Background
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.
Methods
Patients and controls
All patients diagnosed with primary Sjogren syndrome with renal Fanconi syndrome in Peking Union Medical College Hospital (PUMCH) from 1994 to 2014 were enrolled. The diagnosis of pSS was made according to the American–European Consensus Group criteria for pSS [
14]. Fanconi syndrome was defined by the coexistence of hypokalemia, hypophosphatemia, normoglycemic glycosuria, generalized aminoaciduria and hyperphosphaturia [
15]. Clinical records and follow-up data of enrolled patients were carefully reviewed to understand demographic characteristics, symptoms, physical examination and laboratory tests. Laboratory examinations included routine tests: blood, urine, liver and renal function, 24-h urine protein, erythrocyte sedimentation rate (ESR), C-reactive protein and plasma protein electrophoresis. Renal tubular function assay included: blood and urine electrolytes, blood and urine α
1-microglobulin (α
1-MG), β
2-microglobulin (β
2-MG), blood pH, carbon dioxide combining power, urine
N-acetyl-β-amino-glucosidase (NAG), retinol binding protein (RBP) and blood and urine osmotic pressure tests. Immunology assay included: immunoglobulin (IgG, IgA, IgM), rheumatoid factor (RF), blood complement and antinuclear antibodies spectrum. Screening for autoantibodies to SSA/Ro and SSB/La was performed systematically using Ouchterlony double-gel immunodiffusion and western blotting. Other tests included lacrimal and salivary gland secretion test (Schirmer test), salivary scintigraphy, parotid sialography and labial biopsy. The estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease (MDRD) study equation [
16]. Systemic manifestations of these patients were evaluated by Eular Sjogren’s Syndrome Disease Activity Index (ESSDAI) [
17]. Twenty patients with pSS and tubulointerstitial nephritis (pSS + TIN) were included as the control group. TIN clinically manifested as hematuria, leucocyturia, proteinuria (24-h urine protein < 2 g), renal function impairment, distal RTA and hypokalemia, with mainly tubulointerstial impairment in renal biopsies, with or without minor glomerular damage. They had the same workup to exclude proximal tubule injury. Six cases of glomerular minor lesion (GML) were selected as normal controls. These normal patients underwent renal biopsy in the setting of mild isolated hematuria and strong desire for renal biopsy to figure out the etiology, but were not found to have underlying pathology.
Pathologic studies of kidney tissue
Two-micrometer slides were cut from formalin-fixed and paraffin-embedded (FFPE) sections of kidney tissues, stained with hematoxylin and eosin, periodic acid–Schiff, periodic acid–silver metheramine and Masson trichrome for light microscopy in the laboratory of Nephrology Department at PUMCH. At least eight sections were examined for each patient. All sections were examined by an experienced pathologist who was blinded to the patient’s characteristics. Tubulointerstitial injury was evaluated based on the Oxford Classification of IgA glomerulonephritis [
18]. The classification used to describe the degree of lymphocytic infiltration in renal tissue was similar to that used in previous studies of the labial gland in patients with Sjogren syndrome: grade 0 (G0), absent, no lymphocyte infiltration; grade 1 (G1), slight infiltration, scattered lymphocytes infiltrating with an aggregate of fewer than 50 cells; grade 2 (G2), moderate infiltration, focal periductal lymphocytes aggregating in the labial gland, with 50 or more cells per one lesion; and grade 3 (G3), dense infiltration showing EGC-like structures in labial gland and CD21 staining positive [
19].
Immunohistochemistry staining of megalin, cubulin, CD21 and IL-17A
Immunohistochemical (IHC) staining was performed on serial sections using standard methods in five pSS patients with Fanconi syndrome who underwent renal biopsy, five pSS + TIN patients (randomly selected from 20 patients in the control group) and six GML patients. Three-micrometer sections cut from paraffin-embedded tissue were deparaffinized and rehydrated. Sections were heated in a pressure cooker with 0.01 mol/L citrate buffer (pH 6.0) for 5 min to expose antigen and then incubated with the primary antibody (megalin, IL-17A and CD21, rabbit polyclonal antibodies; Abcam, Cambridge, MA, USA; cubilin, goat polyclonal antibody; Santa Cruz, CA, USA) overnight at 4 °C. After incubation with 0.3% H2O2 for 15 min, sections were incubated with the 1:500 HRP-conjugated anti-rabbit or anti-goat IgG (ImmunoReagents, USA) for 1 h at 37 °C. 3,3′-Diaminobenzidine (DAB) was used as a staining substrate. All section images were captured by a Nikon microscope (Eclipse 80i; Nikon, Japan) equipped with a digital photograph camera (DS-U1; Nikon, Japan).
Megalin immunofluorescence staining was done on 3-μm paraffin sections. The slides were incubated with primary antibody (megalin, rabbit polyclonal antibody; Abcam) overnight at 4 °C. Secondary antibody fluorescein-conjugated AffiniPure donkey anti-rabbit IgG (EarthOx, USA) was applied and incubated at 37 °C for 1 h. The micrographs were taken by confocal laser microscopy (Leica, Germany).
The degree of IHC staining was evaluated by calculating the percentage of positive glomeruli. Staining and scoring were performed blindly on coded slides. At least six fields were selected randomly in the renal cortex of each specimen for photo-documentation. Analytical measurements were done using Image Pro Plus 6.0.
Statistical analysis
Continuous variables are presented as the mean ± standard deviation. The variables consistent with normal distribution were compared using Student’s t test, one-way analysis of variance or Pearson’s correlation coefficients; skewed distribution samples were compared using the Mann–Whitney test or Spearman’s correlation. Categorical variables are expressed as percentages and are compared using the chi-square test. Statistical processing was performed using Graphpad Prism 6.0 and p <0.05 was considered statistically significant.
Discussion
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.
Table 2
Comparison of clinical profile between our cases and pSS-related Fanconi syndrome reported in the literature
| Polyuria | + | n.a. | 3.8 | TIN, tubular atrophy | n.a. | n.a. |
| Paralysis, polyuria | + | n.a. | n.a. | TIN | Prednisolone 10 mg/day | n.a. |
| Polyuria, nocturia, weight loss | + | 2.7 mg/dl | 2.9 | Diffuse TIN | Supportive only | Improved |
| n.a. | n.a. | 2.7 mg/dl | n.a. | TIN, tubulitis | n.a. | n.a. |
| Muscle weakness | + | 1.3 mg/dl | 2.5 | n.a. | Prednisolone “low dose” | Improved |
| Weight loss | + | 1.8 mg/dl | 3.5 | Diffuse TIN, proximal tubulitis | Supportive only | Dieda
|
| Polyuria | + | 1.6 mg/dl | 2.4 | Diffuse TIN, proximal tubulitis | Prednisolone 10 mg/day | Improved |
| Muscle weakness | + | 1.3 mg/dl | 2.7 | Diffuse TIN, proximal tubule atrophy | Prednisolone 30 mg/day, 6 months later 12.5 mg/day | Improved |
| n.a. |
| Muscle weakness, respiratory distress | + | 1.4 mg/dl | 2.7 | n.a. | Supportive only | n.a. |
| Renal dysfunction, organizing pneumonia, multiple bone fracture | + | 1.3 mg/dl | 3.0 | n.a. | Mizoribine 50 mg/day | n.a. |
| Hypokalemic paralysis | + | 2.2 mg/dl | 1.6 | Diffuse TIN | Mycophenolate mofetil 1 g/day | Improvedf
|
| Paralysis | + | 2.1 mg/dl | 1.3 | Dense lymphocytic interstitial infiltrate | Supportive only | Improved |
| Paralysis, cardiac arrestc
| + | 1.1 mg/dl | 1.1 | n.a. | Prednisone 40 mg/d iv. in acute phase | Improvedf
|
| Proteinuria, glycosuriad
| + | | 3.07 | TIN | Methylprednisolone | Improvede
|
| Renal dysfunction | + | 1.07 mg/dl | 3.7 | TIN | Prednisolone 40 mg/day | Improvede
|
| Weakness, osteodynia, impaired mobility | _ | n.a. | 1.3 | n.a. | Prednisone 30 mg/day | |
Our cases | Fatigue, anorexia | + | 151 μmol/L | 3.4 | Diffuse TIN, diffuse tubule atrophy, lymphocyte infiltration | Prednisone 50 mg/day | Improvedf
|
| Fatigue, polyuria, anorexia, osteopathy | + | 88 μmol/L | 2.1 | Focal TIN, focal tubule atrophy | Prednisone 40 mg/day | Improvedf
|
| Fatigue, anorexia, osteopathy | + | 176 μmol/L | 3.3 | Diffuse TIN, diffuse tubule atrophy, lymphocyte infiltration | Prednisone 50 mg/day | Improvede
|
| Fatigue, anorexia | + | 305 μmol/L | 2.7 | Focal TIN, focal tubule atrophy | Prednisone 45 mg/day | Improvede
|
| Fatigue, anorexia, polyuria | – | 72 μmol/L | 2.53 | Mild tubulitis | Supportive only | Improvedf
|
| Fatigue, anorexia, polyuria | + | 184 μmol/L | 3.0 | n.a. | Prednisone 35 mg + cyclophosphamide 0.2 g qod | Improvede
|
| Polyuria | – | 120 μmol/L | 3.4 | n.a. | Prednisone 60 mg/day | Improvede
|
| Fatigue, polyuria, anorexia, osteopathy | + | 202 μmol/L | 2.62 | n.a. | Prednisone 55 mg/day | Improvede
|
| Osteopathy | – | 110 μmol/L | 3.2 | n.a. | Prednisone 55 mg/day + metrotraxate 10 mg qw | Improvedf
|
| Fatigue, polyuria, osteopathy | + | 75 μmol/L | 2.88 | n.a. | Prednisone 40 mg/day + cyclophosphamide 0.2 g qod | Improvedf
|
| Hypokalemic paralysis, osteopathy | + | 65 μmol/L | 2.1 | n.a. | Supportive | Improvedf
|
| polyuria, osteopathy | + | 71 μmol/L | 2.34 | n.a. | Prednisone 30 mg/day + metrotraxate 10 mg qw | Improvedf
|
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.
Acknowledgements
Not applicable.