The New Mechanism Of Renal Tubulointerstitial Injury Induced By Proteinuria

Aim:Proteinuria is an important marker of renal disease, and it’s not only the result of glomerular and tubular diseases, but also derectly induce tubular epithelial change, which is able to generate cytokines and chemokine, and lead to tubulointerstitial inflammation and fibrosis. Proteinuria is an independent risk factor for chronic kidney diseases, while the potential mechanisms have not been fully clarified. NLRP3 inflammasome is the best characterized inflammasome, and it can recognize pathogen-associated molecular patterns and danger-associated molecular pattern to activate its downstream signal pathway, which palys an important role in many diseases. Recently, the role of NLRP3 inflammasome in kidney diseases has been raised concern. In the present study, the albumin-overloaded (AO) nephropathy rats, HK-2 cells stimulated by overloaded albumin in vitro and the biopsy samples of non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria are used to investigate whether proteinuria induce NLRP3 inflammasome and TLR2/TLR4-MyD88-NF-κB pathway activation, and its possible mechanisms involved.Methods:Rats were divided into two groups:the control and AO groups, and rats were received daily intraperitoneal injection of 5g/kg BSA for 9 weeks in the AO group. HK-2 cells were stimulated by BSA (20 mg/ml) in vitro, and 30 non-IgA MsPGN patients with different levels of proteinuria were selected. NLRP3 inflammasome, TLR2/TLR4-MyD88-NF-κB pathway and their downstream cytokines, such as IL-1β, IL-18, TNF-a and IL-6 were detected by immunohistostaining, western blot and real-time PCR in these tubular epithelial cells.For further, KCl (150mM), CA074Me (cathepsin B inhibitor) and DPI (ROS inhibitor) were used in HK-2 cells to investigate the possible mechanism of NLRP3 inflammasome activation stimulated by overloaded albumin via detecting the change of IL-1β and IL-18 expression. And BAY 11-7082 (NF-κB inhibitor), siRNA (to silence TLR2 and TLR4 gene) were used in HK-2 cells stimulated by albumin to study whether TLR2/TLR4-MyD88-NF-κB signal pathway was involved in renal injury caused by proteinuria.Results:It was found that the AO group rats had obvious proteinuria, and presented with significant morphological changes including, tubular dilatation or atrophy, proteinaceous casts in proximal and distal tubules, and interstitial inflammation with heavy macrophage and lymphocyte infiltration. Semiquantitative analysis showed there was a significant increase in the tubulointerstitial injury score in the AO group compared to the control group (P<0.05). Immunohistochemical staining indicated that NLRP3, caspase-1, IL-1β and IL-18 were not only expressed in inflammatory cells, but also expressed in proximal tubules in the AO group rats. Western blot and real-time PCR showed that NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA are significantly increased in AO rats and HK-2 cells stimulated by overloaded albumin in vitro compared to the control group (P<0.05, or P<0.01). Immunohistochemical staining also showed that the expressions of NLRP3, caspase-1, IL-1β and IL-18 were upregulated in non-IgA MsPGN patients with high levels of proteinuria. Furthermore, IL-1β and IL-18 expression were positively correlated with the degree of proteinuria (r=0.836, P<0.05, r=0.901, P<0.05). Meanwhile, TLR2/TLR4-MyD88-NF-KB signal pathway was activated, and its downstream cytokines TNF-α and IL-6 were significantly increased in AO rats compared to the control group (P<0.05). Western blot and real-time PCR also showed that TLR2/TLR4-MyD88-NF-κB signal pathway was activated, and pro-IL-1β, pro-IL-18, TNF-a and IL-6 were all increased in AO rats and HK-2 cells stimulated by high dose of albumin in vitro compared to the control group (P<0.05, or P<0.01). Immunohistochemical staining also showed that TLR2/TLR4-MyD88-NF-KB signal pathway was activated, and TNF-a and IL-6 were strongly expressed in non-IgA MsPGN patients accompanying with high levels of proteinuria. There were significant correlations between proteinuria and TNF-a, IL-6 expression in non-IgA MsPGN patients (r=0.813, P<0.05; r=0.899, P<0.05). CA074Me and DPI significantly reduced the secretion of IL-1β and IL-18 in HK-2 cells stimulated by overloaded albumin (P<0.05, or P<0.01), but KC1 (150mM) didn’t affect the secretion of IL-1β and IL-18. NF-κB inhibitor, BAY 11-7082 also obviously inhibited the secretion of IL-1β, IL-18, TNF-a and IL-6 in HK-2 cells stimulated by overloaded albumin (P<0.05, or P<0.01). The enhanced expression of IL-1β, IL-18, TNF-a and IL-6 induced by albumin was significantly attenuated after silence of TLR2 and TLR4 gene by siRNA in HK-2 cells (P<0.05, or P<0.01).Conclusions:1. Proteinuria is able to induce NLRP3 inflammasome activation, which can result in IL-1β and IL-18 secretion by activated form caspase-1 cleavage in tubular epithelial cells.2. Proteinuria activates TLR2/TLR4-MyD88-NF-κB signal pathway, which not only produce TNF-a and IL-6, but also increase pro-IL-1β and pro-IL-18 in tubular epithelial cells.3. Proteinuria activates NLRP3 inflammasome via the release of cathepsin B and increased ROS during production albumin reabsorption in tubular epithelial cells, but not via K+ efflux.4. Proteinuria can enhance inflammatory factors TNF-a and IL-6 and inactive precursor protein pro-IL-1β and pro-IL-18 via NF-κB activation by TLR2 and TLR4 mediated MyD88-dependent pathway.