Aberrant expression of C1q, IL-6 and β-catenin in class VI lupus nephritis

doi:10.21203/rs.3.rs-3439470/v1

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Aberrant expression of C1q, IL-6 and β-catenin in class VI lupus nephritis

https://doi.org/10.21203/rs.3.rs-3439470/v1

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Background:

complement component C1q, interleukin-6 (IL-6) and β-catenin have been implicated in the pathogenesis of lupus nephritis (LN). However, their correlation with the pathological progression and type of LN remain unclear.

Methods:

the concentrations of C1q, IL-6 and β-catenin were evaluated in plasma, urine and kidney tissues in LN patients, non-LN systemic lupus erythematosus (SLEⁿ) patients, and healthy cohorts, as well as C57BL/6, IL-6^-/-, MRL-Fas/^lprand MRL-Fas/^lprIL-6^-/-mice.

Results:

more abundant plasma C1q, IL-6 and urine C1q proteins were determined in LN and SLEⁿ patients compared to healthy cohorts. Of note, the concentration of IL-6 and β-catenin in both plasma and urine, and plasma C1q was even higher in LN patients relative to SLEⁿ subjects. Moreover, positive correlations were determined for C1q and β-catenin proteins between plasma and urine in LN patients. Of importance, both plasma and urine β-catenin, and urine IL-6 were significantly increased in patients with class VI LN patients relative to those who suffered from class I LN. Immunohistochemical study further uncovered that the abundant IL-6 and β-catenin proteins were deposited in both renal glomeruli and tubules, while the C1q was only found in renal glomeruli of patients with class IV LN. Consistent with the clinical findings, experimental studies in MRL-Fas/^lprIL-6^-/- mice also showed a decreased β-catenin in urine, C1q and β-catenin in kidney tissues of MRL-Fas/^lprIL-6^-/- mice compared with MRL-Fas/^lpr mice. Interestingly, mice with deficiency of IL-6 exhibited less degrees of proteinuria and histological lesions, and reduced serum anti-double-stranded DNA (anti-dsDNA) antibody and sizes of spleen and inguinal node, as compared with MRL-Fas/^lpr and C57/BL6 control mice.

Conclusions:

these data suggest a strong correlation among IL-6, C1q and β-catenin in the pathogenesis of type VI LN in SLE patients, indicating that they may be valuable biomarkers for nephrologists to guide treatment and predict prognosis among these patients.

C1q

IL-6

β-catenin

class VI lupus nephritis

Systemic lupus erythematosus (SLE) is characterized by inflammation affecting multiple organs along with the presence of pathogenic autoantibodies ^[1]. Renal disease is one of the most common and severe organ manifestations of SLE ^[2]. It has been reported that lupus nephritis (LN) is a major risk factor for overall morbidity and mortality in SLE patients, with approximately 10% of individuals with LN progressing to end-stage renal disease (ESRD) even after treatment ^[3]. This is partly due to the absence of effective biomarkers for identifying pathological types of LN in SLE patients.

Currently available non-invasive clinical tests are unable to distinguish histologic subtypes of LN, and reliably differentiate active disease from chronic damage either. Therefore, despite procedure-associated risks, biopsy remains the gold standard for diagnosis and evaluation of treatment outcome. Given the disconnect between non-invasive clinical tests and kidney histology, repeat biopsies are sometimes needed to evaluate kidney parenchymal changes caused by therapy, distinguish residual inflammatory activity from irreversible chronic injury, and characterize disease relapse after renal remission ^[4]. In recent years, increasing molecular data are incorporated into clinical diagnostics in nephrology ^{[5, 6]}. Among these molecules, complement C1q was present in 20–50% of unselected SLE patients and their occurrence strongly correlated with both low complement levels and severe LN ^[7–9]. Our previous results also revealed that complement C1q was a reliable serological marker for identification of SLE patients with active LN and active SLE disease ^{[10, 11]}. Consistent with above findings in SLE mouse model, Thanei et al. reported that complement C1q activated the complement system and induced a proinflammatory cytokine response in macrophages by cross-linking of Fcgamma receptor (FcγR) [8]. Indeed, beyond the realm of autoantibodies and immune complex deposition, a compelling body of evidence underscores the pivotal role of cytokines in the intricate tapestry of SLE pathogenesis ^[12].

Among these cytokines, the interleukin-6 (IL-6)/IL-6 receptor (IL-6R) signaling has garnered significant attention due to its robust link with LN activity in individuals with SLE ^{[13, 14]}. In the context of LN, the primary source of IL-6 are the infiltrating inflammatory cells within kidneys, predominantly macrophages and monocytes ^[15]. Blocking IL-6 with anti-IL-6 antibodies was able to reduce kidney pathology in MRL-Fas/^lpr and NZB/NZW mice ^[16], whereas mice with IL-6 overexpression developed mesangial proliferative glomerulonephritis ^[17]. Intriguingly, a study of IL-6-deficient BALB/C mice in pristance-induced lupus, an alternative murine lupus model, revealed that autoantibody development in this lupus model was IL-6-dependent ^[18]. These results were in accordance with the finding of high IL-6 concentrations in sera of SLE patients and other membranoproliferative nephritides, whereas healthy kidneys showed low IL-6 expression ^[15]. In fact, several lines of study have recently suggested that IL-6 correlated with disease activity and might even be a useful biomarker of SLE ^[19].

In addition to the C1q and IL-6, mounting evidence has recently suggested that Wnt/β-catenin signaling was involved in the pathogenesis of SLE ^[20–23]. In this regard, the Wnt/β-catenin pathway induces the stability of β-catenin and transfers into the nucleus upon the activation, ultimately facilitating the expression of genes involved in cell proliferation, survival, differentiation and migration ^[24]. In SLE patients, aberrant Wnt/β-catenin signaling activity was observed in both peripheral B and T cell fractions ^[25]. Both human and LN MRL-Fas/^lpr mouse studies indicated that the Wnt/β-catenin inhibitor Dickkof-1(DKK-1) protein was significantly elevated in the sera of SLE patients compared with control subjects, and the LN MRL-Fas/^lpr mice exhibited a phenotype with an enhanced Wnt/β-catenin activity, accompanied by an increased level of Dkk-1 in the renal tissues and sera, and an increased frequency of apoptotic cells of the renal tubular and renal interstitial tissues^[26]. Such an activated Wnt/β-catenin signaling was further evidenced in human renal tissues of patients with LN by accessing β-catenin at both transcriptional and translational levels using assays including immunohistochemistry staining, qRT-PCR, and western blotting, suggesting that a dysregulated Wnt/β-catenin signaling was involved in the pathogenesis of LN and might play a role in the renal fibrosis ^[27].

Considering the roles of C1q, IL-6 and β-catenin in LN, we sought to investigate which pathological type of LN has the strongest correlation with these biomarkers.

2.1. Ethics Statement. Human blood, urine and kidney samples were collected with a protocol approved by the Ethic Committee for the Conduct of Human Research at General Hospital of Ningxia Medical University (NXMU-GH-2019-487). Written consent was obtained from all individuals for collecting blood, urine and kidney samples and publishing the data according to the Ethic Committee for the Conduct of Human Research protocol. All participants were older than age of 21 years old. The PI of this study maintains human research records, including signed and dated consent documents, for ten (10) years after their acquisition. The Ethic Committee the Conduct of Human Research at Ningxia Medical University approved the consent procedure for this study (NXMU-GH-2019-487).

2.2. Human subjects. Blood samples of 107 consecutive SLE patients, urine samples of 64 consecutive SLE patients were collected from the outpatient rheumatology clinics of General Hospital of Ningxia Medical University from July to December in year 2021. The sections of kidney biopsy of 26 LN patients were achieved tissues in the department of Pathology of General Hospital of Ningxia Medical University July to December in year 2021. The American College of Rheumatology (ACR) criteria were used to diagnose patients with SLE ^{[28, 29]}. All the SLE patients included in this study received standard-of-care pharmacological treatment that did not include biological agents. Plasmas of 69 and urines of 24 gender and age-matched healthy individuals were also collected. These healthy control cohorts were recruited from those who had undergone comprehensive medical screening at outpatient of the General Hospital of Ningxia Medical University and who had no history of chronic diseases, and no family history of autoimmune diseases. The demographics of individuals involved in this study were outlined in Suppl. Tables S1.

2.3. Detection of IL-6, C1q and β-catenin in plasma and urine by enzyme-linked immunosorbent assays (ELISA). The concentrations of plasma and urine IL-6, C1q and β-catenin were measured using commercially available ELISA kits per manufacturer’s instruction. The ELISA kits for IL-6, C1q and β-catenin were products of YOBIBIO lnc. (Shanghai, China). For detection of IL-6, C1q and β-catenin, the plasma and urine were directly detected with stock suspension, and their concentrations were presented as ng/mL through standard curves.

2.4. Generation of IL-6-deficient MRL-Fas^lprmice. MRL-Fas^lprmice, IL-6 knockout BALB/C mice and healthy C57BL/6 mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were bred in a germ-free environment in the animal facility of the University of Ningxia Medical University. IL-6-deficient MRL-Fas^lpr mice (MRL-Fas^lprIL-6^-/-) were generated using a backcross-intercross scheme as described. The genotype of mice was identified by polymerase chain reaction (PCR) amplification of tail genomic DNA using primers for the wild-type gene (sense, 5’-TTC CAT CCA GTT GCC TTC TTG G-3’; antisense, 5’-TTC TCA TTT CCA CGA CGA TTT CCC-3’), IL-6 deficiency gene (sense, 5’-ATT GAA CAA GAT TTG GGA TTG CAC-3’; antisense, 5’-CGT CCA GAT CAT CCT GAT C-3’) and MRL-Fas^lpr gene (sense, 5’-ATT GAA CAA GAT TTG GGA TTG CAC-3’; antisense, 5’-CGT CCA GAT CAT CCT GAT C-3’). Gel analysis identified the IL-6 gene fragments at 480bp and MRL-Fas^lprgene fragments at 350bp, respectively.

2.5. Proteinuria, anti-dsDNA, spleen and lymph nodes. The mice were tested for proteinuria using a urine protein test kit (cat. no. C035-2-1; Nanjing Jiancheng Bioengineering Institute, China). Serum anti-dsDNA was measured using the commercial ELISA kit (cat. no. ml063514; mlbio Inc., Shanghai, China). The location and size of subcutaneous lymph nodes in mice were visually observed, and the evaluation criteria were as follows: 0=no lymph nodes; 1=small lymph nodes, located on one side of the groin; 2=medium-sized lymph nodes, located on both sides of the groin; 3=large lymph nodes, located on both sides of the groin.

2.6. Histopathology, Immunohistochemical (IHC) staining and Immunofluorescent (IF) staining. In order to histologically examine the pathology of the kidneys of IL-6-deficient MRL-Fas^lpr mice, the kidney tissue of mice was fixed in 10% neutral formalin and processed paraffin embedding and section (4µm) for pathohistological analysis by hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson trichrome staining. Glomerular pathology was assessed by scoring each glomerulus on a semiquantitative scale on glomerular cross-section (gcs) ^[2]: 0=normal (35-40 cells/gcs); 1=mild; glomeruli with few lesions showing slight proliferative changes, mild hypercellularity (41-50 cells/gcs); 2=moderate; glomeruli with moderate hypercellularity (51-60 cells/gcs), including segmental and/or diffuse proliferative changes, hyalinosis; and 3=severe; glomeruli with segmental or global sclerosis and/or severe hypercellularity (>60 cells/gcs), necrosis, and crescent formation. 20 glomerular cross-sections were scored for each kidney. Interstitial/tubular pathology was assessed semiquantitatively on a scale of 0-3 in 10 randomly selected high magnification fields. We determined the largest and average number of infiltrates and damaged tubules and adjusted the grading system accordingly: 0=normal; 1=mild; 2=moderate; 3=severe. Perivascular cell accumulation was determined semi-quantitatively by scoring the number of cell layers surrounding vessel walls (0=none; 1=fewer than a cell layers; 2=5-10 layers; 3=more than 10 cell layers). All scoring was performed on blinded slides. In addition, a part of kidney tissue was fixed in 4% paraformaldehyde (PFA) in PBS for 2 days prior to being embedded in an Optimal Cutting Temperature compound (OCT) for immunofluorescence (IF) staining on tissue sections of 4µm thickness. Cryosections were air-dried at room temperature (RT) for 30 min, re-fixed in 4% PFA for 10 min, and finally permeabilized in 0.2% Triton X-100/PBS for 20 min at RT. Sections were then blocked by incubating them in 5% donkey serum in PBS for 1h before being probed with primary antibodies in dilutant buffer (1% donkey serum, 0.03% Triton X-100, and 1mM CaCl₂ in PBS) at 4℃ overnight. The sections were washed and then incubated with fluorescent dye-conjugated secondary antibodies at RT for 2h. The slides were washed in PBS three times for 5 min, mounted with VECTASHIELD Antifaade Mounting Medium with DAPI (Vector Laboratories), imaged under a Leica TCS SP2 A0BS Confocal System, and processed on Leica Confocal Software v.2.6.1 (Leica). For IHC staining, paraffin sections were deparaffinized and rehydrated. After antigen retrieval, slides were blocked with 5% BSA/TBS at RT for 1h; samples were incubated with primary antibodies at 4℃ overnight. Sections were then washed three times with PBS-tween 20 (0.1%) and then incubated with biotin-or HRP-conjugated secondary antibodies for 30min at RT. Antibody staining was then developed with DAB substrate (Zsbio, Beijing, China) for HRP-conjugated secondary antibodies or incubated with Avidin-Biotin Complex Staining Kit (ABC Kit; Vector Laboratories, Burlingame, CA) before DAB development as per the manufacturer’s instructions. Slides were then counterstained with hematoxylin, dehydrated, mounted, and finally visualized under an upright microscope.

2.7. Western Blotting Analysis. The total protein of the mouse kidney tissues was isolated by homogenizing the tissue in Enhanced RIPA Lysis Buffer (Leagene Biotech Ltd., Beijing, China), followed by centrifugation. The homogenized kidney tissue was then centrifuged at 12,000xrpm for 20min at 4℃; the supernatants were collected as total proteins. The concentration of protein was detected using BCA Protein Assay Kit (Kaiji Biotech Ltd., Beijing, China). The proteins (30µg) were resolved by 8%-9% sodium dodecyl sulfate- (SDS-) polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was then blocked in 5% nonfat milk in TBS for 1 hour at room temperature (RT). The protein of interest was probed with its specific antibodies, and the blots were then developed using the enhanced chemiluminescence (ECL) reagent (Amersham Biosciences, Piscataway, NJ, USA) as described elsewhere. The levels of protein expression were semiquantified by optical densitometry using ImageJ Software version 2.0.0 (http://rsb.info.nih.gov/ij/). The ratio between the net intensity of each sample divided by the GAPDH internal control was calculated as densitometric arbitrary (A.U.), which served as an index of the relative expression of the protein of interest.

2.8. Statistical Analysis. All laboratory data were entered into and extracted from PRISM (version 8.0) (GraphPad Software, La Jolla, CA, USA) and/or EXCEL (version 2016) (Microsoft Software, Redmond, WA, USA) and/or SPSS for Windows (version 26.0) (SPSS Inc., Chicago, IL, USA) and/or R, version 4.1.0 (http://www.r-project.org/). One-way ANOVA or Kruskal-Wallis test was employed for comparisons of more than two groups, and the t-test was conducted for comparison between two groups. The association between qualitative variables was evaluated by Spearman correlation. Data was presented as the mean standard error of mean (SEM) or mean±standard deviation (SD). A value of less than 0.05 was considered statistically significant.^{*, #, &}P<0.05; ^{**, ##, &&}P<0.01; and ^{***, ###, &&&}P<0.001.

3.1. Concentrations of C1q, IL-6, and β-catenin proteins in plasma and urine of LN patients.

In order to determine whether IL-6, C1q and β-catenin were correlated with LN activity, plasma and urine concentrations of IL-6, C1q and β-catenin were evaluated in non-LN SLE (SLEⁿ), LN and healthy cohorts (HC). More abundant plasma C1q and IL-6 proteins were determined in SLEⁿpatients compared to HC (P<0.001 and P<0.001, respectively) (Figures 1A and 1B). Moreover, moderately higher levels of plasma C1q, IL-6 and β-catenin proteins were detected in LN patients relative to HC (P<0.001, P<0.001, and P<0.001) (Figures 1 A, 1B, and 1C). Importantly, strikingly higher levels of plasma C1q, IL-6 and β-catenin proteins were found in LN patients compared to SLEⁿ patients (P<0.001, P<0.001, and P<0.001, respectively) (Figures 1 A, 1B, and 1C). Similar to seen in plasmas, significantly higher levels of urine C1q, IL-6 and β-catenin proteins were also observed in LN patients relative to HC (P<0.001, P<0.001, and P<0.001, respectively) (Figures 1D, 1E, and 1F). Moreover, a moderately more abundant urine C1q protein was detected in urine of SLEⁿpatients relative to HC (P=0.019) (Figure 1D), while significantly more abundant urine IL-6 and β-catenin proteins were determined in LN patients compared to SLEⁿ patients (P=0.029 and P<0.001, respectively) (Figures 1E and 1F). These results implied that the aberrant expression of C1q, IL-6 and β-catenin proteins might contribute to LN onset and progression.

3.2. Correlations of C1q, IL-6, and β-catenin concentrations in plasma and those in urine of LN patients

Above data showed IL-6 and β-catenin were more abundant in both plasma and urine of LN patients compared with those of SLEⁿpatients, and a significantly higher level of urine C1q was also detected in LN patients relative to SLEⁿ patients, the correlations of IL-6, C1q and β-catenin proteins in plasma and urine were thus analyzed to predict their potential interactions in the LN pathogenesis. Positive correlations were determined for C1q and β-catenin proteins between plasma and urine in LN patients (R=0.410, P=0.038, R=0.956, and P<0.001, respectively) (Figures 2B and 2C). In addition, there was a positive correlation between urine C1q and IL-6 proteins (R=0.408 and P=0.038, respectively) (Figure 2G). Urine β-catenin protein showed statistically significant associations with urine C1q and IL-6 proteins, respectively (R=0.619, P<0.001, R=0.754, and P<0.001) (Figures 2H and 2I). Unexpectedly, there was no association between plasma and urine in LN patients determined for IL-6 (R=-0.245 and P=0.226, respectively) (Figure 2A). Meanwhile, plasma C1q protein showed no statistically significant association with plasma IL-6 protein (Figure 2D). Plasma β-catenin protein also showed no statistically significant association with plasma IL-6 and C1q proteins, respectively (Figures 2E and 2F). These results suggested that the correlations of C1q, IL-6, and β-catenin concentrations in plasma and urine were associated with each other.

3.3 An elevated C1q, IL-6, and β-catenin proteins in kidneys of LN patients with class IV

According to ISN/RPS 2018 classifation ^[30], LN patients were further divided into 6 different classes (types) based on histopathological features: class I (minimal mesangial LN) was present in 5 patients (19.2%), class II (mesangial proliferative LN) was present in 5 patients (19.2%), class III (focal proliferative LN) was present in 5 patients (19.2%), class IV (diffuse proliferative LN) was present in 5 patients (19.2%), class V (membranous LN) was present in 5 patients (19.2%), and class VI (advanced sclerotic LN) was present in 1 patient (3.8%) (Figures 3A, 3B and 3C). The levels of IL-6, C1q and β-catenin proteins in plasma, urine and kidney tissues were further analyzed of these 6 classes. Significant differences in urinary IL-6 were found in LN patients with class IV and class VI relative to those with class I and class II, respectively (P=0.0002, P=0.003, P=0.0002, and P=0.003) (Figure 3B). Additionally, significantly higher levels of β-catenin in plasma and urine were determined in class IV LN patients compared to class I LN patients (P=0.018) (Figure 3C). A moderately more abundant urine β-catenin protein was detected in class VI LN patients relative to class I LN patients (P=0.017) (Figure 3C), while no significant difference of plasma and urine C1q were found in different pathological classification of LN patients (Figure 3A). Notably, triple immunofluorescent staining demonstrated IL-6 (Alexa Green) was only found to be present in the renal tubules of class I LN patients, while IL-6 was localized in the renal tubules and glomeruli of class II, class III, class IV, class V and class VI LN patients (Figure 3D). The expression of C1q (Alexa Pink) was observed in renal glomeruli of class III, class IV, class V and class VI LN patients, whereas there was no expression of C1q in in renal glomeruli of class I and class II LN patients. In addition, a large amount of grainy C1q was localized in renal tubules in class III, class IV and class V LN patients (Figure 3D). However, β-catenin (Alexa Red) was not detected in the kidney of patients with class V LN, which was almost exclusively expressed in renal tubules of class I LN patients. Interestingly, β-catenin deposition was found in renal tubules and glomeruli were found of class II, class III, class IV and class VI LN patients (Figure 3D). These data suggest that IL-6, C1q and β-catenin have the strongest correlation with pathological type VI of LN in SLE patients.

3.4 IL-6 deficiency delayed the development of LN phenotype in MRL-Fas^lpr mice.

In order to further determine the potential roles of IL-6, C1q and β-catenin in LN, we then generated IL-6-deficient MRL-Fas^lpr (MRL-Fas^lpr IL-6^-/- ) mice and monitored them for a period of 6 months. The concentrations of proteinuria and anti-dsDNA, as well as the size of spleen and inguinal lymph node were measured. In addition, the mortality rate and histopathological changes in kidneys were also analyzed. Marked reduction of proteinuria and anti-dsDNA were found in MRL-Fas^lprIL-6^-/-mice compared with MRL-Fasl^pr mice at the end of experiment of 24 weeks of the study (Figures 4A and 4B). Similar to the characterizations of proteinuria and anti-dsDNA, the degree of increased size (weight) of spleen and the score of inguinal lymph node in MRL-Fas^lprIL-6^-/- mice were obviously alleviated in comparison with MRL-Fas^lpr mice (Figures 4C, 4D, 4E, and 4F). In addition, the mortality rate of MRL-Fas^lprIL-6^-/- mice was 50% during the observation period of 6 months, whereas only 18% of the female MRL-Fas^lpr mice survived to the end of study (Figure 4G). More importantly, there was a reduction in glomerular hypercellularity in MRL-Fas^lprIL-6^-/-mice compared with MRL-Fas^lpr mice (Figures 4H and 4I). Intriguingly, the histological analysis with MASSON staining did not show any obvious abnormality of interstitium in all groups (Figures 4H and 4I). Above results suggested that the deficiency of IL-6 in the MRL-Fas^lprmouse model diminished LN activity, leading to a delayed development of LN in MRL-Fas^lpr SLE mice.

3.5 A declined C1q and β-catenin in kidneys of IL-6-deficient MRL-Fas^lprmice.

To investigate whether C1q and β-catenin were altered in MRL-Fas^lpr IL-6^-/- mice, their abundance in plasma and urine were first evaluated by ELISA. A lower level of urine β-catenin was found in MRL-Fas^lprIL-6^-/- mice compared with MRL-Fas^lpr mice (Figure 5B). Interestingly, there were no significant differences of plasma C1q and β-catenin, and urine C1q between MRL-Fas^lpr mice and IL-6 deficient MRL-Fas^lprmice (Figures 5A and 5B). By immunoblotting assay, less abundant C1q and active-β-catenin (ABC) were observed in MRL-Fas^lprIL-6^-/- mice relative to MRL-Fas^lprmice (Figures 5C and 5D). IHC staining showed a less abundant Wnt3a protein in MRL-Fas^lprIL-6^-/- mice compared with MRL-Fas^lpr mice (Figures 5E and 5F). ABC and C1q double immunofluorescent staining further demonstrated that ABC (Alexa Green) was present in the glomerular and interstitium, whereas C1q (Alexa Red) only was localized in the glomerular. Of note, ABC and C1q proteins were dominantly overlapped in the kidney of in MRL-Fas^lprmice as determined by a clear yellowish fluorescent staining from the overlap of red and green, while such an overlapped yellow area was significantly diminished in MRL-Fas^lprIL-6^-/- mice (Figure 5E). These findings demonstrated extensive co-localization of ABC and C1q, and strongly suggested that the C1q likely interacted with ABC to regulate IL-6 production in LN.

Despite improvements in diagnosis and treatment in SLE patients with LN have been made, LN is still a serious risk factor for the development of ESRD and early mortality and disability in SLE ^[31]. Recent proteomic measures, including anti-C1q antibodies, cytokines, toll-like receptors (TLRs), vascular cellular adhesion molecule-1 (VCAM-1), neutrophil gelatinase-associated lipocalin (NGAL), angiostatin, have been considered helpful to provide a more profound comprehension of SLE pathogenic process ^[32]. Concerning cytokines, baseline serum and urine IL-6 can be used as a sensitive biomarker for SLE activity, as well as predictors of remission of LN ^[33]. This notion was in accordance with the finding of plasma and urine IL-6 were more abundant in LN patients compared to SLEⁿ patients in the current study. Unexpectedly, there was no association between plasma and urine in LN patients determined for IL-6 in our study. Indeed, no correlation between urine and serum IL-6 levels was observed in a previous study ^[34].

With regard to C1q, several studies indicated that the evaluation of serum and urine C1q could be a useful diagnostic and predictive biomarker for SLE patients with kidney involvement ^{[11, 35, 36]}. Consistently, a strikingly higher level of plasma C1q was found in LN patients compared to SLEⁿ cohorts in the current report. Intriguingly, in contrast to high level of C1q seen in plasma, no significant difference of abundance of urine C1q was found between LN patients and SLEⁿ subjects. Interestingly, plasma C1q was positively associated with urine C1q. We reasoned that it might be due in part to the different study populations or sample sizes.

Several lines of evidence have demonstrated that aberrant canonical Wnt/β-catenin signaling is involved in SLE. Wang et al. recently reported β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis at the mRNA expression level, and the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury^[27]. Consistently, significantly higher levels of β-catenin in both plasma and urine of LN patients relative to SLEⁿsubjects. Additionally, plasma β-catenin was positively associated with urine β-catenin. These results suggested that aberrant expression of C1q, IL-6 and β-catenin proteins in both plasma and urine contribute to LN onset and progression.

In this report, in vivo studies demonstrated that IL-6-deficiency in MRL-Fas^lpr mice diminished lupus activity, including marked reduction of levels of proteinuria and anti-dsDNA, the weight of spleen, the score of inguinal lymph node and the mortality rate. Histologically, it resulted in a reduction of glomerular hypercellularity. The ameliorated histological phenotypes of LN in MRL-Fas^lpr mice was consistent with findings in other mouse models of lupus ^{[16, 37, 38]}. Previous studies have demonstrated that the absence of IL-6 resulted in significant reduction of infiltrating macrophages in the kidney, a decrease in renal IgG and C3 deposition, and a reduction of CD4⁺ and CD8⁺ lymphocytes [2]. Intriguingly, in our study, the histological analysis of interstitial/tubular did not show any obvious abnormality in MRL-Fas^lpr group, and this result was different from those published in the study by Cash and colleagues [2]. The difference might be caused by different genetic background, time, diet and environment during the generation of mouse model. In addition, a study of IL-6-deficient BALB/C mice in pristance-induced lupus, an alternative murine lupus model, revealed that autoantibody development in this lupus model is IL-6-dependent [13]. It is true that IL-6-deficiency in MRL-Fas^lpr mice reduced the level of C1q in both urine and kidney compared with MRL-Fas^lpr mice in this study. Additionally, less abundant active β-catenin protein in kidneys was observed in MRL-Fas^lprIL-6^-/- compared to MRL-Fas^lprmice. In this report, an increased Wnt/β-catenin signaling activity was determined in kidneys of (NZB x NZW) mice during progression of LN, which was paralleled by an increased renal and serum levels of Dkk-1 ^[20]. Such an elevated renal Dkk-1 level was also found in MRL-Fas^lprmice relative to C57BL/6 mice by immunoblotting in our study. IF staining results further showed lower levels of C1q and active β-catenin co-localize in kidneys of MRL-Fas^lprIL-6^-/- mice compared with MRL-Fas^lpr mice. These findings further suggest that the interaction between IL-6, C1q and β-catenin plays a critical role in the development of LN.

The histological phenotypes of LN have long been used to guide disease management, but the molecular pathophysiology of six distinct clinico-pathological classes is still poorly understood ^{[4, 34, 39]}. Of the six classes, classes III and IV are especially important because of the high disease activity and poor renal prognosis in LN patients ^[40]. Previous clinical study found markedly elevated levels of cytokines in LN patients with class IV as compared with the controls, including Th2 cytokines interferon-gamma (INF-γ), IL-2 and TNF-α, and Th2 cytokines IL-4, IL-6, IL-10 and IL-13. Only 4 out of 9 cytokines significantly increased in class V patients ^[41]. In an exploratory analysis of 29 patients with active LN, urine IL-6 level was markedly increased in patients with class IV LN relative to those with class II and class V LN. Conversely, serum IL-6 level did not vary with LN class ^[42]. Indeed, we also found that there was no statistical difference in plasma IL-6 level with different LN class, and a significantly higher level of urine IL-6 was determined in class IV LN patients compared to class I and II LN patients. More importantly, the expression of IL-6 was deposited in renal glomeruli and tubules of class IV LN patients. Moreover, a cross-sectional study showed serum anti-C1q antibody level was the highest in LN patients with class IV, and anti-C1q was positively correlated with renal activity indices (AI), including endocapillary hypercellularity, glomerular leukocyte infiltration, karyorrhexis/fibrinoid necrosis, while inversely correlated with chronicity indices (CI) such as glomerular sclerosis and interstitial fibrosis ^[43]. Interestingly, levels of C1q in both plasma and urine had no statistically significant differences in six classes of LN, whereas the expression of C1q was deposited in renal glomeruli and large amounts of grainy C1q was localized in renal tubules of class IV LN patients in our study.

Moreover, an increased expression of β-catenin in the renal tissues of patients with LN compared with control samples, and more abundant β-catenin and AXIN-2 transcripts were also detected in the LN renal tissues relative to controls. Of note, such an increased abundance of β-catenin transcript was positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury ^[27]. In this report, we found significantly higher levels of β-catenin in plasma and urine were determined in class IV LN patients compared to class I LN patients. Even more, active β-catenin deposits in renal tubules and glomeruli were found in class IV LN patients. These findings imply that IL-6, C1q and β-catenin have the strongest correlation with pathological type VI of LN in SLE patients.

Despite the IL-6, C1q and β-catenin have shown the strongest correlation with pathological type VI of LN in SLE patients. There are several limitations to this proof-of-concept study. First, the relatively small number of LN patients was included in this study, which limited our power to detect statistical differences in various clinical manifestations. Second, the outcome data of all enrollments lack follow-up such as a lack of IL-6, C1q and β-catenin proteins testing after receiving standard-of-care therapy. Third, an enhanced and/or reduced activity of Wnt/β-catenin pathway in MRL-Fas/^lpr mice to further uncover the pathogenic role and mechanism of interaction between C1q, β-catenin and IL-6 in the development of LN. Therefore, this finding deserves confirmation in a larger aggregate sample size in the future.

Collectively, despite these potential shortcomings, we clearly demonstrate that a strong correlation among IL-6, C1q and β-catenin in the pathogenesis of type VI LN in SLE patients, indicating that they may be valuable biomarkers for nephrologists to guide treatment and predict prognosis among these patients.

ACR: American College of Rheumatology; AI: activity indices; ANA: antinuclear antibodies; Ccr: creatinine clearance rate; CI: chronicity indices; DKK1: dickkopf-related protein 1; dsDNA: double-strand DNA; ELISA: enzyme-linked immunosorbent assays; ESRD: end-stage renal disease; FcγR: Fcgamma receptor; HC：healthy cohorts；INF-γ: interferon-gamma; IL-6: Interleukin-6; LN: lupus nephritis; NGAL: neutrophil gelatinase-associated lipocalin; PCR: polymerase chain reaction; SEM: standard error of mean; SLE: systemic lupus erythematosus; SD: standard deviation; TLRs: toll-like receptors; TNF-α: tumour necrosis factor alpha.

Ethics approval and consent to participate

All procedures were approved by the Ethic Committee for the Conduct of Human Research at General Hospital of Ningxia Medical University (NXMU-GH-2019-487). Informed consent was obtained from all patients for their data to be used for research.

Consent for publication

All authors approved the final manuscript and the submission to this journal.

Availability of data and material

All data generated or analyzed during this study are included in the published article.

Competing Interests

The authors declare that they have no competing interests. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript.

Funding

This study was supported by grants from the Key Research and Development Program of Ningxia Hui Autonomous Region (Nos. 2023BEG02021; 2023BEG03014), a grant from the Natural Science Foundation of Ningxia (2021AAC03369).

Acknowledgements

Not applicablle.

Authors' information

Authors and Affiliations

Department of Key Laboratory of Ningxia Stem Cell and Regenerative Medicine, Institute of Medical Sciences, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China

Jing Xue, Yuanyuan Jia & Juan Chen

Department of Nephrology General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China

Yu Min

Ningxia Medical University, Yinchuan, Ningxia, China

Zeqin Zhu

Department of Rheumatology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China

Shuhong Chi

Department of Pulmonary and Critical Care Medicine, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China

Juan Chen

Contributions

J. X., and M. Y., collected clinical data, Z. Z. performed the serological analysis. Y. J. collected plasma samples. J. X. analyzed data and drafted the manuscript. J. C., the designed experiments and revised the manuscript. S. C.analyzed data and interpreted data.

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Aberrant expression of C1q, IL-6 and β-catenin in class VI lupus nephritis

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