FIBRONECTIN glomerulopathy is a rare, autosomal dominant, non-amyloid glomerular disease. It was firstly reported by Burgin and his colleagues in 1980.1 Thereafter, a few case reports were published describing familial and sporadic patients with fibronectin glomerulopathy.2-5 It usually onsets in adolescence with proteinuria, microhematuria, hypertension, and slowly progressive renal failure.In pathology, it is characterized by nonimmune lobular glomerulopathy with massive mesangial and subendothelial deposits composed of fibronectin. In 2008,Castelletti et al6 identified three mutations in the gene encoding fibronectin 1 (FN1) in the different pedigrees of fibronectin glomerulopathy.

In this article, we reported a Chinese patient of fibronectin glomerulopathy, which was confirmed by kidney biopsy. Genetic sequencing of FN1 detected a previously described heterozygous mutation causing a substitution of the tyrosine at amino acid 973 by cysteine (Y973C) in the affected patient.

CASE DESCRIPTION

A 29-year-old woman was hospitalized due to systemic edema and hypertension. She had cerebral infarction three years ago in past medical history. In family history, her father had diabetes and hypertension. Findings of physical examination included obesity,elevated blood pressure of 150/90 mm Hg and peripheral edema. Urinalysis revealed massive proteinuria and microscopic hematuria with bland sediment, and 24-hour urine protein was 15.43 g. Serum albumin and creatinine levels were 28 g/L and 79.6 μmol/L ［corresponding to estimated glomerular filtration rate (eGFR)of 86 ml/min·1.73 m2 calculated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation］, with hypercholesterolemia. Antinuclear antibody and anti-double stranded DNA were negative, as well as hepatitis serologic test. No hypocomplementemia or cryoglobulinemia was detected. Serum protein electrophoresis did not show monoclonal protein. Enlarged kidneys (left was 13.4 cm, and right was 13.2 cm in long diameter) were found in ultrasonography.

Light microscopy revealed enlarged and hyperlobular glomeruli with a homogeneous material in the mesangium and subendothelium (Fig. 1A). There was mild mesangial hyperplasia, without endocapillary or extracapillary proliferation, necrosis, or inflammatory infiltration. The deposited material was period acid-Schiff positive (Fig. 1B) and methenamine silver negative (Fig. 1C). Negative result of Congo red staining excluded amyloidosis.

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Immunofluorescence microscopy was negative for immunoglobulin, light chain, or complement components. Electron microscopy showed massive electron-dense deposits in the mesangial and subendothelial space, with some admixture of irregularly arranged fibrils (15 nm in width) (Fig. 1D and 1E). Indirect immunofluorescence staining for fibronectin was performed, and glomerular deposits stained brightly with monoclonal anti-fibronectin antibody (Fig. 1F).

关键技术：一是从铁矿石中经济有效地回收低品位共伴生的钼，解决了铁钼型矿石选钼的技术难题，首次在工业上实现从铁钼型矿石中回收低品位钼并获得合格钼精矿；二是采用先磁后浮的选别顺序，在选铁的同时实现钼的预富集；三是在采用传统的水玻璃分散矿泥并抑制脉石的基础上，用调整剂组合抑制其他矿物并减免离子对钼浮选的不利影响；四是选钼所用的药剂为无毒或低毒的常用浮选药剂，对环境无不利影响；五是在两次精选后在流程中加入粗精矿再磨—擦洗作业，为再次经多次精选获得合格产品创造条件。

In conclusion, we presented a rare case of fibronectin glomerulopathy in China. This case attaches the importance to electron microscopy and anti-fibronectin antibody staining when nonimmune, lobular glomerulopathy with a massive homogeneous material deposit in the mesangium and subendothelium is observed. The identification of mutations in FN1 is an advance in understanding of the molecular genetic mechanisms of fibronectin glomerulopathy.

Figure 1. Pathological changes of kidney with fibronectin glomerulopathy. A. Glomeruli were diffusely enlarged with lobular accentuation and minimal hypercellularity (arrows). (HE staining) B. Period acid-Schiff-positive material expanding the mesangium and subendothelium of an enlarged glomerulus (arrows). C. The deposits in the subendothelium and mesangium (arrows) failed to stain with silver. (Jones methenamine silver stain) D. Electron microscopy showed mesangial and subendothelial deposits (arrows). E. The electron dense deposits as well as 15 nm fibrils (arrows) arranged randomly were observed. F. Intense immunofluorescence staining showed glomerular deposits with antisera against fibronectin ［primary antibody: mouse monoclonal anti-human fibronectin antibody, clone IST-4 (Sigma-Aldrich); secondary antibody: fluorescin isothiocynanate-conjugated goat anti-mouse］. (×200)

Figure 2. Mutation 2918A→G in the fibronectin gene. The sequencing electropherogram that shows the heterozygous mutation c.2918A→G (reference single-nucleotide polymorphism identifier rs137854488) causing a substitution of tyrosine at amino acid 973 by cysteine in the patient.

DISCUSSION

Fibronectin is a multifunctional extracellular matrix glycoprotein involved in cellular adhesion, migration, and cytoskeleton maintenance. It normally exists in a cellular form and a plasma soluble form. The deposit in kidneys of fibronectin glomerulopathy is mainly derived from circulating fibronectin,2 although plasma fibronectin levels are not elevated in these patients.

Functional studies using recombinant proteins suggest that mutations in FN1 impair the process of the assembly of fibronectin into fibrils and the balance between soluble and insoluble form of fibronectin.6 Y973C mutation affects the Hep-Ⅲ heparin-binding domain of fibronectin, located in the fourth and fifth of fibronectin’s type Ⅲ (Ⅲ4-5) repeats. Hep-Ⅲ has the ability to modulate the cytoskeletal response and induce intracellular signaling.9 Y973C mutation introduces an additional cysteine in the fourth type Ⅲ repeat, which might affect protein folding and function through the formation of abnormal disulfide bonds. Although mutations in FN1 may be necessary for fibronectin glomerulopathy to develop, they are not sufficient since not all family members with the mutation develop clinical kidney disease.

Castelletti et al6 sequenced FN1 and found that heterozygous FN1 mutations constituted the underlying genetic abnormality in 6 of 15 unrelated pedigrees(40%). Three heterozygous missense mutations were detected, Y973C, W1925R and L1974R that co-segregated with fibronectin glomerulopathy. The Y973 mutation was found in four pedigrees. The four families were of different ethnic origin and did not share a disease haplotype, thus excluding a founder effect.A heterozygous Y973C mutation was found in our patient. However, the mutation of FN1 was not detected in her parents. Thus, she could be a sporadic case of fibronectin glomerulopathy. Ishimoto et al7 reported a Japanese case of sporadic, elderly-onset fibronectin glomerulopathy, but did not find any missense mutation of FN1 previously reported. In 2015, a retrospective study of ten Chinese patients of fibronectin glomerulopathy showed that only one case had a family history of renal disease.8 However, the mentioned study in China did not sequence FN1 to investigate the potential mutation. Our patient was the first case reported with Y973C mutation in China.

Anti-hypertensive therapy including angiotensin II receptor antagonist, calcium channel blocker, α and β receptor blocker was given as well as statin for hyperlipidemia. At her last evaluation 12 month after diagnosis, serum creatinine level was 86.6 μmol/L (eGFR 81 ml/min·1.73 m2) while serum albumin level was 30 g/L. 24-hour urine protein was unchanged.

The diagnosis of fibronectin glomerulopathy was established on the basis of renal pathology. As an additional investigation, we performed genetic sequencing of FN1 in the patient and her parents. Genomic DNA was extracted from peripheral blood. The whole exons of FN1 were screened by Sanger sequencing (Sangon Biotech Co., Ltd, Shanghai, China). DNA sequencing showed a previously described heterozygous mutation in exon 19 in which an adenine is substituted by a guanine at nucleotide 2918 of the complementary DNA(c. 2918 A＞G) (Fig. 2), leading to replacement of a tyrosine at amino acid 973 by a cysteine (Y973C). No FN1 mutation was detected in her parents.

Aknowledgements

The authors thank Yan Li and Lin Duan, from Laboratory of Nephrology Department, Peking Union Medical College Hospital, for performing immunofluorescence staining for fibronectin, as well as Ren Wei, from Peking Union Medical College, for interpretation of genetic sequencing results.

在三国时期，筝作为一个非常优雅的乐器，在当时引起了学者、隐士和文人普遍的高度关注。直到东汉末年，古筝才逐渐登上高雅殿堂，社会开始广泛认可。

Conflicts of interest statement

The authors have no conflicts of interest to disclose.

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9. Quecine MC, Leite TF, Bini AP, et al. Label-free quantitative proteomic analysis of puccinia psidii uredospores reveals differences of fungal populations infecting eucalyptus and guava. PLoS One 2016; 11(1):e0145343. doi: 10.1371/journal. pone. 0145343.