*2.6. Correlation Analysis between the Different Parameters Studied in SLE Patients*

The correlation between some parameters were studied both in SLE patients, and LN-SLE patients by Pearson coefficient. Correlations of different parameters in SLE patients are presented in Tables 7 and 8. We detected a negative, statistically significant correlation between ImAnOx and 4-HNE, TBARs, MDA, 8-oxo-dG and PCO. OGG-1 correlated negatively with statistical significance with 8-oxo-dG. NT and TT correlated negatively with statistical significance with 4-HNE, nitrotyrosine and PCO. DS correlated positively with statistical significance with TBARs, AGE, nitrotyrosine and PCO. DS/NT correlated positively, while DS/TT corelated negatively with nitrotyrosine and PCO. NT/TT negatively correlated with pentosidine, nitrotyrosine and PCO. sRAGE correlated negatively with pentosidine and AGE. Correlations of different parameters in LN patients are presented in Table 6. ImAnOx and 4-HNE, TBARs, MDA, F2Isoprostan, 8-oxo-dG and PCO. OGG-1 correlated negatively with statistical significance with 8-oxo-dG. NT and TT correlated negatively with statistical significance with 4-HNE, nitrotyrosine and PCO. DS correlated positively with statistical significance with TBARs, F2Isoprostan, 8-oxo-dG, pentosidine, AGE, nitrotyrosine and PCO. DS/NT correlated negatively with nitrotyrosine and PCOm

while DS/TT corelated negatively only with PCO. NT/TT negatively correlated with pentosidine and PCO. sRAGE correlated negatively with pentosidine and AGE.


**Table 7.** Correlation analysis between studied parameters in SLE patients.

4-HNE—4-hydroxy-2-nonenal, TBARs—Thiobarbituric acid reactive substances, MDA—Malondialdehyde, ImAnOX—Total antioxidative capacity, 8-Oxo-dG—7,8-dihydro-8-oxo-2′ -deoxyguanosin, OGG1—8-oxoguanine-DNA-glycosylase, AGE—Advanced glycation end products, sRAGE—Soluble receptor for advanced glycation end products, PCO—Carbonylated proteins, NT—Native thiol, TT—Total thiol, DS—Disulphide, SLE—Systemic lupus erythematosus, LN—Lupus nephritis, *p*—Statistical significance.


4-HNE—4-hydroxy-2-nonenal, TBARs—Thiobarbituric acid reactive substances, MDA—Malondialdehyde, ImAnOX—Total antioxidative capacity, 8-Oxo-dG—7,8-dihydro-8-oxo-2′ -deoxyguanosin, OGG1—8-oxoguanine-DNA-glycosylase, AGE—Advanced glycation end products, sRAGE—Soluble receptor for advanced glycation end products, PCO—Carbonylated proteins, NT—Native thiol, TT—Total thiol, DS—Disulphide, SLE—Systemic lupus erythematosus, LN—Lupus nephritis, *p*—statistical significance.

#### **3. Discussion**

The results of the present study showed that SLE patients have a high risk of inflammatory responses associated with oxidative stress. The assessment of oxidized metabolites pattern and endogenous strategies for suppression or modulating oxidative stress in SLE patients with or without renal impairment could be relevant in differential regulation of cellular response for preventing the progression of the renal manifestations. We determined a large panel of oxidative damage serum metabolites and endogenous protective mechanisms in SLE patients and a control group. The comparative analysis of oxidized metabolites pattern and antioxidative capacity in SLE with all type lupus nephritis, in type IV lupus nephritis, and in SLE without renal impairment aimed to determine possible deficiencies in protective system of the cells and some humoral biomarkers for detection of renal impairment in SLE patients.

The comparative analysis of lipoperoxidation process by simultaneous determination of 4-HNE, TBARs, MDA and F2-isoprostan in SLE patients and controls demonstrated higher levels of lipoperoxidation-derived reactive carbonyl species in SLE and LN compared with the control group. LN patients have statistically significantly higher levels of TBARs and F2-isoprostan, comparative with SLE non-LN patients. These major oxidative degradations were accompanied by low levels of ImAnOx in SLE patients versus controls. Serum antioxidants inactivation was more accentuated in LN patients compared with non-LN patients. ImAnOx capacity of blocking lipoperoxides formation or blocking lipids peroxidation propagation was demonstrated in this paper by negative association between lipid metabolites levels and antioxidant capacity of serum. Based on these results, we could appreciate that ImAnOx diminishing and F2-isoprostan increase are associated with LN development. As a parameter of oxidative deterioration, F2-isoprostan is considered by some publications as a useful oxidative stress biomarker [39,40]. F2 isoprostan varies according to pathologies; it has very low levels in cancer or cardiovascular diseases, very high in respiratory diseases and uro-genital disorders, or its value varies with clinical features [40] or inflammation, such as in SLE [39]. F2-isoprostan does not present specificity for oxidative stress due to the synthesis mechanisms. In our study, serum variability of F2-isoprostan in SLE and LN was caused by the level of oxidative stress, both SLE groups being characterized by a low inflammatory state. F2-isoprostan is useful in identifying patients at risk of developing LN.

Some studies show that lipid peroxidation plays a major role in SLE pathogenesis. SLE patients had higher levels of MDA and 8-OHdG and lower levels of TAS compared with controls. Circulant levels of MDA, 8-OhDG and TAS in patients with SLEDAI over six were statistically significant modified comparative with patients with SLEDAI below six. MDA presented a positive correlation and TAS a negative correlation with SLEDAI. Remarkable also is the high MDA/TAS ratio, in patients with neuro-psychiatric manifestations and vasculitis in SLE, high levels of oxidized low density lipoprotein cholesterol in SLE patients with thrombocytopenia and vasculitis [41]. Increases of MDA and 8-OhdG, and decreases of TAS were associated with disease activity. ROS role in SLE was evaluated by aldehydes specific immune complexes derived from lipids. These data sustain a possible relation between oxidative stress, anti-species carbonyl reactive, pathogenic mechanisms and prognosis in SLE [42]. A recent study evaluated oxidative stress markers, inflammation and biomarkers of activity in SLE and detected significant differences between active and inactive lupus nephritis for MDA, total and oxidated GSH, TAS, CRP, MCP 1, beta 2-microglobulin, urinary protein/creatinine ratio, dsDNA antibodies, anti-C1q antibodies and C3, C4. These data show a redox disequilibrium in patients with lupus nephritis determined by lipid peroxidation, process that affects glomerular basal membrane integrity and renal tubular function in these patients [43].

An interesting finding of our study, relevant for oxidative stress in SLE was represented by elevated oxidative DNA damage levels and defective DNA repair mechanisms via OGG1. Oxidative DNA lesions were overexpressed both in non-LN and LN groups comparative to controls. OGG1 has the ability to catalyze lesions excision of 8-oxo-dG

type and assure genomic integrity in SLE patients. OGG1 capacity of DNA repair and of 8-oxo-dG serum levels modulating in LN patients is highly altered. Based on these results, we consider that 8-oxo-dG/OGG1 ratio could offer information about LN development risk. DNA alteration by oxidative stimuli, DNA repair mechanisms dysfunctions and immune response alteration in cellular defense were intensively studied in the last years [3]. DNA alteration could induce aberrant activation of innate immunity, and, associated with chronic inflammation, could stimulate DDR/R network. ROS and RNS generated by inflammatory cells promote 8-oxo-dG and 8-nitro-dG overexpression resulting in the alteration of the repair mechanisms of DNA lesions. Still, in SLE, there is proved a bidirectional interaction between DDR/R and immune response induced by oxidative stress. DDR/R network and immune innate response act synergistically for the survival of all living organisms. Epigenetically regulated functional abnormalities of DNA repair mechanisms could lead to increased accumulation of DNA lesions. This accumulation facilitates the production of autoantibodies, the generation of damaged cytosolic DNA and micronuclei that can act as powerful stimulators of the immune system by over-expression of the cGAS-STING-IRF3 path and the production of type I IFN, leading to the systemic expression of autoimmune diseases [3,14,32].

Rigorous research of carbohydrate oxidation in this study showed profound alteration of the AGE–RAGE axis in patients with SLE and LN. The harmful effect of AGE on human tissues was proved by excessive production of RAGE ligands in the studied patients. Thus, serum levels of AGE and pentosidine were significantly increased in patients with SLE and LN compared to control. The toxic action of AGE was limited by sRAGE. The protective effect of sRAGE was well expressed in patients with SLE. On the other hand, the ability of sRAGE to block activation of RAGE in patients with LN was affected. The different behavior of sRAGE in SLE with or without LN could be sustained by negative correlation between AGE, and pentosidine and sRAGE in non-LN patients and their positive association in LN patients. It can be interpreted that an increased amount of AGE in patients' blood leads to increased consumption of sRAGE. These findings support the quality of sRAGE as a biomarker to differentiate patients with SLE at risk of kidney injury. Recent findings suggest that the occurrence of SLE depends on the interactions between genetic background and risk factors, such as exposure to oxidative stimuli. Chronic inflammation associated with SLE disturbs the oxidant/antioxidant balance. These processes are related to the excess accumulation of AGE. Lately, the accumulation of AGE in the skin in patients with autoimmune diseases and the expression of corresponding receptors gain the attention of researchers. These AGE compounds could be found in inflamed tissues. The reference data present in the literature show a link between oxidative stress, AGE formation and autoimmune diseases such as SLE [44,45]. The most representative AGEs are pentosidine, carboxymethyllysine (CML) and carboxiethyllysine (CEL). The high levels of AGEs in patients' blood are still being discussed. Some authors indicated that there were no significant differences between SLE patients and controls regarding plasma levels of CML and CEL. AGE levels did not correlate with CRP. The authors suggested that AGE accumulated mainly in tissues and plasma proteins [45,46]. Additionally, the level of pentosidine did not increase in the blood of patients with SLE. Some patients with increased fructosamine showed remarkably high levels of pentosidine. Other authors demonstrated an increase in plasma AGE in patients with SLE. In addition, the levels of CML and pentosidine showed a positive correlation with the SLEDAI score.

Other RAGE ligands, such as S100A12 levels are similar in SLE and healthy subjects, while levels of S100A8/A9 and S100A12 correlate with cardiovascular risk in SLE patients [47–49]. RAGE ligands appear to be mostly related to inflammation, hyperglycation and cellular stress processes; expression of the receptor itself being directly related to inflammation [50,51].

sRAGE can bind to RAGE, blocking its functions and leading to an improved expression of the molecules involved in inflammation, adhesion and RAGE itself [47].

RAGE expression is also enhanced by TNF-α. It remains unknown whether patients with SLE have autoantibodies against RAGE/sRAGE, but sRAGE functions are insufficient studied in this pathology [9]. A low amount of sRAGE has been detected in Sjogren's syndrome, though a possible link between SLE and visual disturbances could be established [47]. Contradictory data on the level of sRAGE have been reported in SLE patients. Some authors have reported increased concentrations of sRAGE in the serum of lupus patients [45]. Additionally, sRAGE positively correlates with SLEDAI [52]. Other studies showed the decreased levels of sRAGE in the plasma of SLE patients compared to control. Notably, SLE patients treated or untreated have similar levels of sRAGE, while subjects who received long-term treatment have a higher concentration of soluble receptor than subjects receiving short-term one. Short-term treatment induces a rapid decrease in sRAGE levels compared to long-term one. It is suggested that the receptor has a different role in the initial and progressive stage of the disease [47,48]. In addition, patients with SLE who have antiphospholipid antibodies (APA) or antiphospholipid syndrome (APS) have shown a decrease in sRAGE plasma levels compared to control [53]. sRAGE level is associated with leukocytes lymphocytes, neutrophils and monocytes number and with C4 levels, but it does not correlate with the presence of autoantibodies. sRAGE therefore participates in the development of inflammation and the recruitment of leukocytes. RAGE and sRAGE have the ability to interact with each other and with multiple ligands, and they are directly related to the immune. These processes increase the oxidative stress in the body. AGE and RAGE with sRAGE deficiency can cause the formation of neoepitopes, and of autoantibodies [47].

Finally, we found that changes in oxidative proteins and TDH regulators play a central role in the cellular response to oxidative stress. In this paper, it was found that 3-nitrotyrosine, carbonylated proteins and disulfides were overexpressed in patients with SLE and LN compared to the healthy population. The circulating level of 3-nitrothyrosine was significantly increased in patients with LN versus patients with SLE non-LN. The ability of TDH to maintain redox homeostasis of cells has also been demonstrated by the inverse association between the concentrations of sulfhydryl groups and oxidized proteins in both SLE and LN. Through their dynamics, the DS/TN, DS/TT, TN/TT ratios can be potential serum biomarkers that have managed to differentiate between SLE patients, those that will develop LN. Our findings suggest that protein oxidation may play a substantial role in the pathogenesis of chronic kidney damage in patients with SLE. These results reconfirm data from previously published studies regarding levels of multiple markers of protein oxidation. SOD and myeloperoxidase activities were increased, while thiol protein levels, glutathione peroxidase and catalase activities were reduced in serum of SLE patients compared to controls. Disease activity markers were positively correlated with erythrocyte sedimentation rate and carbonylated protein levels, 3-nitrosyrosine and CRP, and negatively correlated with protein thiol levels and SOD, glutathione peroxidase and catalase activities in patients with SLE. There were significant differences in serum levels of carbonylated proteins between patients with and without kidney disease [54].

Lipid peroxides (LOOH), advanced oxidation protein products (AOPP), nitrogen oxides (NOx), sulfhydryl groups (−SH), oxidative degradation products of deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) and antioxidant parameter of radicals' uptake (TRAP) participates in SLE immune pathophysiology and modulates disease severity and adhesion molecule expression [55–57]. Another study showed that increased protein oxidation correlated with SLEDAI score and dsDNA levels [58]. Other publications have shown that oxidative stress is involved in the pathogenesis of LN. Higher levels of serum AOPP have been associated with an increased risk of LN. In contrast, neither high levels of dsDNA nor low levels of C3 were independent risk factors for LN [59]. Evaluation of the prooxidant–antioxidant balance (PAB) showed increased values in patients with SLE with alopecia, discoid rash, oral ulcers, arthritis and nephritis. These findings suggest that PAB measurement may be useful to show the state of oxidative stress in patients with SLE [60].

To resume, the present study is the first one in the literature that presents a pattern of oxidative stress markers and endogenous strategies for suppression/modulating oxidative stress in SLE patients. However, some limitations should be noted. Our study followed patients with SLE non-LN and LN, for a period of three years, only with chronic immunosuppressant treatment. Further studies with a larger number of patients with different therapeutic regimens should be developed. Most of the patients included in the study had type IV nephritis. For a better evaluation of oxidant/antioxidant axis in SLE a larger number of patients with all types of nephritis are needed, although it would be very hard to identify these patients because they have minimal symptoms. In LN patients, it is very important to identify as early as possible the patients at risk for LN development, in order to avoid invasive interventions and to establish an effective medical intervention.
