A common feature in the broad phenotype of systemic lupus erythematosus (SLE) is autoantibody production, a function primarily of the acquired immune system (1). However, an inappropriately active innate immune response is implicated in both the initiation and the pathogenic consequences of autoantibody production in SLE. An important antimicrobial factor in the innate immune response is the production of reactive oxygen and nitrogen intermediates (RONI), including superoxide and nitric oxide (NO) (2).
In murine models of lupus, NO production has been shown to increase with the progression of renal disease. Pharmacologic inhibition of inducible NO synthase (iNOS) in these models significantly reduced both NO and reactive oxygen intermediate (ROI) production and also reduced inflammatory glomerular lesions, without affecting the deposition of immunoglobulin or complement (2, 3). These findings suggest that iNOS activity or products of iNOS such as superoxide and NO contribute to the proliferative glomerular lesions in murine lupus nephritis and that their production is distal to immune complex deposition and complement activation. Cross-sectional studies of NO production in human SLE have demonstrated increased levels of markers of systemic and local NO production in association with disease activity (4–12). These studies, while important, were limited by their retrospective cross-sectional design (4, 8–14), lack of control for dietary sources of nitrates, and small study populations (4, 6–12).
The present investigation was conducted to address these limitations by prospectively examining the longitudinal association between production of NO and lupus disease activity in individual patients. We hypothesized that NO production would be 1) increased in SLE patients compared with control subjects, 2) associated with SLE disease activity markers over time, and 3) associated with measures of proliferative lupus nephritis activity and damage as measured by simultaneous renal biopsy and renal outcome assessment.
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The results presented herein demonstrate significant associations between a marker of systemic NO production and measures of disease activity in SLE. There were significant increases in serum NOx levels observed during complement activation, particularly in the setting of proliferative glomerulonephritis. Global NO burden over time (AUC NOx) was associated with progression of renal damage, and serum NOx levels mirrored response to therapy. These findings suggest a pathogenic role of RONI production in human proliferative glomerulonephritis, similar to findings in our prior study of murine lupus nephritis (24, 25).
The novelty of this study lies in: 1) its prospective and longitudinal design, 2) reduction of the confounding effects of exogenous dietary NOx through the use of a low-NOx diet prior to sample collection, 3) study of a large number of patients, a significant proportion of whom were African American, and 4) reduction of the confounding effect of high-dose corticosteroids on iNOS expression during renal biopsy visits.
A previous prospective study did not demonstrate an association between lupus nephritis and serum NOx levels. However, in that study, higher corticosteroid doses were allowed, and simultaneous renal biopsy results were not available (5). Other groups have demonstrated in cross-sectional analyses that patients with active SLE, low complement levels, and high anti-dsDNA levels had elevated levels of serum NOx (10, 12). The longitudinal design of the present study enabled the analysis of within-patient associations between disease activity markers and serum NOx levels, thus avoiding between-patient variability that is inherent in any lupus cohort and that may have reduced the power of previous studies. This is the first study to demonstrate an association between serum NOx levels and proteinuria in humans. This correlation is consistent with findings in studies of murine lupus nephritis, in which overproduction of NO has been shown to parallel, and even precede, the onset of proteinuria (24, 25).
The longitudinal association of systemic NO production with response to therapy and with biopsy-proven proliferative glomerulonephritis is a novel finding that has implications with regard to therapy. Our study population was predominantly African American, while the study cohorts in prior investigations of NO measures in SLE were mostly white or Hispanic. Thus, it is intriguing to postulate that one potential mechanism for the higher rate of renal failure and poorer response to therapy in African Americans with proliferative glomerulonephritis compared with whites (26) is increased RONI production.
In an earlier investigation of the same study cohort, we observed increases in markers of production of systemic peroxynitrite (ONOO, a product of iNOS activity), which were more closely associated with disease activity among African Americans than among whites (27). We have also described an increased prevalence of 2 iNOS gene (NOS2) promoter polymorphisms in African American women with lupus compared with sex- and race-matched controls (28). These polymorphisms were not present in patients of European descent and have been reported to be frequent in malaria-endemic areas (29, 30). One has been associated with increased ex vivo leukocyte NO production (31). Together, these findings suggest the possibility of differential genetic control of NOS2 transcription under inflammatory conditions among African Americans. One confounder in the association between serum NOx levels and treatment response is that the treatment was random and not controlled. Future studies investigating this association should involve subjects participating in large clinical trials, to reduce the number of treatment variables.
While there was a very strong association between serum NOx levels and SLEDAI scores in the bivariate longitudinal analysis, the weaker (though still significant) association in multivariate longitudinal analysis may be due to several factors. First, the clinical variables used in the mixed models were in fact elements from the SLEDAI. Inclusion of colinear variables into multivariate models often results in loss of significance of one or more of those variables. The strong association between SLEDAI scores and serum NOx levels in the bivariate analysis supports this interpretation. Second, there is a clear association between reduced complement levels and NO production, yet not all of the elements of the SLEDAI are associated with significant complement activation. The association with proliferative renal disease, which is dependent on complement activation, is consistent with this notion. However, the association with renal disease may not have been captured by the SLEDAI as well as it would have been with, for instance, the British Isles Lupus Assessment Group index (BILAG), due to lack of sensitivity of the SLEDAI to changes in renal disease activity. However, the BILAG measure was not available at the initiation of this trial and therefore could not be used for longitudinal followup (32).
The association between progression of renal insufficiency and elevated NOx levels over time may result from two general mechanisms. First, reduced renal excretion of NOx may lead to increased serum levels. The demonstration of a lack of association between renal excretion of NOx and serum NOx levels, with a clear association between serum NOx and C3 levels in the same patients, makes it unlikely that decreased NOx clearance is the dominant cause of elevated serum NOx levels in this population. Second, the association between global burden of NOx production over time (AUC NOx) and future renal damage (ΔCr) suggests that glomerular RONI production is a pathogenic mediator in glomerular proliferation and sclerosis. This is further supported by data demonstrating that serum NOx levels did not decrease as quickly in patients whose lupus nephritis did not respond to treatment, compared with responders. Because not all study visits occurred within 4 weeks of the predetermined 90-day visit interval, the AUC analyses could be subject to a time bias. While this bias should be considered, the more significant association between AUC NOx and ΔCr when data were normalized to time suggest that the results obtained were not predominantly a reflection of time bias.
Based on the results of this and previous studies, we propose the following model for potential causes and consequences of pathogenic RONI production in complement-mediated disease manifestations of SLE: In the setting of immune complex deposition and complement activation, complement split products C3a and C5a bind to their receptors. Engagement of C5a and C3a receptors results in elevated transcription of NOS2, increased expression of iNOS, and logarithmic increases in production of RONI such as superoxide and ONOO (33). Evidence of this mechanism has been provided in models of anti–Thy-1–induced nephritis, in which soluble CR1 reduces NOS2 messenger RNA and iNOS protein levels, and in intestinal ischemia-reperfusion models, in which an anti-C5 monoclonal antibody reduces RONI production (34, 35). RONI are known mediators of apoptosis (36). Markers of increased NO production have been observed in cells activated by immune complex deposition and complement activation in lupus, i.e., vascular endothelial cells (8) and glomerular and tubulointerstitial cells (7).
In studies of lupus patients, increased apoptosis was noted among those with proliferative glomerulonephritis in association with increased iNOS expression (7, 37), and in circulating endothelial cells without evidence of increased iNOS expression (38). However, findings of other studies have suggested that iNOS expression induces apoptosis in endothelial cells. In human umbilical vein endothelial cells, ROI induce NF-κB–mediated increases in iNOS expression and apoptosis (39). From these observations, one could hypothesize that RONI play a role in SLE endothelial cell apoptosis as well. ROI can depolymerize glomerular basement membrane heparan sulfate moieties, resulting in loss of the glomerular basement membrane charge barrier and increased excretion of protein into the urine (40). ONOO is capable of altering the activity of multiple enzymes thought to play regulatory roles in glomerular function, such as cyclooxygenase (41), prostacyclin synthase (42), and endothelial NOS (43). ROI are capable of increasing DNA binding of the redox-sensitive transcriptional regulator NF-κB (44). This can result in an increased inflammatory response that, in an adoptive transfer model of autoimmune diabetes, can be inhibited by a superoxide dismutase mimetic (45). Finally, ONOO can increase the antigenicity of dsDNA (46), resulting in a feed-forward loop that can accelerate the initiation and progression of proliferative glomerulonephritis.
The results presented herein suggest that increased RONI production is a pathogenic mediator in complement-mediated disease processes in SLE, particularly proliferative lupus nephritis. This has potential implications regarding the management of and/or monitoring of response to therapy of aggressive proliferative glomerulonephritis. The full effect of reactive intermediates on glomerular pathology cannot be determined without specifically targeting their production in humans. Therefore, future work will evaluate the effect of available reactive intermediate–reducing therapies on markers of iNOS activity relative to treatment response. In addition, novel therapies for inhibiting iNOS activity or scavenging RONI should be studied as adjunctive induction treatment in proliferative lupus nephritis.