Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoantibody production and immune complex (IC) deposition at multiple sites (1–6). Immune-mediated nephritis caused by deposition of pathogenic autoantibodies and ICs in the glomeruli contributes to mortality and morbidity in this disease. Studies of spontaneous lupus nephritis in murine models and experimental anti–glomerular basement membrane (anti-GBM) disease have provided valuable insights into the underlying mechanisms of human lupus nephritis (7–12). The experimentally induced model of anti-GBM disease has proven to be a particularly useful tool for studying the susceptibility of end organs to immune-mediated damage (7–10).
Previous studies by our group revealed that NZW mice exhibit increased susceptibility to experimental anti-GBM–induced glomerulonephritis (EAG) compared with C57BL/6 (B6) mice (10). Several lupus susceptibility loci derived from the NZW/NZM2410 strain have already been bred onto the normal B6 background as congenic intervals (13–21). One such lupus susceptibility interval is Sle3, on chromosome 7 (17, 20, 21). Unlike other congenic strains tested, B6.Sle3 mice (bearing the NZW/NZM2410-derived “z” allele of Sle3 on the nonautoimmune B6 background) exhibit increased susceptibility to EAG compared with B6 mice (8, 21, 22). Further fine mapping using recombinant subcongenics bearing progressively narrowed subintervals of Sle3 revealed the susceptibility genes to be located between D7mit157 and D7mit158 on chromosome 7 (21), an interval harboring the kallikrein (klk) cluster of genes. Microarray and real-time polymerase chain reaction (PCR) studies indicated that the B6.Sle3 congenic as well as several EAG-susceptible strains (such as 129/SvJ, NZW, and DBA/1) had significantly reduced renal expression of kallikreins compared with B6 and BALB/c control strains, following anti-GBM challenge. Furthermore, there were also indications that particular klk alleles may be associated with lupus nephritis in patients with SLE (22).
The above-mentioned studies suggested that kallikreins may be renoprotective in immune-mediated nephritis. The renoprotective effect of kallikrein was further underscored by 2 functional experiments. Delivering the kallikrein-1 (Klk1) gene via adenoviral vector into B6.Sle3 mice (EAG-sensitive strain) attenuated the severity of anti-GBM–induced nephritis (23), while blocking the kallikrein function in BALB/c mice (EAG-resistant strain) using a kinin B2 receptor antagonist (HOE140) rendered these mice sensitive to anti-GBM–induced nephritis (22, 24). However, the studies did not address the question of whether kallikreins could also modulate the renal injury that arises during the course of spontaneous lupus nephritis. Therefore, we established a novel transgenic mouse strain with inducible, kidney-specific kallikrein expression on the background of B6.Sle1.Sle3 of double-congenic mice, using the Cre-ERT2/LoxP system. Importantly, renal-specific expression of Klk1 significantly ameliorated lupus nephritis. The protective effect of Klk1 against lupus nephritis may be related to suppression of oxidative stress and reduction of inflammation.
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In the NZM2410 lupus-prone mouse strain, the interplay of 2 loci, Sle1 and Sle3, leads to the emergence of pathogenic autoantibodies and lupus. Importantly, a key genetic contribution from the Sle3 interval is the kallikrein gene (22, 23). The Sle3 interval in strains such as B6.Sle3, NZW, NZM2410, and (NZB × NZW)F1 harbor a Klk allele that encodes reduced kallikrein production (22). Strains bearing this allele (including NZW, 129/Sv, and DBA/1) exhibit increased anti-GBM–induced nephritis, suggesting that kallikrein may be renoprotective. Evidence for this has emerged from 2 different studies. In one study, adenoviral delivery of kallikrein ameliorated anti-GBM–induced nephritis (23). In another study, blocking this axis using bradykinin B2 receptor antagonists resulted in more severe anti-GBM–induced nephritis (22). Given that renal tubular cells constitute a major source of kallikreins (32–35), we reasoned that local elaboration of kallikreins may confer protection against nephritis. The present study examines whether the above-described model also holds true for spontaneous lupus nephritis. Whereas B6.Sle1.Sle3 mice (which produce less kallikrein because of genetic polymorphisms encoded within the Sle3 interval) exhibit signs of lupus nephritis, deliberate up-regulation of kallikreins within the renal tubules significantly dampened renal disease in this model, as evidenced by both clinical and histopathologic readouts.
These findings resonate well with previous reports that kallikreins may be renoprotective in other models of renal disease (34–45). Kallikreins have been shown to play protective roles against ischemic stroke–induced renal injury in rats by inhibiting apoptosis and inflammation (36–39). Similar protective effects have also been reported in salt-induced hypertensive glomerulosclerosis (40, 41) and gentamicin-induced nephrotoxicity (42, 43). Further evidence of renal protection has been observed in a mouse model of diabetic nephropathy. It has been reported that kallikreins protect against microalbuminuria in experimental type I diabetes mellitus, while a lack of both bradykinin B1 and B2 receptors enhances nephropathy in diabetic mice (44–46).
The mechanisms through which kallikreins may confer renoprotection have not been fully understood. Previous experiments demonstrated that the protection against tissue damage provided by kallikreins could be attributed to pleiotropic effects in inhibiting oxidative stress, apoptosis, inflammation, and fibrosis (47). Several studies have shown that the kallikrein–kinin system can increase nitric oxide (NO) production through stimulating endothelial cell NO synthase activity and can increase cAMP levels in kidney epithelial cells (48–51). In the kidney, proximal tubular epithelial cells are high oxygen–using cells that are vulnerable to oxidative stress. NO and cAMP decrease mitochondrial oxidative metabolism by inhibiting cytochrome c oxidase and activating NADH-ubiquinone oxidoreductase, which in turn suppresses the level of oxidative stress (52–55). The suppression of oxidative stress could also lead to reduced inflammatory cell recruitment and less apoptosis in renal tubular cells. In vitro experiments with cultured renal tubular cells also showed that kallikreins and kinins suppressed H2O2-induced apoptosis and increased cell viability and Akt phosphorylation (39).
In our studies, we observed that kallikreins may be conferring renoprotection through at least 2 distinct pathways: the inhibition of inflammation and redox balancing. Kallikrein induction inhibited the expression of proinflammatory cytokine/chemokines such as IL-1, IL-6, and TNFα, the expression of which has been reported to be elevated in immune-mediated nephritis in both humans and mice (56, 57). In addition, kallikrein treatment in our model also suppressed the production of renal superoxide, with increased NO production. Superoxides (including H2O2) induce reactive oxygen species (ROS) formation, which can have deleterious effects on cells. ROS produced in renal cells have the potential to damage key cellular components including lipids, proteins, and DNA, and the balance of intracellular redox is a key determinant of cell survival, proliferation, differentiation, and apoptosis in nephritis (58). Kallikreins have previously been reported to be able to inhibit H2O2-induced ROS formation via NO production in cultured tubular cells (39).
With respect to the role of NOx in oxidative stress–induced renal damage, reports have been seemingly contradictory. For example, increased NO in lupus glomerulonephritis has been reported to be associated with increased oxidative stress and disease activity (59), as reviewed recently (60, 61). In contrast, NOx has been shown to play a protective role in oxidative-related organ damage in the heart and kidney (62). It is conceivable that these molecules may exert a multitude of effects, possibly being dependent on the specific cell types and molecular contexts. Our data indicated that tamoxifen-induced Klk1 expression is associated with increased NOx production, and this was corroborated by antinitrotyrosine staining (Figures 5C–E). Admittedly, it remains to be formally demonstrated that these resulting changes in the NO pathway are working to subdue disease, as we hypothesize, or are counteracting the protection conferred by kallikreins.
In conclusion, we observed renal kallikreins to be potent modulators of local pathologic reactions in lupus nephritis, a finding that resonates well with the previously reported beneficial roles of renal kallikreins in immune-mediated nephritis (22), diabetic nephropathy (44, 45), and nephritis caused by other triggers (47–51). At the mechanistic level, kallikreins may be modulating renal disease by inhibiting local inflammation, oxidative stress, and apoptosis. These genetic studies suggest that local delivery of kallikreins to the inflamed kidneys may be a viable treatment option for lupus nephritis. Experiments to directly test this hypothesis are therefore warranted.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Mohan and Q-Z Li had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Shao, Raman, Wakeland, Igarashi, Mohan, Q-Z Li.
Acquisition of data. Shao, Yang, Yan, Y. Li, Du, Raman, Zhang, Q-Z Li.
Analysis and interpretation of data. Shao, Yang, Yan, Y. Li, Du, Mohan, Q-Z Li.