Background/Purpose: The role of redox regulation of cell function in the different International Society of Nephrology/Renal Pathology Society (ISN/RPS) classes of lupus nephritis (LN) is not known. Different reactive intermediate (RI) species lead to differential changes in proteins that are important in transcriptional regulation and signaling. RIs are unstable species that can be measured indirectly by their ability to modify protein tyrosines (Tyr) and phenyalanines to form nitroTyr (NTyr, via peroxynitrite), metaTyr (mTyr, via hydroxyl radicals (OH·), usually metabolized from SO and H₂O₂), and chloroTyr (ClTyr, via HOCl). This study was designed to address the hypothesis that different classes of LN have distinct profiles of RI production.

Methods: 62 patients with active LN had a renal biopsy with ISN/RPS classification at screening. Serum and urine samples were collected before induction therapy for active LN. Serum (n=62) was analyzed for nitrate and nitrite (NO_X, a marker of nitric oxide (NO) production) by chemiluminescence analyzer.  In a random subset of patients (n=34), serum proteins were analyzed for NTyr, mTyr, and ClTyr by HPLC with electrochemical detection and reported as the ratio of modified to unmodified Tyr * 1000. Snap frozen murine renal cortical tissue lysates from MRL/lpr and MRL/lpr NOS3 -/- (endothelial NO synthase or eNOS) mice with active proliferative LN were analyzed for superoxide (SO) production by luciginen assay with and without inhibitors of NOS and NADPH oxidase. Results were normalized to untreated MRL/lpr wildtype control tissue. Groups were compared by Wilcoxen rank sum or Student t test, and p < 0.05 was significant.

Results: Those with class IV LN had significantly higher levels of serum NO_X. mTyr was elevated in those with class III and IV LN. In class V LN, NTyr and ClTyr were increased (differences not significant). SO production was increased (to 160%) in MRL/lpr NOS3-/- kidney cortex. This increase was reduced by both L-NMMA (a nonspecific NO synthase inhibitor, 70% of control) and DPI (a nonspecific NADPH oxidase inhibitor, 40% of control).

Levels of markers of RI production in patients with LN by ISN/RPS class
ISN/RPS Class	nitrate + nitrite	mTyr/Tyr*1000	ClTyr/Tyr*1000	Ntyr/Tyr*1000
Pure I	39 (n = 1)	0.6 (n = 1)	0.1 (n = 1)	0.2 (n = 1)
Pure II	30.2 ± 7.2 (n = 9)	0.2 ± 0.1 (n = 4)	0.1 ± 0 (n = 4)	0.3 ± 0.1 (n = 4)
Pure and mixed III	30.4 ± 5.3 (n = 29)	9 ± 5.5 (n = 17)	0.1 ± 0 (n = 18)	0.7 ± 0.3 (n = 18)
Pure and mixed IV	59.6 ± 17.8 (n = 12)	7.8 ± 4.2 (n = 8)	0.6 ± 0.3 (n = 9)	1.1 ± 0.3 (n = 9)
Pure V	24.9 ± 6.3 (n = 10)	0.7 ± 0.4 (n = 4)	13.9 ± 13.7 (n = 5)	21.2 ± 21 (n = 5)
Values reported as means ± standard error. Bolded values are significantly different from other classes.

 Conclusion: These results suggest increased NOS activity in class IV LN. In those with proliferative LN, increased OH• production or reduced antioxidant scavenging appears to be occurring. OH• can indirectly come from NADPH oxidase and NO synthase. Higher levels of SO in MRL/lpr cortical tissue from NOS3 -/- mice suggests that eNOS-derived NO can scavenge SO. Reduction of SO with both a NOS inhibitor and an NADPH oxidase inhibitor suggests that both enzymes produce SO, perhaps in a synergistic fashion, in proliferative LN. Therefore, effective therapy for SO-mediated redox signaling in proliferative LN may need to induce increases in the scavenging effect of eNOS-derived NO or target SO directly with scavengers. Myeloperoxidase (forms NTyr and ClTyr) may be an important enzyme target in class V disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/markers-of-nitric-oxide-and-hydroxyl-radical-formation-are-increased-in-proliferative-lupus-nephritis-and-may-emanate-from-increased-nitric-oxide-synthase-and-nadph-oxidase-production-and-reduced-endo/