Study On The Role Of GRN In The Pathogenesis Of Lupus Nephritis And Its Underlying Mechanisms

Systemic lupus erythematosus (SLE), a chronic inflammatory autoimmune disorder, is a potentially fatal disease characterized by immune complex deposition and the subsequent inflammation that contributes to sever tissue damage. One of the most severe manifestations is lupus nephritis (LN), which remains a cause of substantial morbidity and mortality. LN occurs in up to50%of patients at onset of the disease and over60%of patients during the progression of SLE. Recently reported10-year survival rates of patients with lupus nephritis range from68%to98.2%. A better understanding of the pathogenesis of LN is an important step in identifying more targeted therapeutic approaches. Substantial researches have helped define the pathogenic mechanisms of renal manifestations. Immune complex (IC) glomerular deposits generate release of proinflammatory cytokines and chemokines causing inflammation, leading to monocytes and polymorphonuclear cells chemotaxis. Subsequent release of proteases generates endothelial injury and mesangial proliferation. And then, the presence of ICs also promotes adaptive immune response and causes release of type I interferon which further activates macrophages to release more proinflammatory molecules, resulting in epithelial glomerular proliferation and fibrosis. However, underlying molecular mechanisms that mediate LN still remains unclear so far, thus impeding the advance of efficient therapies toward LN.Recent reports showed that some multifunctional proteins, which were previously studied for their roles in autoimmune and inflammatory diseases, might be involved in the pathogenesis of LN. A promising candidate is granulin (GRN), a glycosylated protein with a repeating cysteine-rich motif, is highly expressed in epithelial cells, certain types of neurons, and macrophages. GRN is originally identified as an autocrine growth factor that regulates cell growth, development, and tissue remodeling. As a multifunctional protein, GRN has also been linked to a variety of physiologic and disease processes, including inflammation, wound healing and regulation of innate immunity. Furthermore, recent studies have shown that GRN is correlated with autoimmune diseases, including rheumatoid arthritis, multiple sclerosis and type-2diabetes. And one report has found that GRN is associated with the disease activity of SLE. However, whether GRN takes responsibility in the pathogenic mechanisms of LN still remains unclear.Our previous study has demonstrated that syngeneic activated lymphocyte derived DNA (ALD-DNA) could function as an auto-antigen to induce SLE syndrome including severe renal manifestations in syngeneic BALB/c mice. Given the emblematical autoimmune syndrome and exclusion of the genetic mutation interruptions in this lupus model, the ALD-DNA-induced mice could be used as an ideal model to explore the pathogenic mechanisms for LN. In our previous work, we’ve found that the ALD-DNA-induced M2b polarization play pivotal role in the pathogenesis of LN. Yet, the potential mechanisms of how ALD-DNA induces M2b polarization remain unclear.Here, we carefully determined the potential role and possible mechanisms of GRN in the pathogenesis of LN using ALD-DNA-induced lupus model, which might provide a novel mechanism accounting for the progression of LN and a clue for developing novel therapeutic strategy against LN.Part1Correlation analysis of serum GRN levels with the severity of lupus nephritis in SLE miceTo study whether GRN was elevated in lupus model, we generated lupus mice according to our previously reported procedure. Briefly, female BALB/c mice were injected with ALD-DNA, and high titers of anti-dsDNA Abs, immune complex glomerular deposition, glomerulonephritis and proteinuria could be detected in this lupus model. Serum GRN levels of lupus model were assayed by ELISA every2weeks and we found significantly increased serum GRN levels in lupus mice as compared with those in control mice4weeks after the initial ALD-DNA injection. Pearson correlation analysis showed that the serum GRN levels were positively correlated with kidney score, indicating that GRN was significantly correlated with the severity of LN. To further examine the correlation of serum GRN levels with the severity of LN, we then analyzed the correlation of serum GRN levels with urine protein levels. Notably, serum GRN levels were also positively associated with urine protein levels. Taken together, these data indicated that serum GRN levels were up-regulated and were associated with the pathogenesis of LN in lupus mice.Part2GRN was critical for the pathogenesis of lupus nephritisWe first up-regulated the expression of GRN by injecting BALB/c mice intramuscularly with pGRN as previously described to evaluate whether GRN was involved in the pathogenic process of LN. The serum GRN levels were significantly elevated in pGRN-treated lupus mice compared with those in pcDNA3.1-treated lupus mice. To investigate the effect of increased GRN on ALD-DNA-induced LN, we then analyzed renal pathology, kidney score and proteinuria in pGRN-treated or pcDNA3.1-treated lupus mice. Exacerbation of renal pathology was found in the pGRN-treated lupus mice as compared with those controls, and the kidney score of pGRN-treated lupus mice was much higher than that of control mice, indicating severer LN resulted from GRN overexpression. Furthermore, distinguished increase of urine protein was observed in the pGRN-treated lupus mice as compared with those controls. Except for the increased levels of urine protein, we also noticed that overexpression of GRN could accelerate the initiation of proteinuria, a strong evidence for the aggravated LN by GRN overexpression. Moreover, serum creatinine, a commonly accepted index of kidney function, was up-regulated in pGRN-treated lupus mice.We then investigated whether down-regulation of GRN could ameliorate LN in lupus model. We down-regulated the levels of GRN in vivo by intravenous injection of lentivirus encoding shRNA directed against GRN (LV-shGRN) or lentivirus encoding nontargeting shRNA (LV-shNC) as control. Decreased serum GRN levels were found in lupus mice injected with LV-shGRN versus those injected with LV-shNC. We then measured the renal tissue H&E staining, kidney score and urine protein to examine whether down-regulation of GRN could ameliorate LN. Notably, we observed that renal pathology was ameliorated in LV-shGRN-injected lupus model versus controls. We further analyzed the kidney score and observed that LV-shGRN-injected lupus mice got lower kidney score compared with control mice, suggesting that down-regulation of GRN could ameliorate LN in lupus model. Moreover, urine protein showed delayed onset as well as remarkable reduction in LV-shGRN-injected lupus mice compared with those of control mice. And we also found the serum creatinine levels were reduced in LV-shGRN-injected lupus mice. These findings presented above collectively showed that GRN could aggravate LN in lupus mice.Part3Mechanisms of GRN in modulating the pathogenic process of lupus nephritisBased on the recent reports and our current research, we speculated that GRN exacerbated lupus nephritis by enhancing macrophage M2b polarization. To test whether GRN could aggravate LN by enhancing ALD-DNA-induced M2b-polarized macrophages, we first isolated CD11b+/F4/80high renal macrophages from pGRN or pcDNA3.1-treated lupus mice, LV-shGRN or LV-shNC-treated lupus mice by FACS. Real time analysis was performed to detect the gene expression levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, MCP-1and Nos2(iNOS),representing M2b-polarized phenotype, as well as Argl, a marker for M2a and M2c. Macrophages from pGRN-treated mice showed much higher expression levels of TNF-α, IL-1β, IL-6, IL-10, MCP-1and Nos2and slightly higher IL-12, and no significant changes in Argl, while administration with LV-shGRN in lupus model considerably inhibited M2b polarization, as renal macrophages from LV-shGRN-injected lupus mice performed lower mRNA levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, MCP-1and Nos2than those of controls, and arginase1levels did not change significantly when GRN was down-regulated. These results demonstrated that GRN exacerbated LN in lupus model by promoting ALD-DNA-induced M2b polarization.We then found that ALD-DNA could effectively enhance the expression of GRN both in mRNA level and protein level in M2b macrophage in vitro. To explore the possible contributions of GRN to ALD-DNA-induced M2b differentiation in vitro, we added purified GRN to the culture medium in which macrophages were maintained, and then stimulated macrophages with ALD-DNA. We found remarkably increased production of TNF-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1compared with those in controls, as well as the expression of iNOS. To test whether GRN was required for ALD-DNA-elicited M2b polarization, we exposed macrophages to an elastase-selective inhibitor and then stimulated macrophages with ALD-DNA. Inclusion of elastase inhibitor interfered with ALD-DNA-triggered M2b polarization. We got the similar results by transfection with specific GRN siRNA. Taken together, we concluded that GRN was essential for ALD-DNA-induced M2b polarization. We also found that the activation of MAPK signaling pathways was required in the effect of GRN on ALD-DNA-induced M2b polarization.Based on the ALD-DNA-induced lupus model, we found that GRN, a multifunctional protein, was involved in the pathogenesis of lupus nephritis and proved that GRN exacerbated lupus nephritis by accelerating ALD-DNA-induced macrophage M2b polarization. Moreover, we also found that MAPK signal activation was required in the effect of GRN on ALD-DNA-induced M2b polarization. Our research might provide insights into the pathogenesis of LN as well as potential therapeutic strategies against LN.