Nephrokeli, a Chinese herbal formula, may improve IgA nephropathy through regulation of the sphingosine-1-phosphate pathway

Abstract

Nephrokeli (NPKL) is a Chinese herbal formula that has been used to treat patients with IgA nephropathy (IgAN) for improvement of proteinuria and kidney injury. However, the mechanism remains unclear. Sphingosine-1-phosphate (S1P) and its receptors S1PR2 and S1PR3 are known to play an important role in kidney disease. Here, we tested whether NPKL is able to regulate the S1P pathway in the kidney of IgAN rats. Four groups of rats were included in the study: Control, IgAN, IgAN treated with losartan, and IgAN treated with NPKL. The IgAN model was generated by injection of bovine serum albumin and staphylococcus enterotoxin B. We found that IgAN rats had increased staining for proliferating cell nuclear antigen (PCNA) in the mesangial area and increased mRNA and protein levels of S1PR2 and S1PR3 in the kidney compared to control rats. Connective tissue growth factor (CTGF), a downstream growth factor in the S1P pathway, was also elevated in the kidney of IgAN rats. Treatment with either NPKL or losartan was able to reduce PCNA staining and the expression of both S1PR2 and S1PR3 in the kidney of IgAN rats. However, NPKL (but not losartan treatment) reduced the expression of CTGF in the kidney of IgAN rats. In addition, we treated rat mesangial cells with sera collected from either NPKL-treated rats or control rats and found that NPKL-serum was able to reduce S1P-induced mesangial cell proliferation and the expression of S1PR2/S1PR3 and CTGF. NPKL also attenuates expression of fibrosis, inflammation, and oxidative stress markers in the kidney of IgAN rats. Our studies provide the mechanism by which NPKL attenuates kidney injury in IgAN rats.

Figure 1. Immunohistochemical staining of PCNA in the glomerulus.

Kidneys from the four different groups of rats were stained with antibody against PCNA and then counterstained with Hematoxylin to show nucleus. The top panel shows representative pictures of immunostaining of glomeruli in kidneys of normal rats (A), IgAN rats (B), IgAN+NPKL rats (C), and IgAN+losartan rats (D). The bottom panel shows quantitation of the PCNA staining, which is expressed as the mean±SD. *p<0.05 when compared with normal rats and ^#p<0.05 when compared with IgAN rats, n = 10.

Figure 2. Regulation of S1PR2 and S1PR3 expression in the kidney of rats by NPKL.

Total RNA and protein were extracted from the kidneys of rats in the different groups. Real time RT-PCR and western blotting were performed to examine S1PR2 and S1PR3 mRNA and protein levels. Panel A displays real time RT-PCR results. Panel B shows representative western blots of S1PR2 and S1PR3. Panel C shows the densitometric data of the western blots. The data are expressed as the mean±SD. *p<0.05 when compared with normal rats and ^#p<0.05 when compared with IgAN rats, n = 10.

Figure 3. Expression of CTGF in the kidney.

Real time RT-PCR and western blotting were performed to examine mRNA and protein levels of CTGF in the kidney of these rats. Panel A displays real-time RT-PCR results. Panel B shows representative western blots of CTGF. Panel C shows the densitometric data of the western blots. The data are expressed as the mean±SD. *p<0.05 when compared with normal rats and ^#p<0.05 when compared with IgAN rats, n = 10.

Figure 4. Effects of NPKL on mesangial cell proliferation.

Mesangial cells were cultured and treated with vehicle or S1P together with sera from control rats (CL-Serum) or NPKL-treated rats (NPKL-Serum). Cell proliferation was assessed by MTT assay kit in these cells at days 1, 2 and 3. *p<0.01 when S1P-treated cells are compared with cells treated with both S1P and NPKL-Serum, n = 4.

Figure 5. Effects of NPKL on the expression of S1PR2 and S1PR3 in mesangial cells.

Mesangial cells were cultured and treated with vehicle or S1P together with sera from control rats (CL-Serum) or NPKL-treated rats (NPKL-Serum) for 24 hours and protein lysates were obtained from these cells for western blot analysis of S1PR2 and S1PR3. Representative western blots from three independent experiments are shown (A) and western blots were quantified by the densitometry (B). *p<0.05 when S1PR-CL-Serum are compared with CL-Serum and ^#p<0.05 when S1PR-NPKL-Serum are compared with S1PR-CL-Serum.

Figure 6. Effects of NPKL on the expression of CTGF in mesangial cells.

Mesangial cells were cultured and treated with vehicle or S1P together with sera from control rats (CL-Serum) or NPKL-treated rats (NPKL-Serum) for 24 hours. Total RNA and protein lysates were obtained from these cells for real-time PCR analysis (A) and western blot analysis (B) of CTGF. Representative western blots from three independent experiments are shown. The western blots were analyzed by densitometry (C). *p<0.01 when compared with cells without S1P stimulation and ^#p<0.05 when compared with cells stimulated with both S1P and NPKL-Serum, n = 3.

Figure 7. Effects of NPKL on the expression of fibrosis, inflammation, and oxidative stress markers in the kidneys of IgAN rats.

Immunostaining was performed in the kidney sections of these rats using the specific antibodies as described in the method. The representative pictures of three different rats in each group are shown.