Puerarin attenuated early diabetic kidney injury through down-regulation of matrix metalloproteinase 9 in streptozotocin-induced diabetic rats

Abstract

Radix puerariae, a traditional Chinese herbal medication, has been used successfully to treat patients with early stage of diabetic nephropathy. However, the underlined mechanism of this renal protective effect has not been determined. In the current study, we investigated the effects and the mechanism of puerarin in Streptozotocin (STZ)-induced diabetic rats. We treated STZ-rats with either puerarin or losartan, an angiotensin II receptor blocker, as compared to those treated with vehicle. We found that both puerarin and losartan attenuated kidney hypertrophy, mesangial expansion, proteinuria, and podocyte foot process effacement in STZ rats. In addition, both puerarin and losartan increased expression of podocyte slit diaphragm proteins such as nephrin and podocin. Interestingly, we found that puerarin treatment induced a more pronounced suppression of oxidative stress production and S-nitrosylation of proteins in the diabetic kidneys as compared to losartan treatment. Furthermore, we found that matrix metalloproteinase-9 (MMP-9), which is known to be activated by oxidative stress and S-nitrosylation of proteins, was also suppressed more extensively by puerarin than losartan. In conclusion, these data provide for the first time the potential mechanism to support the use of puerarin in the treatment of early diabetic nephropathy.

Figure 1. Effect of puerarin on blood glucose level of STZ-induced rats.

Rats were injected with one dose of STZ and treated with or without puerarin for 7 days. Blood glucose level was examined once every day for 7 days. Data are presented as mean ± SE, n = 10.

Figure 2. Blood and urinary albumin levels of STZ-induced rats.

Bloods were obtained at the end of experiment while 24-h urine was obtained at the last day of experiment. A. Urinary albumin was determined by ELISA as described. B. Serum albumin was determined by an automatic clinical chemistry analyzer. Data are displayed as mean ± SE. *p<0.01 STZ rats compared to normal control, #p<0.05 STZ rats compared to STZ rats treated with puerarin, and ^&p<0.05 STZ rats compared to STZ rats treated with losartan, n = 10.

Figure 3. Kidney hypertrophy index and histology.

A. Kidney and body weight of these rats were recorded and kidney hypertrophy was estimated by determination of kidney/body weight ratio. *p<0.01 STZ rats compared to normal control, #p<0.05 STZ rats compared to STZ rats treated with puerarin, and ^&p<0.05 STZ rats compared to STZ rats treated with losartan, n = 10. B. Kidney histology was analyzed after H&E staining. The representative pictures are shown.

Figure 4. Kidney electron microscopy.

A. Kidney samples from the four experimental groups were examined under transmission electron microscope as indicated. Panel A is for normal rats; panel B is for STZ rats, panel C is for STZ+puerarin rats and panel D is for STZ+losartan rats. Magnification 1,800×. B. Quantification of podocyte effacement is shown (n = 5, *p<0.001 STZ rats versus all other groups).

Figure 5. Expression of nephrin and podocin in the kidney.

Total RNA and protein were extracted from the kidney respectively. Total RNA was subjected to real-time RT-PCR analysis (A, B) and protein was used for western blot analysis (C, D) for nephrin (A, C) and podocin (B, D). GAPDH was used as loading control of mRNA levels in real-time PCR and β-actin was used as loading control protein loading for western blot. Data are presented as mean ± SE. *p<0.05 STZ rats compared to normal control, #p<0.05 STZ rats compared to STZ rats treated with puerarin, and ^&p<0.05 STZ rats compared to STZ rats treated with losartan, n = 5.

Figure 6. Assessment of oxidative stress in the kidney.

A. 8-hydroxy-2′-deoxy-guanosine (8-OHdG) levels were determined in the kidney of these rats by ELISA assay as described in the methods and materials. Data are represented as mean ± SE, *p<0.001 STZ rats compared to normal control rats, #p<0.01 STZ rats compared to STZ rats treated with puerarin, ^&p<0.01 STZ rats compared to STZ rats treated with losartan, and ^@p<0.05 losartan treated STZ rats compared to puerarin treated STZ rats, n = 5. B. Immunostaining for S-Nitrosylation of proteins was performed in the kidney of these rats. The representative pictures of each group are shown.

Figure 7. Expression of matrix metalloproteinase 9 (MMP-9) in the kidney.

Kidneys from four experimental groups were collected for immunostaining and real-time PCR. A. Immunostaining of MMP-9 was performed. The representative pictures of each group are shown (a = normal, b = STZ, c = STZ+puerarin and d = STZ+losartan). B. MMP-9 mRNA levels were measured by real-time PCR in the kidney of these rats. Data are presented as mean ± SE, *p<0.01 STZ rats compared to normal control rats, #p<0.05 STZ rats compared to STZ rats treated with puerarin, ^&p<0.05 STZ rats compared to STZ rats treated with losartan, and ^@p<0.05 losartan treated STZ rats compared to puerarin treated STZ rats, #p<0.05 puerarin-treated STZ rats compared to normal control rats, n = 5.

Figure 8. Immunostaining for type IV collagen.

The representative pictures of each group are shown.

Figure 9. Summary schema.

Diabetic milieu such as hyperglycemia induces ROS production and S-nitrosylation of proteins, which in turn activates MMP-9 expression. MMP-9 induces podocyte injury through alteration of GBM components such as degradation of type IV collagen. Puerarin prevents podocyte injury through attenuation of ROS production, inhibition of S-nitrosylation of proteins, and reduction of MMP-9 expression.