Anserine and Carnosine Induce HSP70-Dependent H2S Formation in Endothelial Cells and Murine Kidney.

Anserine and carnosine have nephroprotective actions; hydrogen sulfide (H2S) protects from ischemic tissue damage, and the underlying mechanisms are debated. In view of their common interaction with HSP70, we studied possible interactions of both dipeptides with H2S. H2S formation was measured in human proximal tubular epithelial cells (HK-2); three endothelial cell lines (HUVEC, HUAEC, MCEC); and in renal murine tissue of wild-type (WT), carnosinase-1 knockout (Cndp1-KO) and Hsp70-KO mice. Diabetes was induced by streptozocin. Incubation with carnosine increased H2S synthesis capacity in tubular cells, as well as with anserine in all three endothelial cell lines. H2S dose-dependently reduced anserine/carnosine degradation rate by serum and recombinant carnosinase-1 (CN1). Endothelial Hsp70-KO reduced H2S formation and abolished the stimulation by anserine and could be restored by Hsp70 transfection. In female Hsp70-KO mice, kidney H2S formation was halved. In Cndp1-KO mice, kidney anserine concentrations were several-fold and sex-specifically increased. Kidney H2S formation capacity was increased 2-3-fold in female mice and correlated with anserine and carnosine concentrations. In diabetic Cndp1-KO mice, renal anserine and carnosine concentrations as well as H2S formation capacity were markedly reduced compared to non-diabetic Cndp1-KO littermates. Anserine and carnosine induce H2S formation in a cell-type and Hsp70-specific manner within a positive feedback loop with CN1.

Carnosine Activates Cellular Stress Response in Podocytes and Reduces Glycative and Lipoperoxidative Stress.

Carnosine improves diabetic complications, including diabetic nephropathy, in in vivo models. To further understand the underlying mechanism of nephroprotection, we studied the effect of carnosine under glucose-induced stress on cellular stress response proteins in murine immortalized podocytes, essential for glomerular function. High-glucose stress initiated stress response by increasing intracellular heat shock protein 70 (Hsp70), sirtuin-1 (Sirt-1), thioredoxin (Trx), glutamate-cysteine ligase (gamma-glutamyl cysteine synthetase; γ-GCS) and heme oxygenase-1 (HO-1) in podocytes by 30-50% compared to untreated cells. Carnosine (1 mM) also induced a corresponding upregulation of these intracellular stress markers, which was even more prominent compared to glucose for Hsp70 (21%), γ-GCS and HO-1 (13% and 20%, respectively; all p < 0.001). Co-incubation of carnosine (1 mM) and glucose (25 mM) induced further upregulation of Hsp70 (84%), Sirt-1 (52%), Trx (35%), γ-GCS (90%) and HO-1 (73%) concentrations compared to untreated cells (all p < 0.001). The glucose-induced increase in 4-hydroxy-trans-2-nonenal (HNE) and protein carbonylation was reduced dose-dependently by carnosine by more than 50% (p < 0.001). Although podocytes tolerated high carnosine concentrations (10 mM), high carnosine levels only slightly increased Trx and γ-GCS (10% and 19%, respectively, compared to controls; p < 0.001), but not Hsp70, Sirt-1 and HO-1 proteins (p not significant), and did not modify the glucose-induced oxidative stress response. In podocytes, carnosine induced cellular stress tolerance and resilience pathways and was highly effective in reducing high-glucose-induced glycative and lipoperoxidative stress. Carnosine in moderate concentrations exerted a direct podocyte molecular protective action.