Case of hereditary kidney disease presenting thin basement membrane with a single heterozygous variant of Intersectin 2

Abstract

Objective: Intersectin 2 (ITSN2) is reported to cause hereditary nephrotic syndrome, but the number of cases remains quite small. We observed a case of progressive renal dysfunction and family history for end-stage kidney disease with a known single heterozygous ITSN2 variant. This study aimed to reveal the novel pathological significance of altered ITSN2 expression via a detailed examination.

Patient and Methods: A 52-year-old Japanese woman with mild proteinuria and hematuria visited our center. The patient did not opt for a detailed examination but was instead followed up with conservative treatment consisting of low-dose angiotensin receptor blockers. Serum Cr worsened from 1.15 to 1.79 mg/dL after 7 years when precise diagnosis was performed by renal biopsy and genetic testing.

Results: Kidney biopsy showed a thin basement membrane (TBM) and global glomerulosclerosis in 37.5% (6 out of 16) glomeruli examined. Comprehensive gene panel testing of 121 genes revealed a known ITSN2 variant, assumed to be involved in pathogenesis. No variants in the Alport syndrome genes, which are typically responsible for TBM, were detected.

Conclusion: A possible novel phenotype of the heterozygous ITSN2 variant was identified as a cause of hereditary renal failure. Further investigation of similar cases is required for a better understanding.

Introduction

Chronic kidney disease (CKD) is often irreversible, progressive, and is associated with a higher cardiovascular risk. In advanced stage, renal replacement therapy (RRT, hemodialysis, peritoneal dialysis, or kidney transplantation) is performed to increase chances of survival. End-stage kidney disease (ESKD) requiring RRT in Japan accounted for approximately 350,000 cases by the end of 2020¹⁾. Diabetic kidney disease, nephrosclerosis, chronic glomerulonephritis, and hereditary diseases of the urinary tract such as autosomal dominant polycystic kidney disease (ADPKD) are common causes of ESKD. Recent genetic analyses have revealed a great prevalence of inherited kidney diseases other than ADPKD, including COL4A-related nephropathy, autosomal dominant tubulointerstitial kidney disease (ADTKD), and congenital nephrotic syndrome²⁾.

ITSN2 is an adaptor and a scaffold protein with a unique multidomain structure. By binding to numerous proteins, they assemble multimeric complexes implicated in endocytic membrane trafficking and actin assembly machinery^3,4,5). ITSN2 variants have recently been reported to cause nephrotic syndrome^{6, 7)}. ITSN2 has two major splice variants: ITSN2-S and ITSN2-L. The DH domain of ITSN2-L has a specific activating effect on CDC42, a member of the Rho family of low-molecular-weight G proteins (Rho GTPases)^{4, 8)}. CDC42, together with RhoA and Rac1, is an important regulator of actin cytoskeleton in glomerular epithelial cells. Knockout of CDC42 specifically in glomerular epithelial cells of mice results in congenital proteinuria⁹⁾. Although the loss of function due to altered ITSN2 triggers dysfunction of CDC42, suggested as pathophysiological mechanism of nephrotic syndrome, the details are still unknown. We report a case of CKD with findings of a thin basement membrane (TBM) and a family history of dominant inheritance mode. Panel-based comprehensive genetic testing revealed a known single heterozygous variant in ITSN2 without typical causal variants in COL4A3/4A4/4A5 responsible for Alport syndrome (AS) / thin basement membrane disease (TBMD), which generally presents as the TBM phenotype. In addition, a possible mechanism of CKD development with TBM caused by the ITSN2 variant is discussed.

Case Report

A patient was referred for proteinuria, macroscopic hematuria, and mild renal disorder (unknown details) during a medical checkup at approximately 52 years of age. Three years later, she visited JA Toride Medical Center, with the laboratory results indicating a rise in serum Cr (1.15 mg/dL). During her visit, urinary protein was 0.26 g/day and only 1–4 red blood cells / high-power field (hpf) were observed in urine specimen. In addition, she had mild hyperuricemia, dyslipidemia, and fatty liver, but blood pressure was within the normal limits. She had a family history of kidney diseases. Her younger sister underwent renal transplantation for IgA nephropathy. Moreover, her father, three of his seven siblings, and two of the proband’s five cousins in their 40s–70s were on dialysis (Figure 1). She did not wish for a close examination, including kidney biopsy at that time; instead, she accepted conservative treatment with low doses of angiotensin receptor blocker, 25 mg/day of losartan potassium, and nutritional support. However, her renal function gradually deteriorated, even with well-controlled blood pressure within normal ranges. Medication was initiated for hyperuricemia after 1 year and for dyslipidemia after 3 years. Both were relatively well controlled, with 4–5 mg/dL uric acid levels, 70–120 mg/dL LDL cholesterol (150 mg/dL prior to renal biopsy), 35–50 mg/dL HDL cholesterol, and 250–350 mg/dL triglycerides. Seven years later, at age 59, her serum Cr level gradually rose to 1.79 mg/dL. After obtaining informed consent, the patient was admitted to the hospital for renal biopsy.

Figure 1.

Pedigree of the family showing end-stage kidney disease (ESKD) status.

HD: hemodialysis; DM: Diabetes mellitus. Numbers I, II, and III indicate generation.

On admission, the patient was 149 cm tall and weighed 62.1 kg with 27.6 kg/m² BMI. Her vital signs were as follows: pulse rate, 74 beats/min; blood pressure, 115/76 mm Hg; and oxygen saturation, 99% (room air). Routine physical examinations revealed no apparent abnormal findings on the body surface or in the cardiovascular, respiratory, and abdominal systems. The patient did not exhibit any symptoms of hearing loss.

As shown in Table 1, the patient had slight proteinuria (0.01 g/day) and microscopic hematuria (1–4 red blood cells/hpf). There was also no increase in NAG or β2 microglobulin levels, which is indicative of renal tubular damage. Besides mild anemia (hemoglobin, 11.7 g/dL) and azotemia (1.6 mg/dL of Cr and 41 mg/dL blood urea nitrogen), there were no other remarkable findings in routine screening. Abdominal echography revealed that the maximum axis of the right and left kidney was 102.7 and 97.5 mm, respectively, with no evidence of hydronephrosis.

Table 1. Laboratory data on admission.

Urine		Complete blood cell counts		Serum immunological test
Specific gravity	1.01	WBC	5,740 /μL	CRP	0.29 mg/dL
pH	5.5	Hb	11.7 g/dL	IgG	1,213 mg/dL
Protein	(−)	PLT	19.4 × 104/μL	IgA	147 mg/dL
	0.01 g/day	Serum biochemistry		IgM	222 mg/dL
Occult blood	(+)	TP	7.6 g/dL	C3	99 mg/dL
U-NAG	2.9 IU/L	Alb	4.8 g/dL	C4	I27.8 U/ml
β2MG	1,483 μg/L	BUN	41 mg/dL	Rheumatoid factor	<3.0 U/mL
Urine sediment		Cre	1.6 mg/dL	PR3-ANCA	<1.0 U/mL
WBC	<1 /HPF	Na	139.1 mEq/L	MPO-ANCA	<1.0 U/mL
RBC	1–4 /HPF	K	4.66 mEq/L	Antinuclear antibody	<40
		Ca	9.6 mg/dL	free light chain	(−)
		P	4.7 mg/dL	HBs-Ag	(−)
		UA	4.3 mg/dL	HCV-Ab	(−)
				T-SPOT	(−)

WBC: white blood cell; RBC: red blood cell; CRP: C-reactive protein.

Kidney biopsy

The renal biopsy specimen contained 16 glomeruli, six of which demonstrated global sclerosis (Figure 2a) and three were surrounded by interstitial fibrosis. Spike formation, increased mesangial cells, adhesions or glomerular crescent formation were not observed. The tubular dilation and interstitial fibrosis were moderate and sclerotic changes were prominent in the small arteries (Figure 2b). Immunofluorescence staining for IgG, IgA, IgM, C3c, C4, C1q, and fibronectin was negative. Electron microscopy revealed thinning of the basement membrane (155–233 nm width) (Figure 2c). There was no fusion or rupture in the basement membrane, and no remarkable foot process effacement in podocytes. A few high-density deposits were observed in the paramesangial regions.

Figure 2.

Histological slide of kidney tissue.

a) Periodic acid-Schiff (PAS) staining, ×40. Prominent global glomerulosclerosis observed in 37.5% (6 out of 16) glomeruli. Some glomeruli dropped out, showing mild atrophy of the interstitial tissue. b) Elastica van Gieson (EVG) staining, ×400. Fibrous thickening of the intima of the interlobular artery and mild proliferation of endothelial cells. c) Electron microscopy showing thinning of the wall of the glomerulus. Scale bar shows 1.0 μm.

Genetic testing

Because of the patient’s family history of ESKD and observed histology of TBM, genetic testing was planned to explore the possibility of AS/TBMD and other genetic disorders. The patient provided written informed consent for utilization of clinical information and DNA to identify genetic risk variants for kidney function, and for the results to be presented or published without her private information. This study was approved by the Institutional Review Board of Tokyo Medical and Dental University (approval number #G2000-080). Panel-based comprehensive genetic screening of 121 genes responsible for inherited kidney diseases was conducted¹⁰⁾. A single heterozygous variant of ITSN2 (NM 147152:c.1016C >G:p. Ser339Cys) was identified. This variant was previously reported as one of the homozygous variants responsible for inherent nephrotic syndrome by Ashraf et al⁷⁾. There were no other causal variants of COL4A5, COL4A4, COL4A3, MYH9, or LMX1B, which are known to cause a thin basement membrane phenotype.

Evaluation for systemic vascular lesions

Owing to the accelerated sclerosis of the kidney without an explainable cause, we conducted whole-body surveillance of the vascular system, including echocardiography of the carotid arteries, Ankle Brachial Pressure Index (ABI) analysis, Computed Tomography (CT) of the chest and abdomen, and brain Magnetic Resonance Imaging (MRI) aged 60–61. Bilateral carotid arteries showed multiple carotid plaques, with the maximum intima media thickness (maxIMT) of plaques in the right and the left carotid arteries being 1.5 mm and 1.9 mm, respectively. Regional calcification in the aortic arch, abdominal aorta, and right common iliac artery was found on the CT scan. Bilateral ABIs were in the normal range, and there were no abnormal findings on brain MRI.

Two years after the renal biopsy, her serum Cr level rose to 2.2 mg/dL and conservative and alimentary therapies were still continued.

Discussion

In 2018, ITSN2 gene variant was first reported as one of the causative genes of steroid-sensitive nephrotic syndrome (SSNS)⁷⁾. An analysis of 2,000 cases of nephrotic syndrome, including 800 SSNS, a homozygote of ITSN2 (p.Ser339Cys) was identified in SSNS cases from Arab consanguineous marriage families. In a collaborative study with Tohoku University, a sibling case of nephrotic syndrome with ITSN2 variant has been reported in Japan⁶⁾. In a previous case study, two compound heterozygous variants of frameshift deletion (p.Ile1214Serfs*2) and the same missense variant (p.Ser339Cys) as in this case were detected. Whether a single heterozygous variant possesses pathological meaning should also be considered carefully, since the clinical course may be different from the above reported cases. The patient did not manifest nephrotic syndrome, and the disease onset was in the middle of her life presumably. The pathological significance of the variant is classified as variant of unknown significance (VUS) [PM2/BP4] as per 2015 ACMG criteria, which is not a clear basis for pathogenic variants as well. Similar to other reports, Ashraf et al.⁷⁾ did not mention the details of kidney tissue findings. Although TBM was observed, considering that both sexes in this family exhibit equal incidence of ESKD, it was unlikely that the genetic cause was COL4A5 mutation (major cause of AS with X-linked inheritance). Furthermore, comprehensive genetic testing did not detect variants in AS/TBMD genes. Recent work has demonstrated that AS is mediated through the activation of mesangial actin dynamics with a Rac1/CDC42 cross-talk mechanism, resulting in invasion of the sub-capillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, which activates focal adhesion kinase in podocytes and causes the upregulation of pro-inflammatory cytokines and metalloproteinases driving the progression of glomerulonephritis¹¹⁾. Although little is known about the association between this signal and basement membrane thinning, it is possible that ITSN2 dysfunction has some form of morphological effect through the decreased activity of CDC42. Other typical hereditary kidney diseases with a few urinary findings and a certain family history of ESKD include autosomal dominant tubulointerstitial kidney disease (ADTKD) due to UMOD or MUC1 variants¹²⁾. However, in this case ADKTD was less likely because of the lack of pronounced tubular dilatation and interstitial fibrosis, the presence of atypical TBM in ADTKD, and the fact that genetic testing did not detect variants in causal genes. Ideally, to confirm genotype-phenotype correlations, genetic analysis for relatives with kidney dysfunction should be performed, but unfortunately, this could not be performed because they were already estranged from the proband.

Although 37.5% (in 6 of 16 glomeruli) global glomerulosclerosis was detected by histological findings, she had no previous history of hypertension. There was a possibility of renal damage caused by lifestyle-related diseases or obesity, but glomerular hypertrophy was less evident, and sclerotic changes in small arteries were more prominent than those expected from other risk factors. According to a previous report¹³⁾, from 16 (9–16 ranged) glomeruli obtained in biopsy at the age of 59 (55–59 range), the number of glomeruli with global sclerosis is expected to be 2, less than the six observed glomeruli in this case, suggesting accelerated renal atherosclerosis. In addition to the kidney, the patients maximum IMT of the carotid arteries was above normal (expected) range, along with aortic calcification. It was reported that max IMT in Japanese non-cardioembolic stroke patients under 65 years of age with dyslipidemia of 1.14 ± 0.29 mm was affected by age, smoking, serum HDL cholesterol levels, and diabetes mellitus¹⁴⁾. In the group with serum HDL cholesterol levels below 50 mg/dL, max IMT was 1.22 ± 0.3 mm¹⁴⁾. In this case, max IMT was 1.5 mm on the right and 1.9 mm on the left, much above expected levels considering her age and HDL cholesterol level. Calcification of the aortic arch was detected in 30.1–37.9%^15,16,17) and of the abdominal aorta in 33.0–37.8% of Japanese women aged between 60 and 69 years^{16, 17)}. Only 5.9% of Japanese aged 60–69 years (the same age category as the patient) had calcification of both the arch and abdominal aorta¹⁵⁾. In this case, regional calcifications were found in the aortic arch, abdominal aorta, and right common iliac artery, which also suggested accelerated atherosclerosis. Reports of marked vascular calcification in mice overexpressing CDC42¹⁸⁾ and that ITSN2 and CDC42 are involved in aging and atherosclerosis are on record^19,20,21). CDC42 activity was presumed to be reduced in this case, and it is possible that altered CDC42 activity affected atherosclerosis in the kidney. In addition, AS is complicated by focal segmental glomerulosclerosis (FSGS) lesions²²⁾. Although FSGS was not identified in this case, it is conceivable that fragility of basement membrane is associated with susceptibility to glomerulosclerosis.

Conclusion

We report the case of a 52-year-old woman with progressive renal dysfunction with TBM and conspicuous global glomerulosclerosis, and with a family history of ESKD. Comprehensive gene-panel testing revealed a known single heterozygous variant of ITSN2, assumed to be involved in pathogenesis. To elucidate the role of ITSN2 in kidney diseases, studies on more cases are warranted.