Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a K i of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.

Objective To assess the influence of serum carnosinase (CN1) on the course of diabetic kidney disease (DKD). Methods hCN1 transgenic (TG) mice were generated in a BTBR Ob/Ob genetic background to allow the spontaneous development of DKD in the presence of serum carnosinase. The influence of serum CN1 expression on obesity, hyperglycemia, and renal impairment was assessed. We also studied if aggravation of renal impairment in hCN1 TG BTBR Ob/Ob mice leads to changes in the renal transcriptome as compared with wild-type BTBR Ob/Ob mice. Results hCN1 was detected in the serum and urine of mice from two different hCN1 TG lines. The transgene was expressed in the liver but not in the kidney. High CN1 expression was associated with low plasma and renal carnosine concentrations, even after oral carnosine supplementation. Obese hCN1 transgenic BTBR Ob/Ob mice displayed significantly higher levels of glycated hemoglobin, glycosuria, proteinuria, and increased albumincreatinine ratios (1104 ± 696 vs 492.1 ± 282.2 μg/mg) accompanied by an increased glomerular tuft area and renal corpuscle size. Gene-expression profiling of renal tissue disclosed hierarchical clustering between BTBR Ob/Wt , BTBR Ob/Ob, and hCN1 BTBR Ob/Ob mice. Along with aggravation of the DKD phenotype, 26 altered genes have been found in obese hCN1 transgenic mice; among them claudin-1, thrombospondin-1, nephronectin, and peroxisome proliferator-activated receptor-alpha have been reported to play essential roles in DKD. Conclusions Our data support a role for serum carnosinase 1 in the progression of DKD. Whether this is mainly attributed to the changes in renal carnosine concentrations warrants further studies. Jiedong Qiu, Thomas Albrecht and Shiqi Zhang contributed equally to this work. An abstract of this work has been awarded a poster prize on the congress of the German Diabetes Association (DDG) 2018 and on the International Congress on Carnosine and Anserine (ICCA) 2017 and has been presented on the congress of the European Association for the Study of Diabetes (EASD) 2018.