A case of tubulointerstitial nephritis with infiltration of neutrophils and interleukin-17-positive cells associated with Behçet’s disease

Abstract

Behçet’s disease (BD) is a non-infectious inflammatory condition characterized by neutrophilic infiltration. In addition to primary symptoms, including oral and genital ulcers, ocular involvement, and skin lesions, BD can also affect various organs. However, renal involvement, particularly in tubulointerstitial nephritis, has rarely been described. Herein, a rare case of acute tubulointerstitial nephritis in a patient clinically diagnosed with BD is reported. The renal lesion presented with other symptoms of BD and fever, and was considered to be BD-related due to the presence of neutrophilic infiltration and its responsiveness to BD-directed therapy. Alterations in T-helper (Th) 1, Th2, and Th17 cytokine profiles are associated with BD activity. Interleukin (IL)-17 plays a central role in neutrophil activation, and recent studies have demonstrated a strong correlation between IL-17A levels and BD activity. In the present case, elevated serum IL-17A levels and infiltration of IL-17A-positive cells into the renal tissue reflected an active phase of BD and a BD-associated renal lesion.

Introduction

Behçet’s disease (BD) is a non-infectious inflammatory condition characterized by oral aphthous ulcers, genital ulcers, and eye and skin lesions. There is no specific biomarker for BD, and its diagnosis is based on symptoms and clinical findings [1, 2]. Renal lesions in BD are less frequent than the main symptoms and most are nephropathies due to AA amyloidosis caused by chronic systemic inflammation. Other reported cases have included proliferative glomerulonephritis, membranous nephropathy, crescentic glomerulonephritis, and immunoglobulin A nephropathy. Reports describing acute tubulointerstitial nephritis in patients with BD are scarce, as are studies investigating the direct relationship between BD and tubulointerstitial nephritis [3, 4].

BD is characterized by neutrophil hyperactivity. An imbalance in T lymphocyte responses and associated cytokine abnormalities have been implicated in the pathogenesis of the disease. Altered T-helper (Th) 1, Th2, Th17, and T regulatory (Treg) cytokine profiles have been linked to BD activity, although these findings remain inconsistent across studies. Among these, interleukin (IL)-17 plays a central role in the activation and mobilization of neutrophils, and some recent studies have reported a strong correlation with BD activity [5, 6].

We encountered a case of interstitial nephritis with neutrophil infiltration in a patient who was clinically diagnosed with BD. Because BD-associated nephritis is rare and often challenging to diagnose, we measured serum Th1/Th2/Th17/Treg cytokine levels and performed immunostaining of renal tissues for CD4, CD8, and IL-17A. In this case, elevated serum IL-17A levels and the presence of IL-17A-positive cells in the renal tissue supported the conclusion that renal lesions are associated with BD. Herein, we report a case of tubulointerstitial nephritis associated with BD.

Case report

A 40-year-old Japanese woman was referred to the authors’ department for unexplained fever and pyuria. She experienced acneiform eruptions since childhood and painful oral ulcers in adulthood. Two weeks before the fever, she developed a vulvar ulcer that did not improve with amoxicillin and potassium clavulanate, and worsened as the fever worsened. Valacyclovir treatment did not yield improvement. Urinalysis revealed > 100 white blood cells per high-magnification field and positive urinary eosinophils (Fig. 1). Contrast-enhanced computed tomography revealed wedge-shape, poorly defined areas in both kidneys (Fig. 2A). Ultrasonography revealed no obvious aneurysms, stenosis, or infarction in renal arteries. Subsequent treatment with cefmetazole was ineffective; accordingly, drug-induced interstitial nephritis was suspected.

Fig. 1.

Clinical course. The day of fever onset was defined as day 0. Erenumab, naratriptan hydrochloride, loxoprofen sodium hydrate, and vonoprazan fumarate were taken by the patient for migraine headache(s) several times irregularly before the fever. CT, computed tomography; AMPC/CVA, amoxicillin and potassium clavulanate; VAC, valaciclovir; CMZ, cefmetazole; PSL, prednisolone

Fig. 2.

Renal imaging. A Contrast-enhanced computed tomography revealing wedge-shaped poorly defined areas in both kidneys. B Gallium scintigraphy revealing diffuse uptake in both kidneys, but no abnormal uptake in other organs

The patient had no history of renal disease except for pyelonephritis when she was 24 years of age, with no abnormal laboratory findings. For 2 years, she had been taking sodium valproate daily, and irregular erenumab, naratriptan hydrochloride, loxoprofen sodium hydrate, and vonoprazan fumarate for migraine headache(s).

After the fourth week of fever, the vulvar ulcer exhibited slight improvement; however, the high fever persisted, thus prompting another full examination. Physical examination results were normal except for aphthous ulcers in the mouth and vulva. An ophthalmological examination did not reveal uveitis or other abnormalities. Body temperature was 38.4℃, blood pressure was 105/96 mmHg, and heart rate was 96 beats/min. Gallium scintigraphy revealed diffuse uptake in both kidneys, but no abnormal uptake in other organs (Fig. 2B).

Blood tests revealed an increased neutrophil count, markedly increased C-related proteins, and mild renal dysfunction (Table 1). Urinalysis revealed elevated levels of tubulointerstitial markers such as β₂-microglobulin and N-acetyl-β-D-glucosaminidase. After discontinuation of cefmetazole, the number of white blood cells in the urine decreased. White blood cells in the urine consisted mainly of neutrophils and some lymphocytes, and eosinophils in the urine disappeared. Urine and blood cultures were negative. Serological test results for human immunodeficiency virus, hepatitis B, hepatitis C, herpes, and cytomegalovirus were negative. No autoantibodies or complement or immunoglobulin abnormalities were detected. Drug-induced lymphocyte stimulation tests (DLST) were negative for amoxicillin, valproic acid, loxoprofen, and vonoprazan. The human leukocyte antigen (HLA) types include A24, A26, B35, and B54, of which A26 has been suggested to be associated with BD. Further examination of serum levels of Th1/Th2/Th17/Treg cytokines revealed the following: interferon-gamma (IFN-γ), ≤ 0.1 IU/mL; IL-4, 14.0 pg/mL (control, ≤ 6.0 pg/mL); IL-6, 17.9 pg/mL (control ≤ 5.8 pg/mL); IL-10, ≤ 2 pg/mL; and IL-17A 45 pg/mL (control ≤ 31 pg/mL), indicating mild increases in IL-4, IL-6 and IL-17A. Assays for cytokines other than IL-17A were outsourced to a commercial entity (SRL Inc., Shinjuku, Japan), and IL-17A levels were measured using a commercially available ELISA kit (KE00203, Proteintech, Rosemont, IL, USA).

Table 1.

Laboratory data

	Normal range	At the time of renal biopsy	2 weeks after treatment
White blood cells (/µl)	3300–8600	8920	7980
Neutrophils (%)	40.0–70.0	85.7	74.1
Lymphocytes (%)	16.5–49.5	5.1	17.6
Monocytes (%)	2.0–10.0	8.7	7.7
Eosinophils (%)	0.0–8.5	0.3	0.5
Basophils (%)	0.0–2.5	0.1	0.1
Hemoglobin (g/dL)	11.6–14.8	8.6	9.8
Platelets (× 104/µl)	15.8 −34.8	30.7	30.8
Total protein (g/dL)	6.6–8.1	6.0	6.0
Albumin (g/dL)	4.1–5.1	2.0	2.7
BUN (mg/dL)	8.0–20.0	8.0	14.8
Creatinine (mg/dL)	0.46–0.79	0.87	0.62
eGFR (mL/min/1.73m2)	60.0	57.9	83.9
Uric acid (mg/dL)	2.6–5.5	2.5	3.0
Sodium (mmol/L)	138–145	141	140
Potassium (mmol/L)	3.6–4.8	3.5	3.7
Chloride (mmol/L)	101–108	103	102
Calcium (mg/dL)	8.8–10.1	8.0	8.7
Phosphorus (mg/dL)	2.7–4.6	3.3	–
CRP (mg/dL)	− 0.15	19.06	0.50
Urine pH		7.5	7.0
UWBC (/HPF)	− 4	10–19	1–4
URBC (/HPF)	− 4	1–4	< 1
UTP (g/gCr)	− 0.15	0.36	0.05
Uβ2MG (µg/gCr)	− 300	1594	193
NAG index (U/gCr)	1.6–15.0	44.3	10.4
Epithelial cell casts (/WF)		1–4
Waxy casts (/WF)		1–4

BUN Blood nitrogen urea; eGFR Estimated glomerular filtration rate; CRP C-related protein; UWBC Urinary white blood cells; URBC Urinary red blood cells; HPF High power field; UTP Urinary total protein; Uβ2MG Urinary β2-microglobulin; NAG N-acetyl-β-D-glucosaminidase; WF Whole field

A biopsy was obtained from the left kidney, which exhibited a wide area of contrast failure. Immunoglobulin or complement deposits were not observed. Light microscopy revealed interstitial fibrosis with localized inflammatory cell infiltration and diffuse edematous expansion (Fig. 3A). The inflammatory cells were predominantly lymphocytes with prominent neutrophils and plasma cells, but no eosinophils (Fig. 3B-D). No abnormalities, including vasculitis or thrombosis, were observed in twenty glomeruli. Additional immunostaining of the remaining renal sections revealed more CD4-positive than CD8-positive cells, with prominent IL-17A-positive cells (Fig. 4).

Fig. 3.

Light microscopy images of a renal biopsy specimen. A interstitial fibrosis with inflammatory cell infiltration and diffuse edematous expansion (Masson trichrome staining) are evident. B, C and D inflammatory cell infiltration in tubular interstitium is localized, with prominent lymphocytes, neutrophils (arrows) and plasma cells, but no eosinophils (hematoxylin–eosin staining). Scale bar: 100 μm

Fig. 4.

Immunostaining for CD4, CD8 and interleukin (IL)-17A of renal biopsy specimen. CD4-positive cells are more numerous than CD8-positive cells, and IL-17A-positive cells are prominent (incomplete serial sections). Scale bar: 50 μm

Based on these clinical course (Fig. 1) and pathological findings, the patient was diagnosed with BD and associated acute tubulointerstitial nephritis. After starting oral medication with 30 mg prednisolone and 1 mg colchicine, the fever reduced, and the oral ulcers exhibited signs of improvement within a few days. The levels of C-related proteins, tubulointerstitial markers, and renal function returned to normal within approximately 2 week (Table 1).

Discussion

The patient had a long history of oral ulcers and recently developed genital ulcers. Approximately two weeks later, fever was observed, and approximately three weeks later, pyuria was detected. Urinary leukocytes changed from eosinophils to neutrophils, and renal biopsy revealed tubulointerstitial nephritis accompanied by neutrophilic infiltration. As the patient's oral ulcers and genital ulcers fulfilled the international diagnostic criteria for BD [7], a diagnosis of BD was established. The basic pathogenesis of BD is neutrophil hyperfunction with neutrophil-infiltrating lesions during the active phase of the disease. Neutrophil hyperfunction can be induced by several factors such as genetic factors, infection, and allergic constitution [2]. The prevalence of HLA-B51 antigen is significantly higher among patients with BD, and the HLA-A26 allele found in this patient has been reported as a secondary disease susceptibility gene [2, 8]. Some external causes of BD point to viral or bacterial infections [1, 9]; however, the infection could not be ascertained in this case. Renal findings appeared along with other clinical manifestations of BD, including neutrophilic infiltration, which improved with prednisolone and colchicine, the main treatment modalities. Therefore, we concluded that the patient had BD-associated tubulointerstitial nephritis.

Renal involvement in BD is rare, and tubulointerstitial nephritis has scarcely been reported [3]. To our knowledge, only five cases of biopsy-proven tubulointerstitial nephritis in patients with BD have been reported, as summarized by Akpolat [3] and Belmouaz [10]. In the first case [11], tubulointerstitial nephritis developed prior to the typical manifestations of BD (oral ulcers and uveitis); however, the renal pathological findings were unclear and investigation for other causes was inadequate. In the subsequent three cases [12–14], the available clinical and pathological data were insufficient to definitively attribute tubulointerstitial nephritis to BD. Only the fifth case provided a comprehensive description of both the clinical course of BD and the renal pathology [10].

Belmouaz et al. [10] reported a case of a 46-year-old North African woman who presented with more severe clinical and pathological features than those in the present case. She had bilateral acute uveitis, oral ulcers, headache, abdominal pain, arthralgia, and acute renal failure with Fanconi syndrome (serum creatinine 1.9 mg/dL). Renal biopsy revealed polymorphic inflammatory cell infiltration (lymphocytes, neutrophils, histiocytes, eosinophils, plasma cells) in the interstitium. The patient also exhibited severe proximal tubular damage consistent with Fanconi syndrome, with predominant CD8-positive T lymphocyte infiltration. In contrast, in our case, the infiltrating inflammatory cells were mainly lymphocytes and neutrophils, with no eosinophils, and CD4-positive cells were more abundant than CD8-positive cells. The interstitium was generally edematous; however, the inflammatory cells were localized, suggesting a stage beyond the peak of inflammation. Tubular structures were relatively preserved and were not complicated by Fanconi syndrome. The difference in renal pathology between our case and that of the patient described by Belmouaz et al. [10] may depend on the phase of BD activity and the timing of the renal biopsy.

T lymphocytes (Th1, Th2, and Th17) and various proinflammatory cytokines also play major roles in neutrophil hyperfunction. Th17 immune responses, which have recently received attention, predominantly produce IL-17 to induce neutrophil recruitment and promote inflammation. Serum IL-17A levels have been reported to be elevated during the active phase of BD [6, 15, 16]. In the present case, elevated serum levels of IL-17A and the infiltration of IL-17A-positive cells in the kidneys suggested the active phase of BD and associated tissue involvement. The ‘mild’ elevation of serum IL-17A levels could result from the passing of peak of disease activity. The Th1/Th2 balance varies among individuals and across different phases of the disease. Some reports suggest that Th1 cytokines (e.g., IFN-γ, tumor necrosis factor-alpha) are more involved than Th2 cytokines [17, 18], although Th2 cytokine (e.g., IL-4, IL-13) levels are increased in some cases of BD [19, 20]. Our findings regarding serum cytokines were consistent with the previous study, reporting elevated levels of IL-17 and IL-6, and decreased levels of IL-10 and IFN-γ during the active phase in patients with BD [21]. IL-6 promotes differentiation of Th17 cells [22], thereby contributing to a Th17-dominant immune response. Reduced IL-10 levels imply Treg cell dysfunction and facilitate Th-17 cell activation [23].

Interestingly, urinary eosinophils were initially believed to reflect drug-induced allergic interstitial nephritis. However, no reliable evidence was obtained in the DLST, and they subsequently changed to neutrophils. The level of IL-4 (a Th2 cytokine), which induces eosinophils, was also increased in the present case, and it remains possible that an allergic mechanism was involved in the onset of nephritis. Immune checkpoint inhibitors (ICIs) are a well-recognized cause of drug-induced interstitial nephritis with neutrophilic infiltration, and IL-17 involvement has also been reported [24, 25]. However, while other drugs may have been reported to induce diverse inflammatory cell infiltrates beyond eosinophils, reports clearly establishing IL-17 involvement remain scarce, with only one study addressing proton pump inhibitor-induced interstitial nephritis [26]. Therefore, interstitial nephritis characterised by Th-17 cell activation and neutrophil infiltration is difficult to explain by non-ICI drugs alone, and is highly likely to arise within the immunological background of BD, involving a complex Th cytokine network. Furthermore, since elevated levels of Th2 cytokines have also been reported in patients with BD [19], the phenomenon of urinary leukocytes shifting from eosinophils to neutrophils may reflect a series of clinical processes explainable by BD.

In conclusion, we report a case of acute tubulointerstitial nephritis with infiltration of neutrophils and IL-17A-positive cells in a patient with BD and describe it as a rare BD-related kidney lesion.

Author contributions

The original draft was written by NU and KT, and all authors reviewed the draft. The final manuscript was read and approved by all authors. Immunostaining of renal biopsy specimen was performed by KT.

Declarations

Conflict of interest

The authors have declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from participants included in the article.