Leptin relieves ischemia/reperfusion induced acute kidney injury through inhibiting apoptosis and autophagy

Abstract

Objectives: Acute kidney injury (AKI) can be caused by ischemia/reperfusion (I/R), nephrotoxin, and sepsis, with poor prognosis and high mortality. Leptin is a protein molecule that regulates the body's energy metabolism and reproductive activities via binding to its specific receptor. Leptin can inhibit cardiomyocyte apoptosis caused by I/R, but its effect on I/R kidney injury and the underlying mechanisms are still unclear. This study aims to investigate the effect and mechanisms of leptin on renal function, renal histopathology, apoptosis, and autophagy during acute I/R kidney injury.

Methods: Healthy adult male mice were randomly divided into 4 groups: a sham+wild-type mice (ob/+) group, a sham+leptin gene-deficient mice (ob/ob) group, an I/R+ob/+ group, and an I/R+ob/ob group (n=8 per group). For sham operation, a longitudinal incision was made on the back of the mice to expose and separate the bilateral kidneys and renal arteries, and no subsequent treatment was performed. I/R treatment was ischemia for 30 min and reperfusion for 48 h. The levels of BUN and SCr were detected to evaluate renal function; HE staining was used to observe the pathological changes of renal tissue; TUNEL staining was used to observe cell apoptosis, and apoptosis-positive cells were counted; Western blotting was used to detect levels of apoptosis-related proteins (caspase 3, caspase 9), autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3 I, LC3 II], mTOR-dependent signaling pathway proteins [phosphate and tension homology (PTEN), adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (AKT), extracellular regulated protein kinase (ERK), phosphorylated PTEN (p-PTEN), phosphorylated AMPK (p-AMPK), phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK)].

Results: There was no significant difference in the levels of BUN and SCr between the sham+ob/+ group and the sham+ob/ob group (both P>0.05). The levels of BUN and SCr in the I/R+ob/+ group were significantly higher than those in the sham+ob/+ group (both P<0.05). Compared with the mice in the sham+ob/ob group or the I/R+ob/+ group, the levels of BUN and SCr in the I/R+ob/ob group were significantly increased (all P<0.05). There was no obvious damage to the renal tubules in the sham+ob/+ group and the sham+ob/ob group. Compared with sham+ob/+ group and sham+ob/ob group, both the I/R+ob/+ group and the I/R+ob/ob group had cell damage such as brush border shedding, vacuolar degeneration, and cast formation. Compared with the I/R+ob/+ group, the renal tubules of the mice in the I/R+ob/ob group were more severely damaged. The pathological score of renal tubular injury showed that the renal tubular injury was the most serious in the I/R+ob/ob group (P<0.05). Compared with the sham+ob/+ group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, the ratio of LC3 II to LC3 I was significantly increased, and the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/+ group (all P<0.05). Compared with the sham+ob/ob group, the protein levels of caspase 3, caspase 9, PTEN, and LC3 II were significantly up-regulated, and the ratio of LC3 II to LC3 I was significantly increased, while the protein levels of p-mTOR, p-PTEN, p-AMPK, p-AKT, and p-ERK were significantly down-regulated in the I/R+ob/ob group (all P<0.05). Compared with the I/R+ob/+ group, the levels of p-mTOR, p-PTEN, p-AMPK, p-AKT were more significantly down-regulated, while the levels of caspase 3, caspase 9, PTEN, and LC3 II were more significantly up-regulated, and the ratio of LC3 II to LC3 I was more significantly increase in the I/R+ob/ob group (all P<0.05).

Conclusions: Renal function and tubular damage, and elevated levels of apoptosis and autophagy are observed in mice kidneys after acute I/R. Leptin might relieve I/R induced AKI by inhibiting apoptosis and autophagy that through a complex network of interactions between mTOR-dependent signaling pathways.

Keywords: acute kidney injury; apoptosis; autophagy; ischemia/reperfusion; leptin.

图1

PCR产物凝胶电泳鉴定ob/ob小鼠的基因型 Figure 1 Identification of genotype in ob/ob mice by PCR product gel electrophoresis

图2

各组小鼠BUN(A)和SCr(B)水平的比较 Figure 2 Comparison of level of BUN (A) and SCr (B) in mice of each group *P<0.05 vs the sham+ob/+ group; †P<0.05 vs the sham+ob/ob group; ‡P<0.05 vs the I/R+ob/+ group.

图3

各组小鼠肾组织病理变化 Figure 3 Pathological changes of kidney in mice of each group Representative histology of sham+ob/+ group (A), I/R+ob/+ group (B), sham+ob/ob group (C), and I/R+ob/ob group (D) shown by HE staining (×400). Percentage of injured tubules evaluated by counting the renal tubules with signs of injury (E). *P<0.05 vs the sham+ob/+ group; †P<0.05 vs the sham+ob/ob group; ‡P<0.05 vs the I/R+ob/+ group.

图4

各组小鼠肾组织TUNEL染色及TUNEL阳性细胞计数 Figure 4 TUNEL staining and quantification of TUNEL positive cells of renal tissues in mice of each group A: Representative images of TUNEL staining (×400); B: Quantification of TUNEL positive cells. *P<0.05 vs the sham+ob/+ group; †P<0.05 vs the sham+ob/ob group; ‡P<0.05 vs the I/R+ob/+ group.

图5

蛋白质电泳图示各组小鼠肾组织凋亡和自噬相关蛋白质的表达 Figure 5 Protein electrophoresis showing expression of apoptosis and autophagy related proteins in renal tissues of mice in each group

图6

各组小鼠肾组织mTOR依赖的信号通路蛋白质的表达水平 Figure 6 Histogram of protein electrophoresis results showing expression levels of apoptosis and autophagy related proteins in renal tissues of mice in each group *P<0.05 vs the sham+ob/+ group; †P<0.05 vs the sham+ob/ob group; ‡P<0.05 vs the I/R+ob/+ group.