analyzed through immunofluorescence and western blotting. The same drug combination was also tested in streptozotocin (STZ)-treated DBA/2J mice, a model of human DN. In mice, K63Ub was evaluated by IHC, while renal fibrosis was evaluated by Sirius red and Collagen III expression. Urinary albuminuria was measured by ELISA.

Results
We observed that the association of the specific K63Ub inhibitor with Ramipril was able to block hyperglycemia-induced EMT in HK2 cells by significantly reducing α-sma expression, when compared to single drugs alone (p&lt;0.05).To demonstrate the efficacy of these drug combinations in reducing the progression of renal damage in DN we firstly confirmed the increased accumulation of K63 Ub proteins in DBA/2J STZ-treated mice (p=0.01). Interestingly, increased K63Ub in diabetic mice was also associated to increased tubular-interstitial fibrosis (p&lt;0.05). Treatment of STZ-mice with the specific K63Ub inhibitor was able to reduce both K63Ub proteins accumulation and renal fibrosis, evaluated on kidney samples by IHC against Collagen III (p≤0.05) and by Sirius Red staining (p≤0.05) when compared to both untreated mice and mice treated with ramipril. Importantly, treatment with the K63Ub inhibitor alone did not reduce albuminuria (STZ-mice: 561.29±390.56; STZ+K63Ubinhibitor: 724.25±690.89; p=n.s.), while the drug combination including the specific K63Ub inhibitor and Ramipril, significantly reduced both K63Ub-related fibrosis and albuminuria (p=0.01), demonstrating an addictive and synergic effect of these molecules when used in combination.

Conclusion
Our data demonstrated and confirmed the importance of K63Ub in the progression of renal fibrosis in vitro and in vivo. We proposed and patented a novel combination of drugs that ameliorates both fibrosis and proteinuria in DN. Novel treatment regimens could represent an important goal for reducing the incidence of ESRD related to diabetes complication.