Modulation of cyclins and p53 in mesangial cell proliferation and apoptosis during Habu nephritis

Abstract

Background: Mesangial cell (MC) proliferation and apoptosis are the main pathological changes observed in mesangial proliferative nephritis. In this study, we explored the role of cyclins and p53 in modulating MC proliferation and apoptosis in a mouse model of Habu nephritis.

Methods: The Habu nephritis group was prepared by injection of Habu toxin. Mesangiolysis and mesangial expansion were determined by periodic acid-Schiff (PAS) reagent staining. Immunohistochemical analysis of PCNA and KI67, and TUNEL staining were used to detect cell proliferation and apoptosis, respectively. Expression levels of cyclins and p53 were examined by Western blotting.

Results: PAS staining showed that mesangial dissolution appeared on days 1 and 3, and mesangial proliferation with extracellular matrix accumulation was apparent by days 7 and 14. Both PCNA and KI67 immunohistochemical analysis showed that MC proliferation began on day 3, peaked on day 3 and 7, and recovered by day 14. TUNEL staining results showed that MC apoptosis began to increase on day 1, continued to rise on day 7, and peaked on day 14. Western blot analysis showed that cyclin D1 was upregulated on day 1, cyclins A2 and E were upregulated on days 3 and 7, and p53 was upregulated on days 3, 7 and 14. There was no change in the expression levels of Bax or p21.

Conclusion: We explored the tendency for MC proliferation and apoptosis during the process of Habu nephritis and found that cyclins and p53 may modulate the disease pathology. This will help us determine the molecular pathogenesis of MC proliferation and provide new targets for disease intervention.

Keywords: Cell cycle; Cyclins; Mesangial proliferative nephritis; p53.

Fig. 1

Mesangiolysis and MC proliferation with mesangial expansion were the major pathological changes during Habu nephritis. a PAS staining showed that mesangial dissolution appeared on days 1 and 3 with fibrinoid material deposition, and mesangial proliferation with extracellular matrix accumulation was apparent by days 7 and 14 with extracellular matrix accumulation. Control mice were injected with an equal volume of normal saline and killed at day 0 (Con group). b The glomerulus cell count results showed that cell number decreased on day 1, than began to increase and peaked on day 7, and recovered on 14, four mice per group, data are mean ± SD; *represents P < 0.05 compared to Con

Fig. 2

Renal function damage by examining serum creatinine and blood urea nitrogen levels and the urine albumin/creatinine ratio. The Serum creatinine (a) and blood urea nitrogen (b) increased on days 3 and 7 but decreased by day 14. six mice per group,data are mean ± SD, *represents P < 0.05 compared to Con. C. The urine albumin/creatinine ratio increased rapidly on day 1, peaked on day 3 and decreased by day 7, four samples per group (four mice samples pooled for each), data are mean ± SD, *represents P < 0.05 compared to Con, **represents P < 0.01 compared to Con

Fig. 3

Mesangial cell proliferation was detected by PCNA and KI67 immunohistochemistry. a Immunohistochemistry graph of PCNA. b PCNA positive rates (mean ± SD) in each group, n = 3 mice per group, (10–15 glomeruli detected for each mice). The rate increased on day 1, peaked by days 3 and 7, and subsequently decreased at day 14. c Immunohistochemistry graph of KI67. d Double staining of KI67 immunohistochemistry with PAS. e KI67 positive rates (mean ± SD) in each group. The trend of KI67 was similar to PCNA results. Four mice per group, *represents P < 0.05 compared to Con, **represents P < 0.01 compared to Con

Fig. 4

Mesangial cell apoptosis was detected by TUNEL staining. A TUNEL staining graph. B. Apoptosis rates calculated by the percentage of TUNEL-positive cells relative to the total number of glomerulus cells. The apoptosis rate increased from days 1 to 7 and peaked on day 14, three mice per group (30 glomeruli detected for each mice),data are mean ± SD, *represents P < 0.05 compared to Con, **represents P < 0.01 compared to Con

Fig. 5

The expression of cell cycle regulatory proteins in the glomeruli of Habu nephritis was examined by Western blotting. a Positive cell cycle regulatory proteins including cylins A, D1 and E, and negative regulatory proteins including p53, p21 and Bax were examined by Western blotting. b The expression trends of positive cell cycle regulatory proteins in Habu nephritis. Cyclin D1 was mainly up-regulated during the mesangiolysis phase (days 1 and 3), cyclins A and E were upregulated on day 3, peaked on day 7, normal by day 14. c The expression trends of negative cell cycle regulatory proteins in Habu nephritis. p53 increased from days 1 to 7 and peaked on days 7 and 14. However, there was no change in the expressions of Bax or p21 during Habu nephritis (the intensities of these protein bands were presented as their ratios to theβ-actin levels and data from the control group were arbitrarily set as 1.0). Data are mean ± SD, *represents P < 0.05 compared to Con, **represents P < 0.01 compared to Con