3.1. Renal injury was attenuated in Empa treated mice.
The Schematic diagram of the experimental procedure was shown in Figure 1A. Remarkable increase of body and kidney weight was detected in KK-AY group and these changes were rescued partially in Empa treated mice (Figure 1B, C, E). Meanwhile, the urinary NGAL (ng/24h), and urinary NAG (U/24h), two markers of tubular injury increased obviously in KK-AY group at the 10th week though no significant change of urinary microalbumin (μg/24h) was found (Figure 1F, G, H). With the prolongation of high-fat diet breeding, an obvious increase of 24h urinary microalbumin (ug/24h) was detected in KK-AY group at 16th week, and these trends were reversed in the Empa group (Figure 1 F, G, H).The HE stains of kidney showed obvious tubular vacuolation and dilatation in KK-AY group, and the Empa alleviate these alterations partially (Figure 1D).
3.2. The apoptosis in KK-Ay mice kidney were alleviated in Empa treated mice.
Apoptosis is a form of programmed cell death which has been proved to be connected to DKD. In present study, the number of TUNEL positive cells increased dramatically in KK-AY group, and this trend was reversed significantly in Empa group (Fig. 2B). Proteins related to cellular apoptosis were also measured by Western Blotting. We found that in KK-AY group, the expression of pro-apoptotic proteins such as BAX, Cleaved-CASPSE 3 increased significantly, whereas the anti-apoptotic protein BCL-2 reduced obviously, all these changes were reversed in Empa group (Fig. 2A, D). Cytochrome c (CYT), which is released from mitochondria to cytoplasm in the process of apoptosis plays a critical role in the activating of CASPASE. Here we found that the CYT in mitochondria decreased obviously in the KK-AY group and, in the Empa group the mitochondrial CYT recovered (Fig. 2C, D). These above findings suggest that the diabetic environment result in increased apoptosis in kidney and these changes were partially reversed in Empa group.