| IgA nephropathy characterized by deposition of IgA in the glomerular mesangium, is one of the most common form of primary glomerular nephritis. The etiology is unknow and the prognosis is bad. Until now, no effective therapy was performed for this disease. Every year,about 1%～2% of patients enter ESRD, needing renal replacement therapy. Male youngsters are inclined to suffer from IgAN,which brings a big burden on social and economic development. Thus,the etiology and progressive mechanism of this disease have become one of the greatest research issue for medical workers all over the world especially for nephrologists.Many reports have suggested the relationship between tonsils and the mechanism of IgAN. Physical, chemical and inflammation of tonsils often worsen the urinary findings in patients with IgAN. Many long-term follow-up studies showed that tonsillectomy can improve the urinary findings,keep stable renal function, but the detailed mechanism remains to be elucidated. Compared with the patients with chronic tonsilitis, abnormalities of the tonsilar mononuclear cells and their secretory products in the patients with IgAN have been demonstrated by many studies. TMCs from IgAN have stronger activities to antigen stimulation compared to non-IgAN and can produce more inflammation cytokines. In patients with IgAN,the ratio of tonsillar immunocytes become apparently inverted,sub-group ratio of lymphocytes and apoptosis is abnormal. Cells that secrete IgA,pIgA become enriched and can produce underglycosylation IgAl which has a defect in galactosylation and sialic acid and is the same type as deposited in kidney mesangial cells. TMCs from tonsils in patients with IgA nephropathy can also produce more IgA and TGF-β, IFN-γand other cytokines. MCP-1 which can influence glomerular sclerosis and IL-8 which can inhibit the proliferation of tubules are also increased.Can the immulogical molecules secreated by TMCs in patients with IgA nephropathy influence the growth and differentiation of human kidney residential cells(such as renal mesangial cells and tubular epithelial cells), other than regulate the local immunity in the tonsils? Are there any differences between the tonsils with or without IgA nephropathy? There hasn't been any report answering these questions. Previous studies have shown that when glomerular mesangial cells were cocultured with the supernatant of PBMCs(periferal blood monouclear cells, PBMCs) in patients with IgA nephropathy, the expression of ICAM-1 was upregulated. Compared with chronic tonsilitis without IgA nephropathy, the supernatant of TMCs can upregulate the expression of TGF-β1, PAI-1, IL-6 in human glomerular mesangial cells. The function of one single cytokine has been reserched broadly, but in vivo, the coexistence of different cytokines can demonstrate multiple, overlap,antagonist or synergetic effect. In order to mimic the environment in vivo and observe the products of TMCs in patients with IgA nephropathy,we collect the tonsils in patients with or without IgA nephropathy,separate the TMCs,collect the supernatant, coculture with HMC cells and HK-2 cells directly, and collect the supernatant of HMC cells cocultured with supernatant of TMCs, and then coculture it with HK-2 cells. Observe the collection between these two kind of cells and the roles that these different supernatants may have played in the proliferation and differation of renal mesangial cells and tubular epithelia cells and the possible mechanism, thus further understand the possible roles that tonsillar immunity might have played in the onset and the progression of IgA nephropathy.This reserch has two parts.Part I The effect of cultured supernatant of TMCs in patients with IgA nephropathy on the apoptosis of human mesangial cellsObjective:To investigate the effect and its related mechanism of the supernatant of TMCs in patients with IgA nephropathy on human mesangial cells, explore the possible role of the products of TMCs in patients with IgAN might have played in the mechanism of IgAN.Methods:13 patients with IgA nephropathy and 13 patients without IgA nephropathy together with chronic tonsilitis were enrolled. TMCs were seperated and cultrued ex vivo. Each group was divided into 2 groups:as PHA stimulated or not. After PHA stimulated for 72 hours, collect the supernatant from the stimulated and unstimlated group. Coculture the human glomerular mesangial cells with these supernatant. Flow cytometer read the apoptosis rate of mesangial cells. Bcl-2, Bax, Fas mRNA were measured with RT-PCR. The lever of TGF-β1 in the supernatant was measured with ELISA.Results:Coculture the human mesangial cells with the supernatant from the PHA stimulated TMCs in patients with IgAN,compared with control, the PHA stimulated non IgAN group, unstimulated IgAN and non-IgAN, the apoptosis rate was statistically higher (P<0.05). Coculture the human mesangial cells with the supernatant from the PHA stimulated TMCs in patients with IgAN, Bcl-2/β-actin mRNA was lowered to 25% of the negative control, Bax/Bcl-2 was upregulated to 4.3fold of the negative control, has a statistical meaning (P<0.05)compared with the negative control, unstimulated IgAN and non-IgAN, PHA stimulated non-IgAN group. Coculture the human mesangial cells with the supernatant from the PHA stimulated TMCs in patients with IgAN, Fas/β-actin mRNA was upregulated to 2.38 fold of the negative control, much higher than the negative control, unstimulated IgAN and non-IgAN, PHA stimulated non-IgAN group(P<0.05). The expression of TGF-β1 in the supernatant from the TMCs in patients with IgAN was obviously higher than the non-IgAN group. But between the PHA stimulated and unstimulated groups, the diffrence was not statistical(P>0.05).Conclusion:1.The supernatant from the PHA stimulated TMCs in patients with IgAN have apoptotic effect on human mesangial cells. The mechanism may relate to the decreased Bcl-2 gene expression, the upregulated ratio of Bax/Bcl-2 and the upregulated expression of Fas gene expression.2. The expression of TGF-β1 in the supernatant from the TMCs in patients with IgAN was obviously elevated, but the PHA ex vivo stimulation can't promote TMCs to secrete TGF-β1. It seems the level of TGF-β1 in the supernatant of TMCs doesn't correlate with the apoptotic effect of the supernatant has on human mesangial cells. Part II The effect of cultured supernatant of TMCs in patients with IgA nephropathy on the proliferation,apoptosis and EMT of human tubular epithelia cellsObjective:To investigate the effect and its related mechanism of the supernatant of TMCs and its mesangial supernatant in patients with IgA nephropathy on the proliferation,apoptosis and EMT of human tubular epithelia cells, explore the possible role of the products of TMCs in patients with IgAN in the mechanism of IgAN.Methods:As a 20% concentration, add the supernatant with or without PHA stimulating in patients with or without IgAN, into the culture medium in culture with human glomerular mesangial cells for 24 hours,collect the mesangial supernatant. Coculture the HK-2 cells with different concentrations of supernatant from the TMCs in patients with or without IgAN, at the time of 12 hours and 24 hours, apply MTT to test HK-2's proliferation. After coculture the HK-2 cells with the supernatant of the TMCs and their mesangial supernatant, flow cytometer was used to read the apoptosis rate of HK-2 cells. Bcl-2, Bax, E-cadherin,a-SMA mRNA were measured with RT-PCR. E-cadherin and a-SMA protein were measured with Western Blot.Results:Coculture the HK-2 cells with supernatant from the PHA stimulated TMCs and its mesangial supernatant in patients with IgAN for 24 hours,compared with the negetive control, PHA stimulated non IgAN group, unstimulated IgAN and non-IgAN group, the surviving rate was statistically lower(P<0.05),which continued to go down with the higher concentration and longer time period and the apoptosis rate statistically increased(P<0.05). Coculture HK-2 cells with the supernatant from the PHA stimulated TMCs in patients with IgAN, Bcl-2/p-actin mRNA was lowered to 40% of the negative control, Bax/Bcl-2 was upregulated to 2.41 fold of the negative control, has a statistical meaning (P<0.05)compared with the negative control, unstimulated IgAN and non-IgAN group. E-cadherin/β-actin mRNA was lowered to 43% of the negative control, has a statistical meaning (P<0.05)compared with the negative control, unstimulated IgAN and non-IgAN group(P<0.05). a-SMA was upregulated, has a statistical meaning (P<0.05)compared with the negative control.unstimulated IgAN and non-IgAN, PHA stimulated non-IgAN group(P<0.05).Coculture HK-2 cells with the mesangial supernatant derivied from the PHA stimulated TMCs in patients with IgAN, Bcl-2/β-actin mRNA was lowered to 47% of the negative control, has a statistical meaning (P<0.05)compared with the negative control. Bax/Bcl-2 was upregulated to 2.23 fold of the negative control, has a statistical meaning (P<0.05)compared with the negative control, unstimulated IgAN and non-IgAN, PHA stimulated non-IgAN group(P<0.05). E-cadherin/β-actin mRNA was lowered to 66% of the negative control, andα-SMA was upregulated, has a statistical meaning (P<0.05)compared with the negative control,unstimulated IgAN and non-IgAN, PHA stimulated non-IgAN group(P<0.05). The western blot shows that coculture the HK-2 cells with the supernatant from the PHA stimulated TMCs in patients with IgA nephropathy, E-cadherin/β-actin was lowered to 50% of the negative control, compared with control group, unstimulated IgAN and non-IgAN group,a statistical meaning was found(P<0.05).α-SMA was increased compared with control(P<0.05). Coculture HK-2 with the mesangial supernatant derived from the PHA stimulated TMCs in patients with IgA nephropathy, E-cadherin/p-actin was downregulated to 47% of the negative control, andα-SMA was upregulated, which has a statistical meaning (P<0.05)compared with negetive control, PHA stimulated non IgAN, unstimulated IgAN and non-IgAN group.Conclusion:Coculture the HK-2 cells with the supernatant of the TMCs and their mesangial supernatant in the PHA stimulated group in patients with IgA nephropathy, the growth of HK-2 was inhibited, the apoptotic rate was elevated. The mechanism may be related to the down regulation of Bcl-2, and upregulation of Bax/Bcl-2. These supernatant can also induce the EMT of HK-2, down regulate gene and protein expression of E-cadherin, upregulate gene and protein expression ofα-SMA. Renal mesangial cells and tubular epithelial cells may have a cross-talk relationship in the IgA nephropathy.
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