The pathogenesis of tubulointerstitial nephritis and uveitis (TINU) syndrome remains unknown, but T cell-mediated immune response has been postulated to play a role. On the other hand, TINU syndrome is characterized by hypergammaglobulinemia and high serum immunoglobulin G (IgG) levels, suggesting an involvement of humoral immunity. We describe a case of TINU syndrome in a 13-year-old girl with multiple tubular dysfunctions including renal glucosuria, tubular proteinuria, phosphaturia, uricosuria, and concentrating and acidifying defect. IgG antibody from her serum was reactive against 125-kDa human kidney protein. Immunofluorescence study using mouse kidney revealed that the antibody was against cortical renal tubular cells. The antibody disappeared as the renal symptoms resolved. We suggest that IgG antibody may contribute to tubular dysfunction in some patients with TINU syndrome.
Tubulointerstitial nephritis and uveitis (TINU) syndrome was originally described by Dobrin et al1 in 1975. Since then, more than 50 patients have been reported. Although its cause remains unknown, evidence suggests that TINU syndrome is a cell-mediated immune disease. On the other hand, TINU syndrome has sometimes been associated with hypergammaglobulinemia and elevated serum immunoglobulin (IgG) levels, suggesting a humoral-mediated immune response.2 Recently, antitubular cell antibodies have been demonstrated in patients with Sjögren syndrome and systemic lupus erythematosus who have renal tubular acidosis.3,,4 In the present study, we examined whether a similar antibody may be detected in a patient with TINU syndrome.
A 13-year-old girl presented with photophobia. A diagnosis of bilateral anterior nongranulomatous uveitis was made. Because her serum creatinine was 1.1 mg/dL, and glucosuria and proteinuria were noted, she was referred to our hospital. She reported fatigue, abdominal pain, and a 1.8-kg weight loss over the previous month. She denied having fever, joint pain, nocturia, preceding infection, or medication use. Her medical history was unremarkable. On examination, her height was 157 cm, weight was 42.2 kg, and blood pressure was 116/74 mm Hg. Physical examination was unremarkable.
Laboratory findings showed 10.4 g/dL hemoglobin, 31.6% hematocrit, and white blood cell count 5900/mm3 with 76.7% neutrophils, 15.2% lymphocytes, and 2.7% eosinophils. The erythrocyte sedimentation rate was 73 mm in the first hour. C-reactive protein was 1.61 mg/dL; serum creatinine, 0.9 mg/dL; blood urea nitrogen, 11.4 mg/dL; uric acid, 1.8 mg/dL; total protein, 8.5 g/dL; albumin, 4.6 mg/dL; sodium, 140 mEq/L; potassium, 3.3 mEq/L; chloride, 103 mEq/L; calcium, 9.4 mg/dL; and phosphorus, 2.6 mg/dL. Fasting blood glucose was 96 mg/dL. Venous blood gas analysis revealed pH 7.338, Pco2 46.1 mmHg, and HCO3− 25.0 mEq/L. Serum gammaglobulin was 1600 mg/dL (normal: 800–1500 mg/dL), and IgG 1860 mg/dL (normal: 635–1770 mg/dL), immunoglobulin A, 395 (normal: 190–340 mg/dL), and immunoglobulin M 237 mg/dL (normal: 37–154 mg/dL). Serum complement levels were normal, and antinuclear antibody, anti-DNA antibody, rheumatoid factor, antineutrophil cytoplasmic antibody, circulating immune complexes, and Coombs' test were negative. Anti-streptolysin O titer was <57 IU/mL. Antibodies for hepatitis B and C, toxoplasma,Treponema pallidum, and cytomegalovirus were not detected. Tuberculin test was negative. Serum angiotensin converting enzyme was normal.
Urinalysis showed a pH of 7.5, specific gravity 1.010, 2+ glucosuria (7.5 g/d) and 2+ proteinuria (0.93 g/d); there were 21 to 50 white blood cells/high-powered field and 3 to 5 red blood cells/high-powered field with sparse granular casts. Thirty to forty percent of urine white blood cells were eosinophils as detected by Hansel's stain. Urinary β2-microglobulin was 77.8 mg/L (normal: <1.0 mg/L), α1-microglobulin 146 mg/L (normal: <10 mg/L), and N-acetyl-β-D-glucosaminidase/creatinine 41.4 (normal: <5). Urine protein electrophoresis showed a tubular pattern. Creatinine clearance was 108 mL/min per 1.73 m2. Fractional excretion of sodium was 0.59% (normal: <2%), fractional excretion of potassium, 15.6% (normal: 4%–16%); urinary calcium excretion, 0.5 mg/kg/day; fractional excretion of uric acid, 36.9% (normal: 7.6 ± 3.75%); and the percentage of tubular reabsorption of phosphate, 69.7%. There was no aminoaciduria. Urine osmolality after 14-hour water deprivation was 469 mOsm/L. Furosemide 1 mg/kg was administered intravenously, and urine pH was followed until 4 hours. The minimal urine pH was 5.8 at 2 hours. Chest radiograph and cardiac echogram were normal. Gallium scintigraphy revealed uptake in the eyes and kidneys.
A percutaneous renal biopsy was consistent with acute interstitial nephritis. The edematous interstitium was infiltrated with lymphocytes, plasma cells, and eosinophils (Fig 1). There were breaks in the tubular basement membranes with infiltration of the tubular epithelial cell layer by lymphocytes and plasma cells. Glomeruli were normal. Immunofluorescence studies showed no immunoglobulins, fibrinogen, or complement.
She was treated with topical steroids. Serum creatinine normalized (0.6 mg/dL) spontaneously, and anemia, blood abnormalities, and most of the urine abnormalities disappeared within 6 months. Urine concentration defect and β2-microglobulinuria resolved after 1 year.
Sera from the reported patient (acute phase and after complete recovery), a patient with sarcoidosis who had similar multiple tubular dysfunctions and hypergammaglobulinemia as the reported case, and a healthy participant were tested.
Purification of IgG
Eight hundred μL of serum was incubated with 200 μL antihuman IgG agarose (Sigma, St Louis, MO) at 4°C overnight. After washing with phosphate buffered saline (PBS) 3 times, half of the agarose was used for assay for antibody binding kidney proteins as described below. IgG was eluted from the remaining agarose with 300 μL eluting buffer (0.1 M glycine, 0.15 M NaCl, pH 2.4) for immunohistochemical studies. Eluted fractions were immediately neutralized by the addition of sodium hydroxide. Purification of IgG was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Assay for Antibody Binding Kidney Proteins
Normal kidney tissue was obtained from a kidney explanted because of tumor nephrectomy. Tissue was homogenized in solubilization buffer containing 20 mmol/L HEPES (pH 7.2), 1% Triton-100, 10% glycerol, 20 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, and 10 μg/mL leupeptin. Insoluble material was removed by centrifugation (10 500 × g, 10 minutes). The protein content in lysate was measured with a DC protein assay (Bio-Rad Laboratories, Tokyo, Japan). One mg kidney lysate was incubated with 100 μL IgG bound agarose at 4°C overnight. After washing with PBS 3 times, 25 μL Laemmli's buffer was added and boiled. Bound proteins were separated by SDS-PAGE and visualized by Coomassie blue staining.
Mouse cryostat kidney sections were incubated with IgGs. For the secondary antibody, affinity purified goat antihuman IgG (H & L) (American Qualex, San Clemente, CA) was used. The fluorescence signal of labeled specimens was observed first with a Zeiss Axivert microscope and then analyzed by a laser confocal microscope (Zeiss LSM 510, Zeiss, Germany). Digitized images were produced with a Mirus Film Printer Galleria (Mirus Industries Corporation, Santa Clara, CA) using Raster Plus 95 software (version 1.01) (Graphx, Inc, Woburn, MA).
Figure 2 shows SDS-PAGE gels showing kidney proteins reacting with IgGs. Antibody recognizing approximately 125-kDa protein was present in IgG from the reported patient, but was barely detectable in a healthy participant and a patient with sarcoidosis (Fig 2A). This antibody disappeared after recovery (Fig 2B).
Localization of this antibody in the kidney was detected by indirect immunofluorescence with the use of mouse renal tissue. IgG from the reported patient reacted with renal tubular cells in the cortex but not with those in the medulla (Fig 3A, B). The staining was diffuse in the proximal tubules and cortical distal tubules. Secondary antibody alone showed yellow staining attributable to autofluorescence (Fig 3C). The intracellular localization of the antigen was in the cytoplasma. IgG from a patient with sarcoidosis or a healthy participant did not show any specific staining (data not shown).
We reported a patient with TINU syndrome who had antitubular cell autoantibody. The antibody disappeared at recovery. T cell-mediated immune response has been suggested to underlie the pathogenesis of the disease. Thus, granuloma formation is reported in various organs including bone marrow, lymph nodes, and the liver.1,,5 The presence of T cells in the renal interstitial infiltrates has been demonstrated.6,,7 In contrast to increased immune reactivity at the inflammatory sites, patients with TINU syndrome exhibit anergy to skin tests. Furthermore, decreased lymphokine secretion by peripheral blood mononuclear cells of patients with TINU syndrome has been demonstrated.5 These findings are consistent with active local immunity and subsequent peripheral suppressed immune response. On the other hand, circulating immune complexes8,,9 and autoantibodies such as rheumatoid factor,1,,10 antinuclear antibody,11 and antineutrophil cytoplasmic antibodies12,13 have been reported in patients with TINU syndrome. These reports, together with our finding, suggest that humoral-mediated process may be operative at least in some patients with TINU syndrome.
Autoantibodies to tubular cells have been demonstrated in the sera from patients with Sjögren syndrome,3 systemic lupus erythematosus,4 thyroid disease,14 and vasculitis.15 We reported for the first time the presence of antitubular antibody in a patient with TINU syndrome. It remains to be clarified whether the antitubular cell antibody may have developed after tubular antigen exposure by injury, or may have participated in the pathogenesis of TINU syndrome by inducing tubular injury.
The identity of 125-kDa protein also remains to be clarified. It was present in the cytosol but not in the nucleus of cortical tubular cells. Whether this 125-kDa protein can be found in the eye is not known. It has been postulated that autoimmune reaction against common antigens in the kidney and uvea leads to tubulointerstitial nephritis and uveitis. Also, the role of autoimmunity in the pathogenesis of uveitis has been suggested. Thus, antibodies to RNP, Ro, dsDNA, ssDNA, histones, and others have been demonstrated in sera from patients with uveitis.16
The reported patient had both proximal (tubular glucosuria, proteinuria, uricosuria, phosphaturia) and distal tubular dysfunctions (concentrating and acidifying defect), whereas the antibody reacted with only tubular epithelia in cortex. Therefore, the observed distal tubular dysfunctions might be attributable to injury to cortical distal tubules, a segment participating in both urine concentration and acidification.
Immunofluorescence study of renal biopsy specimen was negative for IgG. Although in previous studies, autoantibodies to tubular cells have been recognized by localization of immunoglobulins, this traditional technique is probably not sensitive. With the assay used in the present study, more patients with TINU syndrome may be detected who have antitubular cell antibodies.
This study was supported by the Pharmacia-Upjohn Fund for Growth and Development Research.
- Received October 2, 2000.
- Accepted December 11, 2000.
Reprint requests to (M.A.) Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail:
Dr Sakamoto is currently at the Fourth Department of Internal Medicine, Kitasato University School of Medicine.
- TINU =
- tubulointerstitial nephritis and uveitis •
- IgG =
- immunoglobulin G •
- PBS =
- phosphate-buffered saline •
- SDS-PAGE =
- sodium dodecyl sulfate polyacrylamide gel electrophoresis
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- Copyright © 2001 American Academy of Pediatrics