Astragaloside IV ameliorates renal injury in db/db mice

Abstract

Diabetic nephropathy is a lethal complication of diabetes mellitus and a major type of chronic kidney disease. Dysregulation of the Akt pathway and its downstream cascades, including mTOR, NFκB, and Erk1/2, play a critical role in the development of diabetic nephropathy. Astragaloside IV is a major component of Huangqi and exerts renal protection in a mouse model of type 1 diabetes. The current study was undertaken to investigate the protective effects of diet supplementation of AS-IV on renal injury in db/db mice, a type 2 diabetic mouse model. Results showed that administration of AS-IV reduced albuminuria, ameliorated changes in the glomerular and tubular pathology, and decreased urinary NAG, NGAL, and TGF-β1 in db/db mice. AS-IV also attenuated the diabetes-related activation of Akt/mTOR, NFκB, and Erk1/2 signaling pathways without causing any detectable hepatotoxicity. Collectively, these findings showed AS-IV to be beneficial to type 2 diabetic nephropathy, which might be associated with the inhibition of Akt/mTOR, NFκB and Erk1/2 signaling pathways.

Figure 1. Urinary albumin excretion (μg/24h) after 8 weeks and 12 weeks treatment in each group (n = 6–8).

The db/db mice showed significantly more urinary albumin excretion than wild type, and AS-IV therapy was found to ameliorate this to a considerable extent (Fig. 1a). Creatinine clearance (μl/min) calculated at 12 weeks indicated an obvious increase in db/db mice, but AS-IV treatment was not found to reduce it (Fig. 1b). *P < 0.05 and ***P < 0.001 when compared to wild type. ^#P < 0.05 and ^###P < 0.001 when compared to the db/db group.

Figure 2. The db/db group displayed higher blood glucose than the wild type group throughout the experiment and higher glycated HbA1c, urinary glucose, and serum insulin at 12 weeks.

There was no significant difference in these indexes after AS-IV therapy (Fig. 2a–d). n = 8 per group. **P < 0.01 and ***P < 0.001 when compared to wild type group.

Figure 3. Throughout the experiment, db/db mice manifested heavier body weight than wild type and, at the end of the study, they showed heavier kidneys (Fig. 3a,b).

In mice treated with AS-IV, body weight and kidney weight both showed declining trends but not to significant degree (Fig. 3a,b). n = 8 per group. ***P < 0.001 when compared to wild type group.

Figure 4. At 12 weeks, db/db mice displayed larger GTA and GTV (n = 6 per group), thicker GBM and wider FPW (n = 3 per group) relative to wild type mice.

AS-IV treatment reversed these alterations approach to normal condition (Fig. 4a–d). PAS staining and EM images were used to depict these characteristics in each group (Fig. 4e). Scale bars, 20 μm for PAS images, 200 nm for EM. **P < 0.01 and ***P < 0.001 relative to wild type group. ^#P < 0.05 and ^###P < 0.001 relative to the db/db group.

Figure 5. The mesangial matrix fraction (n = 6 per group) was significantly higher in db/db mice (Fig. 5a).

Fibronectin content in the renal cortex was also visibly higher, as indicated by Western blot analysis (n = 4 per group) and immunohistochemistry (Fig. 5b–d). AS-IV treatment was found to reduce mesangial matrix fraction and fibronectin level but not to a significant degree (Fig. 5a,b). *P < 0.05 and ***P < 0.001 relative to the wild type group.

Figure 6. In db/db mice, urinary NAG, NGAL and TGF-β1 excretion increased significantly (Fig. 6a–c).

n = 6–10 per group. The proximal tubular area, lumen and wall (n = 6 per group) became larger and TBM (n = 3 per group) became thicker than in wild type mice (Fig. 6d–h). Figure 6a–c showed AS-IV treatment significantly reduce urinary NAG, NGAL and TGF-β1. The proximal tubular area, lumen, and wall became smaller (Fig. 6d–f). The TBM also became thinner than db/db mice (Fig. 6g). The representative images of tubular PAS staining and TBM are shown in Fig. 6h. Scale bars, 20 μm for PAS images, 200 nm for EM. **P < 0.01 and ***P < 0.001 relative to the wild type group. ^#P < 0.05 and ^##P < 0.01 relative to the db/db group.

Figure 7. Mice in the db/db group showed more renal cortical expression of p-Akt(Ser473), p-mTOR(Ser2448), p-NF-κB p65(Ser536) and p-Erk1/2(Thr202/Tyr204) than wild type mice, as indicated by Western blot respectively.

AS-IV treatment suppressed activation of these proteins (Fig. 7). n = 4–9 per group. *P < 0.05, **P < 0.01 and ***P < 0.001 relative to the wild type group. ^#P < 0.05 and ^##P < 0.01 relative to the db/db group.

Figure 8. Compared to wild type mice, the db/db mice showed visibly higher ALT and slightly but not significantly lower AST (Fig. 8a,b).

There were no significant differences in ALT and AST between the db/db and AS-IV treatment groups (Fig. 8a,b). n = 8 per group. **P < 0.01 relative to the wild type group.