To observe the effects of calcium oxalate crystals on oxidative damage, apoptosis and crystal adhesion in mice by regulating Klotho gene.

A total of 30 8-week-old C57BL/6 male mice in the Animal Laboratory of Xinjiang Medical University were selected to establish the kidney stone model of overexpressing/silencing Klotho gene. The models were randomly divided into Ctrl, model, model+ vector, shKlotho+ model and Klotho+ model groups. Western blotting and polymerase chain reaction (PCR) were used to detect the expression of protein-grade messenger RNA (mRNA), and enzyme-linked immunosorbent assay (ELISA) was used to detect the level of oxidative stress protein. The pathological changes of kidney were observed by hematoxylin eosin staining and the adhesion of calcium oxalate crystals was observed by Von Kossa staining. Apoptosis in renal tissue was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The statistical difference between the two groups was tested by t-test, and the statistical difference between multiple groups was analyzed by single factor analysis of variance.

The model of calcium oxalate stone in mice was established in vivo and verified by Von Kossa staining. In model group, the expression of CAT, GSH and HO-1 decreased significantly, the expression of lactate dehydrogenase (LDH) [(353.89±51.37) U/L vs. (179.07±25.87) U/L] increased significantly, and that of GSH decreased significantly in Ctrl group. The difference was statistically significant (F=25.785, 39.313, 38.321, 139.411, P<0.01). HE and Von Kossa staining showed that part of renal tubules were degenerative and necrotic in model group, and the deposition and adhesion area of calcium oxalate crystals in Klotho+ model group was smaller than that in shKlotho+ model group [(9.50±0.54)% vs. (2.16±0.75)%]. The results of TUNEL staining showed that apoptosis in renal tissue of model group increased significantly. The level of apoptosis in shKlotho+ Model group was significantly higher than that in Klotho+ Model group [(13.33±0.52)% vs. (3.83±0.75)%]. In model group, the mRNA and protein expression of cysteinyl aspartate-specific protease (Caspase)-3, the key protein of apoptosis, was significantly up-regulated [(2.15±0.29) ng/ml], while the mRNAexpression of B-lymphomatoma 2 (bcl-2) was significantly down-regulated [(0.48±0.05) U/L], indicating the occurrence of apoptosis. The mRNA and protein levels of E2 related factors (Nrf2), HO-1 [(0.24±0.04) U/L] and NQO-1 [(1.01±0.11) U/L] in shKlotho+ Model group were lower than those in Klotho+ Model group (F=139.461, 140.063, 115.110, P<0.01).

Klotho inhibits oxidative damage, apoptosis and crystal adhesion of calcium oxalate stone mice through Keap1-Nrf2-ARE signal pathway.

Calcium oxalate stone; Oxidative stress; Klotho