Ectopic Germinal Cener In Primary Sjogren Syndrome With Renal Tubulointerstitial Injury: A Preliminary Study

BackgroundPrimary Sjogren syndrome (pSS) is a systemic autoimmune disease involving multiple organs, characterized by autoimmune epithelitis. Renal involvement is predominated by tubulointerstitial injury that manifested as interstitial nephritis and tubular acidosis. However, clinicopathologic studies of large sample are sparse and mechanisms underlying renal damage remain to be elucidated. Ectopic germinal center (EGC) is located in lymphoid-like structures that formed in inflamed target organs of autoimmune disease. It is the site in which B cells proliferate and diffrentiate to produce antigen-spicific antibodies. It has been proved that EGC formation in labial salivary gland is correlated with pSS pathogenesis, clinicalpathologic feature and prognosis, but evidences in other target organs including kidney are lacking. In the process of labial gland EGC initiating and maintainance, recruitment of plasmacytoid dendritic cell (pDC) and myeloid dendritic cell (mDC), chemotaxis and activation of B cells by CXCR5 and interleukein-21 (IL-21) are key events. Interleukine-17 (IL-17) contributes to both EGC inflammation and labial epithelial cell injury, and is hypothesized as the linkage. But these details are still not clear in renal tissue. Megalin and cubilin are multi-ligand protein reabsorption receptors on the brush border of proximal renal tubule epithelical cells. Their decreased expression is known to be the reson of heritary Fanconi syndrome, but their role in pSS related Fanconi, and the connection with EGC is unclear.Purpose1. To retrospectively study clinicopathological characteristics, treatment and prognosis of pSS with renal tubulointerstitial injury (pSS-TIN), and identify parameters that correlated with clinial features and outcome;2. To evaluate lymphocyte infiltration and EGC formation in renal interstium, to compare the frequency of pDC and mDC in paripheral blood and renal tissue, to observe and semi-quantify the expression of CXCR5 and IL-21, assessing their association with clinical features;3. To observe EGC formation in pSS with Fanconi syndrome (pSS-Fanconi), and evaluate the association between IL-17A expression and the alteration of Megalin/Cubilin;MethodThe clinical record and follow-up data of 64 patients with pSS-TIN comfirmed by renal biopsy in Peking Union Medical College Hospital from January 1993 to June 2015 were reviewed. Primary end-point is defined as end stage renal disease or death, and secondary end-point is defined as complete or partial effectively treated. Pathological features including glomerular sclerosis, tubular atrophy, interstitial fibrosis and lymphocytes infiltration were evaluated semiquantatively. Immunohistochemistry staining of CD21, BDCA-2, DC-SIGN, CXCR-5, IL-21, IL-17, Megalin and Cubilin, and immunofluorence staining of Megalin were performed to evaluate their expression pattern and prevalence. Renal tubule cell abnormalities were assessed by electron microscopy.Flow cytometry was performed to evaluate the ratio of pDC (Lin-1"HLADR+CD123+) and mDC (Lin-1-HLADR+CD11c+) in paripheral blood, in the pSS with renal involvement (pSS-Renal), pSS without renal involvement (pSS-only) and health control group respectively.Continuous variables are displayed as mean ± standard deviation and compared using student’s t-test, one-way analysis of variance or Pearson’s correlation coefficients. Categorical variables are expressed as percentage and compared with chi-squatre test. Kaplan-<eier analysis and Cox proportional hazard model were also used. A P-value of <0.05 was considered significant. Statistical analysis was performed with the SPSS software (version 17.0).Results1. The clinical and pathological characteristics of pSS-TIN patientsThe enrolled 64 pSS-TIN patients were mostly female (M/F:1/9) and middle-aged (41.1±12.7). They often present sicca syndrome, polyurea and paralysis at the onset. Comparing with patients of pSS with membranous nephropathy (pSS-MN), the systemic manifestation of lymphadenopathy, pulmonary involvement, arthralgia, leukocytopenia and thrombocytopenia are less prevalent, but anemia, IgG elevation, acidosis and hypokalemia take a higher percentage. The eGFR averaged 56.3±29.5ml/min/1.73m2, which negatively correlated with age and ESR. Serum HCO3- is negalively correlated with anti-Ro/SSA. Pathologically they are mainly characterised by tubule atrophy (score 2.12±0.87) and interstitial fibrosis (score 2.13±0.93), with lymphocted infiltration of different degree. Multi-regression revealed age, glomerular sclerosis index and tubulointerstitial leision were independently correlated with eGFR. Patients generally received corticosteroid (93.8%) and some with immunosuppressant (45.3%). Median follow-up time was 38.0 (15.0,78.5) monthes. eGFR recovered to 72.3±26.7ml/min/1.73m2 in 1 year and remained stable. Renal function of 59.4% were completely improved and 18.8% were partialy improved. eGFR at renal biopsy is the independent risk fator of long term prognosis.2. Evaluation of EGC formation and preliminarily investigation of mechanismDense lymphocyte infiltration focuses were present in 65.6% pSS-TIN patients. Comparing with patients with scattered lymphocytes infilgration, the ratio of destal renal tubule acidosis was higher (75.7% vs.41.2%, p=0.013) and serum HCO3- was lower (17.3±4.9 vs.20.6±4.4mmol/L, p=0.023).15.6% pSS-TIN had EGC marked by CD21, which was correlated positively with eGFR. There were BDCA2+pDC and DC-SIGN+ mDC infiltration near EGC. The percentage of pDC and mDC in peripheral blood of pSS-TIN were lower than health control group, while mDC was also lower than pSS-only group. In the presence of EGC, the expression of pDC and CXCR5+ cells were all increased, and CXCR5+ cells were mainly detected near the adjacent vessels. pSS-TIN also showed an elevated level of IL-21 than TIN of other etiology.3. The preliminary investigation of relation between EGC and proximal tubule injuryUnder electron microscopy, pSS-Fanconi patients showed diminished brush border, defected tight junction, mitochondrial degeneration and autophagosome accumulation of proximal tubule cell. They showed a significant decrease of Megalin and Cubilin expression. With the presence of EGC, IL-17A+ cells were also detected, and IL-17A staining of renal tubule was correlated with Megalin down-expression.ConclusionThe following were observed under the condition of this study:1. pSS-TIN generally present with renal function defect, acidosis and electrolytes abnormalities; age, glomerular sclerosis index and tubulointerstitial lesion were independently correlated with eGFR; patients show good response to steroid and immunosuppressant treatment, eGFR at renal biopsy was the independent risk factor of long term prognosis,2. pSS-TIN patients had EGC in renal interstitium; pDC and mDC were decreased in periphery and recruited to renal EGC; CXCR5 expression increased around vessels adjacent to EGC; IL-21 expression increased comparing to tubulointerstitial nephritis of other etiology,3. pSS-Fanconi showed decreased Megalin and Cubilin, which is related to EGC formation, IL-17A+ cell infiltration and IL-17A expression.