Ectopic germinal center and megalin defect in primary Sjogren syndrome with renal Fanconi syndrome

Primary Sjogren syndrome (pSS) is a chronic autoimmune epithelialitis targeting exocrine glands, with possible multisystem involvement [1]. Characteristic pathological changes are focal lymphocytic infiltration around the epithelial ducts and production of autoantibody by hyperactive B cells [2]. Renal involvement is observed in pSS, with both tubular and glomerular abnormalities reported. Tubulointerstitial nephritis (TIN) as a result of periepithelial inflammation is a predominant feature of pSS, often with evidence of a distal renal tubule acidosis (RTA) [3‐5]. Fanconi syndrome, the result of proximal tubule epithelial cell (PTEC) injury leading to proximal RTA (type II RTA), hypophosphatemia, hypouricemia, aminoaciduria, glycosuria and urine loss of low molecular weight proteins, is a rare manifestation of pSS. To date, fewer than 20 cases have been reported and the underlying pathogenesis or mechanism remains unclear [6].

Ectopic germinal centers (EGCs), nonlymphoid collections of mature B lymphocytes, have been observed in the labial glands of pSS patients, believed to be the result of chronic inflammation [7]. The presence of CD21⁺ follicular dendritic cells is one of the hallmarks of EGCs [8, 9]. EGCs are suggested to be the site of immune stimulation and have been identified in other autoimmune diseases, such as rheumatoid arthritis and Grave’s disease. Self-reactive T lymphocytes and antibodies contribute to the process of tissue destruction and disease progression [10]. Recent studies suggest that Th17 cells, a subset of CD4⁺ T cells, may directly contribute to lymphoneogenesis in labial glands of pSS patients [11], but it is unclear whether a similar process occurs in the kidney of pSS patients with Fanconi syndrome. Inhibition of receptor-mediated endocytosis has been proposed as the mechanism of Fanconi syndrome in other disease states. Megalin and cubilin are multiligand protein receptors expressed at the brush border membrane and involved in endocytosis in PTECs. Megalin-knockout mice and cubilin-deficient dogs demonstrate deficient endocytosis, reproducing low-molecular proteinuria and vitamin D deficiency, which are the main characteristics of human Fanconi syndrome [12, 13].

In this study, we report the clinical and pathological characteristics and therapeutic outcomes of 12 patients with pSS and Fanconi syndrome. We describe the presence of EGCs in the renal interstitium, the prevalence of Th17/IL-17 expression, and alterations in megalin and cubilin expression, to investigate their possible correlation.

The most common renal manifestation of pSS is a distal RTA, with proximal tubular acidosis seldom reported [20]. Fanconi syndrome, general dysfunction of the proximal tubule, is a relatively rare clinical manifestation of pSS. By searching PubMed with ((Sjogren syndrome) or (autoimmune epithelialitis)) and ((renal tubule) OR Fanconi), about 20 cases were identified [6, 21‐35]. Wang et al. [6] and Shi and Chen [33] have summarized SS-related Fanconi syndrome cases that had been reported. We made a supplement to their summarization. We reported 12 cases of pSS + Fanconi syndrome cases with detailed clinical profile and follow-up records, to our limited knowledge, which is the largest sample in a single center. As presented in Table 2, similar clinical and pathological characteristics were observed in our study. In our series, 58% patients had impaired renal function with moderate TIN, and showed good response to glucocorticoid therapy. Both tubular function injury and eGFR were improved following treatment with steroids.

Few studies have focused on the mechanism of Fanconi syndrome or PTEC injury in pSS. We observed the downregulation of megalin and cubilin in PTECs, which suggests defective endocytosis. Megalin and cubilin are endocytic receptors coexpressed in the proximal tubule, located on the brush border and endocytic vesicles. They bind and mediate the endocytosis of a variety of ligands, including enzymes or enzyme inhibitors, lipoproteins, hormones, signaling proteins, immune or stress response-related proteins, receptors and vitamin carrier proteins as well as drugs and toxins [12, 36]. Megalin and cubilin are associated with key processes to fulfill the classic reabsorption function of proximal tubules: the integrity of cell structure, such as polarity, brush border and endocytic apparatus; apical multiligand receptors megalin and cubilin; and intact transport system consisting of microvilli, clathrin-coated pits, early endosomes, late endosomes and lysosome. The other processes include energy production by mitochondria and basolateral Na⁺-K⁺-ATPase as the driving force for Na⁺-coupled transport [13, 37‐39]. In cystinosis, a known cause of Fanconi syndrome, alterations in megalin activity have been noted on the brush border, endosomes and lysosomes by immunofluorescence under electron microscopy [40]. Megalin and cubilin have also been shown to be critical in Fanconi syndrome from other causes. Mutations in the gene low-density lipoprotein receptor-related protein 2 (LRP2), encoding the protein megalin, have been identified in Donnai-Barrow (DB) syndrome and Facio-Oculo-Acustico-Renal (FOAR) syndrome. These patients show prominent low-molecular weight proteinuria, with malabsorption of vitamin D-binding protein, retinol-binding protein and albumin [41]. Elegant experiments have suggested urinary megalin deficiency implicating abnormal tubular endocytic function in Fanconi syndrome related to Dent’s disease and Lowes syndrome [42]. In these genetic disorders, the down-expression of megalin/cubilin caused by impaired endosome–lysosome trafficking has also been shown [43, 44]. On the other hand, megalin-mediated endocytosis of excessive protein is pathogenic in light-chain tubulopathy. It has been shown that silencing megalin and cubilin genes may inhibit myeloma light chain uptake, suppressing inflammation in PTECs, and reducing the nephrotoxic effects [45]. The mechanism of drug-induced Fanconi syndrome is not fully understood at this point in time [46]. However, as far as we can see, the mechanism of Fanconi syndrome caused by pSS has not yet been discussed. The innovation of our study is to show that defect endocytosis in PTECs mediated by megalin and cubilin may contribute to the reabsorption impairment in patients with pSS and Fanconi syndrome. A trend toward loss of megalin expression was observed in pSS with TIN, but this was not significant compared with the decreased expression of megalin noted in patients with pSS and Fanconi syndrome. We propose that proximal tubule impairment was less severe in TIN with general tubule injury, because no symptoms of endocytic receptor defect were observed.

The mechanism of megalin and cubilin alteration remains poorly understood, although past studies have indicated that renal ischemia and reperfusion injury, inflammation and drugs may all be causative. Lipopolysaccharide has been shown to downregulate megalin and cubulin expression in vitro and vivo [47]. In this study, we first observed the inverse relationship of megalin and IL-17 expression with associated ectopic germinal center (EGC) formation in the kidney. IL-17 is a proinflammatory cytokine secreted by Th17 cells, which comprise a distinct subset of CD4⁺ T cells that play a role in autoimmune disease [48]. Th17 cells are believed to activate follicular dendritic cells and stromal cells via surface molecule LTα1β2 and they secrete IL-17, stimulating stromal cells, fibroblasts and tissue epithelial cells to produce chemokines [11]. IL-17 secretion is induced by IL-21, produced by Tfh cells, a component of EGCs. IL-17 then acts in conjunction with IFNγ to recruit lymphocytes for the formation of the germinal center [49]. In addition, we observed that renal proximal tubule cells can express IL-17. It is reasonable to assume that epithelial cells may be an important component of immune response in TIN, as has been indicated in renal transplant rejection. EGCs are highly organized lymphoid aggregates that form in tissue sites which are not typically associated with lymphoid neogenesis [50]. TIN is characterized by organized infiltration of anatomically distinct and adjacent T-cell and B-cell compartments, with the presence of follicular dendritic cells (DCs) [9]. As the infiltrating cells, DCs have been shown to be necessary and sufficient in the formation of EGCs [51‐53], Depletion of DCs leads to disappearance of existing follicular germinal center structure [52]. We observed that circulating pDCs and mDCs were reduced in pSS patients with TIN, compared with patients with pSS alone. In addition, immunohistochemical staining of BDCA-2 and DC-SIGN revealed increased pDCs and mDCs in renal interstitium. Combining these findings, we assume that in the setting of local inflammation in pSS, DCs can be recruited to renal tissue from peripheral blood (unpublished data). CD21 remains the most reliable marker of follicular dendritic cells. In another study of our group, we find that in pSS with membranous nephrology, all G3 patients (30.5% of 36 patients) showed positive CD21 staining with varying degree, and typical EGC structure was seen in nine patients. The relationship of IL-17 and germinal center activity has been observed previously. In an experimental autoimmune encephalomyelitis murine model, Th17 cells also directly elicit formation of ectopic lymphoid follicle formation, by surface molecule podoplanin and secretion of IL-17 [54]. In the formation of inducible bronchus associated lymphoid tissue (iBALT), IL-17 secreted by Th17 cells contributes to the initial stage [55]. In the labial glands with pSS, germinal centers developed in 25.1 ± 5.0% of patients [56]. Both Sakai et al. and Fei et al. reported that the majority infiltrating cells in the salivary glands of SS patients were CD4⁺ T cells, with a predominant expression of IL-17 [56, 57], which could be modified by immunosuppressive treatment [57]. Consistent with their findings, we observed infiltrating cells expressing IL-17A present in renal interstitium (we did not perform CD4 and IL-17A double-staining to clarify Th17 cells due to limited pathological sections). There is also evidence to suggest that EGC formation may contribute to progression of disease in lupus nephritis [58, 59]. Therefore, the presence of DCs and EGCs in the interstitium of the kidney in patients with pSS suggests that the severe renal interstitial inflammation, with Th17 infiltration and IL-17 secretion, may be correlated to megalin and cubilin impairment. We suppose that local inflammation may subsequently mediate brush border destruction, characterized by decreased megalin and cubilin expression, leading to the reabsorptive dysfunction of PTECs and Fanconi syndrome.

There are several limitations of our study. First, not all patients underwent bicarbonate loading test, and patients with isolated proximal tubule acidosis could not be identified. Second, as a retrospective study, instead of the mechanism we only suggested a correlation between EGC formation and defected endocytosis characterized by megalin/cubilin down-expression. It is possible that megalin/cubilin deficiency is a reflection of generalized proximal tubule injury, because up to 45.2% of pSS patients were present with elevated excretion of β₂-microglobulin. Third, a different degree of inflammation may not explain the whole picture of why only certain pSS + TIN patients present with proximal tubule defect. Whether this is due to specific local antibody remains to be elucidated by further study.

Collectively, we reported 12 cases of pSS with Fanconi syndrome. They present with proximal tubular defect, decreased eGFR and tubulointerstitial nephritis, and they show good response to glucocorticosteroids therapy. Down-expression of megalin and cubilin may cause the endocytosis reabsorption dysfunction of proximal renal tubules. We suggest that this is related to Th17 infiltration, IL-17 expression and formation of ectopic germinal centers.