Objective: To study the effects of serum IgA(1) from patients with IgA nephropathy (IgAN) on the phosphorylation of extracellular signal-regulated kinase (ERK) and proliferation of human mesangial cells (HMC).
Methods: Serum was taken from 10 IgAN patients and 10 healthy persons. IgA(1) was isolated with jacalin column, and then heated to become aggregated form (aIgA(1)). Primary HMCs were cultured and the cells of third or fourth generation were used in the test. HMCs were incubated with aIgA(1) from IgAN patients or from healthy persons. To do blocking test 50 micro mol/L PD98059 was added into the culture of HMC of the 3 groups while the HMCs were in the relatively stationary phase. The phosphorylation of ERK was evaluated by Western blot; cell cycle was examined by DNA fluorescence labeling and flow cytometry. HMC number was counted.
Results: 1) Both the aIgA(1) from patients with IgAN and that from healthy persons induced the phosphorylation of ERK, DNA synthesis and proliferation of HMC in a time-dependent manner and with a similar trend, however, the effects of aIgA(1) from patients with IgAN were much stronger and longer. The incubation times for the maximum effect on phosphorylation of ERK were 15 minutes, 24 - 36 hours, and 48 - 72 hours in the IgAN patient group, healthy person group, and the control group with a ratio of phosphorylated ERK/total ERK of 49.5% +/- 10.1%, 30.7% +/- 4.4% and 10.5% - 12% respectively (P < 0.05). 2) After 18-hour incubation, the percentages of cells at the stage S were 3.48% +/- 0.54%, 6.64% +/- 0.96%, and 7.85% +/- 0.71% in the control group, healthy person group, and IgAN patient group. 24 hours after incubation, the percentages of cells at stage S-G(2)-M were 9.4% +/- 1.86% in the control group, significantly lower than that in the healthy person group (14.5% +/- 0.7%, P < 0.05) and that of the patient group (17.2% +/- 0.3%, significantly higher than the other 2 groups, P < 0.01 and P < 0.05). At the peak time, the cells at stage S-G(2)-M accounted for 33.0% +/- 0.3% and 30.7% +/- 0.7% in the patient group and healthy person group respectively (P < 0.05) with the HMC count of (57.8 +/- 2.5) x 10(4)/ml and (50 +/- 1.5) x 10(4)/ml (P < 0.05) respectively. After 48, 72, and 96-hour incubation with aIgA(1) the HMC counts were (51.7 +/- 1.6) x 10(4)/ml, and (56.4 +/- 2.6) x 10(4)/ml, and (58.8 +/- 1.8) x 10(4)/ml, and (65.8 +/- 2.9) x 10(4)/ml, (60.0 +/- 1.7) x 10(4)/ml and (66.8 +/- 2.0) x 10(4)/ml in the patient group with and without pre-incubation of PD98059 respectively (P < 0.05). However, the HMC count was not significantly changed in the healthy person group. Blocking of ERK with PD98059 could inhibit the effect of aIgA(1) from patients with IgAN on the proliferation of HMC but could not inhibit the effects of aIgA(1) from healthy controls.
Conclusion: IgA(1) induces the phosphorylation of ERK, DNA synthesis and proliferation of HMC, and the effects of IgA(1) from patients with IgAN are stronger than that from healthy persons.