Urinary Modified C-Reactive Protein is Closely Associated with Tubulointerstitial Lesions in Lupus Nephritis

Objective Modified C-reactive protein (mCRP) is known to be involved in the upregulation and amplification of the local inflammatory response. This study investigated the circulating and local levels of mCRP and their relevance to clinicopathological features in patients with lupus nephritis. Methods Ninety-five patients with renal biopsy-proven lupus nephritis and 30 normal controls were enrolled in this study. Plasma and urinary mCRP were screened by enzyme-linked immunosorbent assay (ELISA). The renal deposition of mCRP was detected by immunohistochemistry and immunofluorescence staining. A human proximal tubular epithelial cell line (HK2 cells) was incubated with purified IgG from lupus nephritis, and the production of CRP by HK2 cells was further evaluated. Results Plasma and urinary levels of mCRP increased significantly in patients with lupus nephritis compared with normal controls (P = 0.013, P < 0.001, respectively). The urinary mCRP levels were associated with interstitial inflammatory cell infiltration (r = 0.514, P < 0.001) and interstitial fibrosis (r = 0.270, P = 0.008). The ROC–AUC of the urinary mCRP levels for diagnosing tubulointerstitial lesions was 0.766. The urinary mCRP levels were closely associated with poor outcomes (HR: 1.204, 95% CI: 1.029–1.409, P = 0.020). However, no correlations were found of the plasma mCRP levels with clinicopathological data or the prognosis of lupus nephritis. CRP was mostly deposited in the renal tubules in patients with lupus nephritis, and the expression of CRP was significantly correlated with tubulointerstitial lesion indices. Immunofluorescence staining showed that mCRP could colocalize with IgG in tubules. Lupus nephritis-derived IgG could induce CRP production by HK2 cells. Conclusion Urinary mCRP levels were significantly increased, and urinary mCRP might be a biomarker for tubulointerstitial lesions in patients with lupus nephritis. Renal CRP could be produced by tubular epithelial cells after stimulation by lupus nephritis-derived IgG, and the local presence of mCRP might play a critical role in the development of tubulointerstitial lesions.


Introduction
Systemic lupus erythematosus (SLE) is a prototypical multisystem autoimmune disease, and lupus nephritis is a common and severe manifestation of SLE that can result in irreversible renal impairment. In addition to glomerular injury, tubulointerstitial lesions could also be prominent in lupus nephritis [1]. of tubulointerstitial lesions [6]. However, there is a lack of reliable and appropriate biomarkers reflecting tubulointerstitial lesions in lupus nephritis patients.
Plasma native C-reactive protein (nCRP) has been widely recognized as a highly conserved acute phase reactant, primarily produced by hepatocytes in response to tissue injury or infection that elicits an inflammatory response [7,8]. Thus, it is regarded as a nonspecific marker for ongoing inflammation in clinical practice. nCRP belongs to the pentraxin family, which consists of five identical nonglycosylated globular subunits [9]. When nCRP in the local inflammatory area encounters activated membranes [10][11][12], neutrophil extracellular traps [13], or acidic pH [14], biochemical forces contribute to dissociation of the pentamers into monomers. The dissociated conformation is termed modified CRP (mCRP), which exerts potent proinflammatory actions on endothelial cells, macrophages, and neutrophils and can upregulate and amplify the local inflammatory response [15][16][17].
Levels of nCRP can increase dramatically following an acute-phase stimulus. However, despite evident inflammation and tissue damage, SLE fails to elicit major nCRP production [18]. However, increased levels of plasma mCRP have been found in patients with skin-related inflammatory autoimmune disorders, such as eczema, psoriasis, and urticaria, whereas the levels of plasma nCRP in patients did not differ significantly from those in normal controls [19]. In addition, the expression of CRP mRNA and the synthesis of nCRP have been reported in renal tubular epithelial cells [20]. Schwedler et al. [21] detected tubular mCRP deposition in renal biopsies from patients with diabetic nephropathy, and local mCRP expression was correlated with the severity of histologically detectable lesions.
Thus, we investigated the circulating and local levels of mCRP in patients with lupus nephritis, and their correlations with clinicopathological features and prognosis were evaluated.

Materials and Methods
2.1. Patients and Samples. Complete clinicopathological data of 95 patients with renal biopsy-proven lupus nephritis diagnosed from January 2016 to July 2019 at Peking University First Hospital were collected for analysis. All patients fulfilled the 1997 American College of Rheumatology revised criteria for SLE [22]. The enrollment of patients with lupus nephritis is shown in Figure S1.
Plasma and morning urine samples from all of the patients were obtained on the day of renal biopsy before initiation of immunosuppressive treatment. Plasma and urine samples from 30 healthy volunteers, matched for age and sex, were selected as normal controls. The first plasma exchange samples from five patients with active lupus nephritis were collected upon presentation. Plasma samples from five healthy volunteers served as normal controls.
Renal biopsies of 20 patients with lupus nephritis from Peking University First Hospital were selected for the pathological study. Among these patients, four had severe tubulointerstitial nephritis, and another 16 had no (n = 5) or mild to moderate (n = 11) tubulointerstitial injury. Renal tissues from normal areas of nephrectomized kidneys from five patients with solitary renal cell carcinoma were collected as the normal controls.
All plasma and urine were stored in aliquots at −80°C until use, and repeated freeze-thaw cycles were avoided. Informed consent was obtained from each patient for blood and urine sampling and renal biopsy. The research was performed in accordance with the principles of the Declaration of Helsinki and was approved by the local ethical committees (number 2014 (749)).
The patients were followed-up in outpatient lupus clinics. The primary endpoint was defined as death, and the secondary endpoints were defined as end-stage renal disease (ESRD), renal transplant, or doubling of serum creatinine levels.

Detection of Plasma and Urinary mCRP Levels Using
ELISA. According to previous research [19], mouse monoclonal antihuman CRP antibodies (clone 8, Sigma-Aldrich, St. Louis, MO, USA) were coated onto microtiter wells (Thermo Fisher Scientific, Waltham, MA, USA) at 1 : 1,000 in coating buffer (10 mM sodium carbonate/bicarbonate, pH 9.6) overnight at 4°C. All the following steps were conducted at 37°C. The plates were blocked with 1% bovine serum albumin (BSA) in TBS (10 mM Tris, 140 mM NaCl, 2 mM Ca, pH 7.4) (blocking buffer) for 1 hr. Diluted samples in blocking buffer were added to the wells and incubated for 1 hr. Then, the sheep antihuman CRP polyclonal antibody (BindingSite, Birmingham, UK) diluted to 1 : 2,000 in blocking buffer was added to the wells and incubated for 1 hr. The plates were incubated with HRP-labeled donkey antisheep IgG (H + L) (Abcam, Cambridge, MA, USA) for 1 hr. After incubation, the wells were developed with a 3,3′, 5,5′-tetramethylbenzidine (TMB) and stopped with 1 M H 2 SO 4 . The results were measured with a microplate reader at 450 and 570 nm. Serial concentrations of urea-denatured mCRP standards from 0 to 20 ng/ml were used to develop a standard curve.

Detection of Renal Expression of CRP by Immunohistochemistry
Assay. Renal tissue samples were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Deparaffinized slides were heated in a pressure cooker with EDTA buffer (pH 9) for 5 min. After heat-mediated antigen retrieval, the slides were immersed in freshly prepared 3% hydrogen peroxide for 30 min at room temperature to quench endogenous peroxidase activity. To block nonspecific staining, the slides were incubated with 3% BSA in phosphate-buffered saline (PBS) at room temperature for 1 hr. The primary anti-CRP antibodies (Abcam), which are supposed to recognize nCRP and mCRP according to the product description, were directly added to each slide and incubated overnight at 4°C. The secondary antibodies and staining were performed using a commercial kit (ZGSB-BIO, Beijing, China). The negative control used PBS instead of primary antibody. Image-Pro Plus analysis, software 6.0 (Media Cybernetics, Dallas, TX, USA), was used to evaluate the mean optical density of CRP staining of renal tubules.

Colocalization of mCRP and IgG in the Kidney by
Immunofluorescence Staining. Fresh-frozen sections were fixed in cold acetone for 20 min, followed by blocking with 3% BSA for 1 hr at room temperature. The sections were incubated with primary anti-CRP antibodies (Sigma) overnight at 4°C. The blank control used PBS instead of primary antibody. After extensive washing, rhodamine (TRITC)-conjugated goat antimouse IgG (H + L) (Sigma) was used as a secondary antibody for 1 hr at 37°C. Then, the sections were stained with Alexa Fluor 488-conjugated goat antihuman IgG (H + L) (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 hr at 37°C. After DAPI staining, the sections were examined using a confocal microscope (Olympus Viewer 1000, Tokyo, Japan).

Purification of IgG.
IgG was purified from the plasma exchange of five lupus nephritis patients and the plasma of five normal individuals by a protein G affinity column on an AKTA-FPLC system (GE Biosciences, South San Francisco, CA, USA) [29].
2.9. Analysis of the Levels of nCRP and mCRP in the Cell Supernatant by ELISA. The normal human proximal tubular epithelial cell line (HK2 cells) from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured in DMEM-F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin antibiotics (Gibco) at 37°C and 5% CO 2 .
After incubation of HK2 cells with purified IgG (100 μg/ml) for 24-72 hr, the supernatant was collected and centrifuged at 2000 × g for 10 min to remove cell debris, and the levels of nCRP and mCRP were measured by homemade ELISA according to a previous study [19].

RNA Extraction and Polymerase Chain Reaction (PCR).
After incubation of HK2 cells with purified IgG for 24-72 hr, total RNA was extracted from the HK2 cells using the RNeasy Mini Kit (TIANGEN Biotech, Beijing, China). Complementary DNA (cDNA) was synthesized from total RNA (1 μg) using high-capacity cDNA reverse transcription kits (Vazyme Biotech, Nanjing, China) in accordance with the method provided by the manufacturer. Quantitative real-time PCR was performed using SYBR Green Master Mix reagent (Thermo Fisher Scientific). The housekeeping gene GAPDH served as an internal control. The human CRP primer sequences were: forward 5′-GTCACAGTAGCTCCAGTACACA-3′; and reverse 5′-AAAGTTCCCACCGAAGGAATC-3′. The ratio of CRP mRNA to GAPDH mRNA was analyzed using the 2^− ΔΔCt method.
2.11. Statistical Analysis. SPSS software, version 26.0 (SPSS Inc., Chicago, IL, USA), was applied for statistical analysis. Continuous variables are expressed as the mean AE standard deviation (SD) or median (interquartile range (IQR)), and categorical variables are expressed as ratios. The associations between continuous variables were performed using Pearson's correlation analysis or Spearman's rank correlation analysis. Statistical analysis was performed with the independent-sample t-test, the Mann-Whitney U test, one-way ANOVA or the Kruskal-Wallis test as appropriate. Receiveroperating characteristic (ROC) curves were constructed, and the area under the curve (AUC) was used to distinguish tubulointerstitial lesions. Kaplan-Meier curves were employed to test patient prognosis. Univariate survival analysis was performed using the log rank test. A two-sided P <0:05 was defined as statistically significant.

Results
3.1. General Data of Patients with Lupus Nephritis. The detailed general data of patients with lupus nephritis are shown in Table S1. Twenty-two (23.2%) were male, and 73 (76.8%) were female, with a mean age of 32.5 AE 13.0 years old. According to the ISN/RPS classification system of lupus Mediators of Inflammation nephritis, two patients were classified as class II (2.1%), 27 as class III (28.4%, including 11 with class V + III), 51 as class IV (53.7%, including 16 with class V + IV), and 15 as class V (15.8%).

Associations between Plasma mCRP Levels or Urinary mCRP
Levels and Clinicopathological Features of Lupus Nephritis. No correlations were found between the plasma mCRP levels and clinicopathological data of lupus nephritis patients (Table 1). Moreover, there were no associations between plasma mCRP levels and urinary NGAL levels (r = 0.063, P ¼ 0:545) or urinary KIM-1 levels (r = 0.063, P ¼ 0:549) in patients with lupus nephritis (Figure S2 A1, B1).

Co-localization of mCRP and IgG in Patients with Lupus
Nephritis. Double immunofluorescence assays showed that mCRP was expressed in tubules of the kidneys obtained from patients with lupus nephritis, and IgG was deposited in the same areas. The renal staining of mCRP and IgG was partially merged. The staining of mCRP was scarcely seen in the tubules of samples from normal controls ( Figure 5).

CRP Production and Secretion by HK2 Cells In Vitro.
To explore the ability of renal tubular epithelial cells to synthesize CRP, we incubated HK2 cells with purified IgG for 24-72 hr. As shown in Figures 6(a) and6(b), the levels of both nCRP and mCRP in the supernatant of HK2 cells treated with IgG from patients with lupus nephritis for 24-72 hr were significantly higher than those exposed to normal IgG. The above results were also verified by quantitative real-time PCR (Figure 6(c)).

Discussion
Tubulointerstitial lesions are frequently observed in lupus nephritis, and increasing evidence has shown that tubulointerstitial lesions are a potent predictor of poor outcome [3,4]. Thus, the importance of tubulointerstitial lesions in lupus nephritis has been widely recognized. However, the pathogenesis of tubulointerstitial lesions in lupus nephritis is not entirely clear, and there is a lack of reliable and appropriate biomarkers reflecting tubulointerstitial damage in patients with lupus nephritis. Our study identified for the first time that the levels of mCRP in plasma and urine increased significantly in lupus nephritis patients compared with normal controls. Further     Mediators of Inflammation analysis showed that urinary mCRP levels were significantly correlated with urinary NGAL and KIM-1 levels, which are sensitive indicators of tubulointerstitial injury in lupus nephritis [30]. Regarding renal histopathology evaluation, urinary mCRP levels were also correlated with some tubulointerstitial lesion indices, of which interstitial inflammatory cell infiltration was the strongest. However, no correlations were found between the plasma mCRP levels and the clinicopathological data of lupus nephritis. Thus, our results indicated that urinary mCRP levels, rather than circulating mCRP levels, might be a biomarker of tubulointerstitial injury in lupus nephritis. Furthermore, urinary mCRP levels were valuable for predicting tubulointerstitial lesions (ROC-AUC > 0.700) in our study. More importantly, it was found that the levels of plasma mCRP were not associated with the prognosis of lupus nephritis, whereas urinary mCRP levels were closely associated with poor outcomes according to the univariate survival analysis. These findings supported that urinary mCRP levels could be a useful biomarker in predicting prognosis in lupus nephritis. Notably, our hospital is a tertiary referral center, and some patients were already in very critical condition at the time of admission; thus, the incidence of endpoint events could have been higher. Based on the above research, we raised the possibility that renal locally produced mCRP might be a major source of urinary mCRP in lupus nephritis. To verify our hypothesis, we detected CRP expression in renal biopsies from lupus nephritis patients using an immunohistochemistry assay. Nonspecific staining of renal tissues appeared under the conditions of using the anti-mCRP-3H12 antibodies (residues : CRP production and secretion by HK2 cells: (a) the levels of nCRP; (b) mCRP in the supernatant of HK2 cells treated with IgG from patients with lupus nephritis for 24-72 hr were significantly higher than those exposed to normal IgG; (c) the expression of CRP mRNA in HK2 cells stimulated with IgG from patients with lupus nephritis for 24-72 hr was significantly higher than that in HK2 cells exposed to normal IgG. The above experiments were repeated three times. nCRP, native C reactive protein; mCRP, modified C reactive protein; LN, lupus nephritis.

Mediators of Inflammation
199-206) [31], provided by the cooperative laboratory, or the commercial monoclonal anti-CRP antibody clone 8, so the primary anti-CRP antibodies from Abcam, which are supposed to recognize both nCRP and mCRP, were chosen. Our staining results showed that CRP was mainly detected in the tubule areas of lupus nephritis patients and patients with autoimmune-related tubulointerstitial nephritis, but was negative in the glomeruli. The mean optical density of CRP was significantly correlated with tubulointerstitial lesion indices, such as interstitial inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis in lupus nephritis. These results supported the strong expression of CRP in renal tissues during tubulointerstitial injury. A previous study showed that tubular CRP staining was increased with declining renal function and increasing severity of histological lesions in patients with advanced diabetic nephropathy [21]. In addition, our research showed that CRP staining in tubules was significantly correlated with urinary mCRP levels in patients with lupus nephritis. Thus, the renal expression of mCRP might be a major source of urinary mCRP in lupus nephritis. Moreover, double staining by immunofluorescence revealed that mCRP was indeed deposited in tubules, and the colocalization of mCRP and IgG in patients with lupus nephritis could be detected. Previous studies, including ours, indicated that the circulating level of autoantibodies against mCRP is prevalent in patients with lupus nephritis and has been associated with tubulointerstitial lesions [32,33]. A previous study reported that antidouble stranded DNA antibodies can bind to tubular epithelial cells, stimulate the secretion of the proinflammatory cytokines IL-6, interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) [34] and promote the recruitment of inflammatory cells and the development of interstitial inflammation. In addition, Kinloch et al. [35] found that vimentin was highly expressed by tubulointerstitial inflammatory cells and that antivimentin antibodies might be involved in the in situ adaptive immune mechanism of tubulointerstitial inflammation. Thus, we speculated that the local presence of mCRP antigen not only was a biomarker for tubulointerstitial injury, but also participated in the pathogenesis of lupus nephritis. Anti-mCRP autoantibodies and mCRP might form immune complexes in situ in the tubulointerstitium, resulting in tubule injury, interstitial inflammation, and fibrosis, but the exact role of mCRP in the pathogenesis of lupus nephritis must be further explored.
More importantly, under exposure to lupus nephritisderived IgG, increased nCRP and mCRP levels were detected in the supernatant of tubular epithelial cells. Previous studies have indicated that lupus nephritis was associated with disturbed apoptosis and impaired clearance, which might lead to the disruption of tolerance against autoantigens and the generation of autoantibodies [36,37]. Our results showed that these autoantibodies could also induce CRP antigen expression by tubular epithelial cells and might be involved in the progression of lupus nephritis. However, our study had some limitations. First, whether mCRP is dissociated from nCRP produced by renal tubular epithelial cells or directly synthesized in situ remains to be further studied. Second, the exact role of mCRP in the pathogenesis of tubulointerstitial injury in lupus nephritis requires further exploration.
In conclusion, our work showed that urinary levels of mCRP were significantly increased and that urinary mCRP might be a biomarker for tubulointerstitial lesions in patients with lupus nephritis. In addition, renal CRP could be produced by tubular epithelial cells, and the local presence of mCRP might play a critical role in the development of tubulointerstitial lesions.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declared that they have no conflicts of interest. Table S1: General data of patients with lupus nephritis. Figure S1: Enrollment of patients with lupus nephritis at Peking University First Hospital. HBV, hepatitis B virus. Figure  S2: Associations between plasma mCRP levels or urinary mCRP levels and tubulointerstitial injury features of lupus nephritis. (A1, B1) The association between plasma mCRP levels and urinary NGAL levels and KIM-1 levels in patients with lupus nephritis. (A2, B2) The associations between urinary mCRP levels and urinary NGAL levels and KIM-1 levels in patients with lupus nephritis. mCRP, modified C reactive protein, NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1. Figure S3: Immunohistochemistry staining of CRP in renal biopsies of patients with autoimmune-related tubulointerstitial nephritis. (a(A)) CRP staining was markedly positive in the tubules of patients with lupus-related tubulointerstitial nephritis (arrow). (a(B)) CRP staining was barely seen in the tubules of normal kidneys (×400). Scale bars 100 μm. (b) The mean optical density of CRP in patients with lupus-related tubulointerstitial nephritis and normal controls. CRP, C reactive protein; LN, lupus nephritis. (Supplementary Materials)