Biallelic cubilin pathogenic variants as a cause of « benign » proteinuria: implications for clinical management

INTRODUCTION

Discovering isolated proteinuria in a patient should lead to an appropriate work-up, including biological analysis, kidney ultrasound, and, in some cases, kidney biopsy. Proteinuria may be glomerular (mainly albuminuria with or without microscopic hematuria), tubular (low molecular weight proteins such as α1-microglobulin, retinol-binding protein, or β2-microglobulin), or both. Regardless of the cause of proteinuria, blockers of the renin-angiotensin system (RAS), such as angiotensin receptor blockers or angiotensin-converting enzyme (ACE) inhibitors, are usually prescribed to decrease proteinuria and therefore slow down kidney damage. Indeed, proteinuria is a well-established risk factor for progressive kidney disease^[1,2]. Increased glomerular capillary pressure may induce excessive filtration of plasma proteins and podocyte dysfunction. The increased reabsorption of plasma proteins by proximal tubular cells may be toxic and lead to their apoptosis and interstitial fibrosis^[1,3]. This paradigm recently changed with the discovery of pathogenic variants in the cubilin (CUBN) gene^[4] that may cause nondetrimental proteinuria. We report the case of two siblings with such variants and discuss their clinical management.

CLINICAL CASE

An 8-year-old girl of Turkish origin with no relevant medical history presented to the Pediatric Nephrology clinic for nocturnal enuresis. An initial check-up revealed isolated mild proteinuria (protein/creatinine ratio 1.2 g/g), consisting predominantly of albuminuria. The estimated glomerular filtration rate (eGFR) was normal, as was the ultrasound of the kidneys. A kidney biopsy was performed but it only contained a single glomerulus with a normal appearance by light microscopy. Immunofluorescence was negative. Enalapril was started at 2.5 mg/day. She remained on this medication for 15 years, and proteinuria remained stable at 0.7 g/g to 1.2 g/g despite an increase in enalapril dosage up to 15 mg/day. Enalapril was poorly tolerated, with frequent episodes of orthostatic hypotension. eGFR remained completely normal.

Her younger brother was also found to have moderate isolated proteinuria (0.9 g/g) during a check-up for a hyperactive bladder. His kidney biopsy showed eight normal glomeruli by light microscopy. Immunofluorescence was negative. Enalapril was started, and proteinuria remained stable at 0.6-0.9 g/g over the course of eight years, with preserved eGFR. Both parents have normal urinalysis without proteinuria. The first genetic testing was performed in 2018 in both siblings with a panel including 20 genes related to proteinuria. Results were not contributive, as a single variant of uncertain significance (VUS) of the ACTN4 (actinin alpha 4) gene was found in the sister but not in her brother. Updated genetic testing performed in 2022 [next generation sequencing (NGS) panel enriched for 40 genes important for proteinuric renal diseases]^[5] revealed in both siblings the presence of a pathogenic nonsense variant [American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) class 5] together with a potential splice-disrupting variant (ACMG/AMP class 3) in the CUBN (NM_001081.4):c.703 C > T (p.Arg235ter) and c.10363-3A > G (p.?). CUBN is dominated by 27 contiguous CUB domains, and variants occurring within the C-terminal (after the CUB8 domain) are usually associated with isolated proteinuria. Here, p.R235* led to truncation before CUB8, but none of the patients presented with low levels of vitamin B12 or megaloblastic anemia. Enalapril was therefore discontinued in both patients.

DISCUSSION

The proximal tubule reabsorbs large amounts of low-molecular-weight proteins but also albumin and electrolytes from the glomerular filtrate. Megalin (LRP2), CUBN, and amnionless (AMN) protein are located in the apical part of proximal tubular cells and are responsible for receptor-mediated endocytosis of proteins filtered through the glomerular barrier^[3]. Cubilin has been shown to have an essential role in albumin reabsorption and is encoded by the CUBN gene [Figure 1]^[6]. Biallelic pathogenic variants in the CUBN gene cause Imerslund-Gräsbeck syndrome (OMIM 261100), also called selective vitamin B12 (cobalamin) malabsorption with proteinuria^[6]. In this syndrome, which results in megaloblastic anemia responsive to parenteral vitamin B12 therapy, half of the patients present with mild proteinuria and normal eGFR. The megaloblastic anemia arises from a defect in the receptor for the vitamin B12-intrinsic factor complex in ileal enterocytes. CUBN and AMN proteins represent the two subunits of this receptor. In patients with Imerslund-Gräsbeck syndrome, proteinuria can persist for decades^[7,8]. Most pathogenic variants in CUBN are located in the N-terminal half of the gene [Figure 2].

Figure 1. Albumin reabsorption in proximal tubular cells. Cubilin is located in the apical part of proximal tubular cells and is responsible for receptor-mediated endocytosis of albumin filtered through the glomerular barrier. After the decoupling of cubulin-albumin ligation, albumin is released from the basolateral cell surface into the circulation.

Figure 2. Cubilin protein structure. Cubilin (CUBN) is a 460 kDa glycoprotein with no transmembrane domain. It acts as a receptor for intrinsic factor-vitamin B12 complexes. There are 27 CUB domains. The intrinsic factor and the vitamin B12 binding region are located in domains 5 to 8. The precise location of the binding sites for albumin remains unclear, but it is supposed to be in the CUB domains near the C-terminal area. IGS: Imerslund-Grasbëck syndrome; IF: intrinsic factor.

Recently, biallelic pathogenic variants in the C-terminal domain of CUBN were described as leading to isolated chronic proteinuria^[4]. Indeed, Bedin et al.^[4] identified 39 patients with biallelic CUBN variants among 2,216 individuals with suspected genetic kidney disease, including proteinuric patients. Proteinuria ranged from 0.5-3 g/day with an average age at discovery of 10.9 years. When measured, albuminuria represented more than half of proteinuria, and β2-microglobulin urine levels were low or absent. Kidney biopsies were available in 19 patients and did not show any specific lesion in 11 patients. Four kidney biopsies underwent electron microscopy (EM) evaluation; two were normal and two revealed glomerular synechiae. The use of ACE inhibitors did not lower proteinuria, which remained stable over the years. eGFR was normal in all patients, even those older than 50 years. Bedin et al.^[4] also identified a phenotype-genotype correlation. Indeed, variants located after the CUB8 domain (included in the vitamin B12/intrinsic factor binding region) lead to isolated proteinuria, whereas variants located before the CUB8 domain result in Imerslund-Gräsbeck syndrome, a finding suggesting that there are separate binding sites in cubilin for vitamin B12-intrinsic factor (VitB12-IF) and albumin; however, the precise location of the binding sites for albumin remains unclear [Figure 2]. The latter should thus bind to more carboxy-terminal CUB domains. However, the isolated proteinuria caused by the p.R235* variant located before CUB8 and leading to premature truncation of cubilin illustrates how it remains complex to determine with certainty the resulting phenotype. In addition, four specific C-terminal variants have previously shown strong associations with albuminuria in genome wide association (GWAS)^[9-13]. These CUBN variants were associated with higher eGFR in the study by Bedin et al.^[4].

In another recent cohort, Domingo-Gallego et al.^[14] identified 15 patients with mild proteinuria (0.5-1.8 g/day) having homozygous or compound heterozygous pathogenic variants in the C-terminal region of the CUBN protein. In most cases, proteinuria was detected incidentally, as in our patients. They confirmed the glomerular nature of proteinuria, normal kidney histology, lack of response to RAS blockade, and preserved eGFR in adulthood. Six children from Turkey were also identified with biallelic CUBN pathogenic variants located in the C-terminal domain of the protein^[15]. One child had a second kidney biopsy three years after the first normal kidney biopsy. This second biopsy revealed one area of periglomerular fibrosis among 27 glomeruli. Yang et al.^[16] also reported glomerulosclerosis and effacement of foot processes in podocytes on EM in three children with CUBN pathogenic variants. The authors suggested a role for podocyte dysfunction together with a defect in the reuptake of albumin in the proximal tubule of patients with CUBN mutations. These data need to be confirmed. Indeed, these structural changes in podocytes were absent in two children on EM^[17]. These authors showed that CUBN variants induce changes in the scaffolding capabilities of cubilin protein in vitro. These changes reduce the interactions between CUB and AMN, leading to an aberrant localization of AMN in the cytoplasm of proximal tubular cells instead of the cell membrane. This may interrupt the receptor-mediated endocytosis that re-uptakes filtered albumin.

In conclusion, in the absence of functional cubilin in proximal tubular cells, albumin reabsorption is incomplete, and this leads to mild albuminuria. This mechanism acts downstream of the glomerular barrier, and does not affect the intraglomerular pressure, and is thus not expected to damage podocytes. This explains why anti-proteinuric agents such as ACE inhibitors do not succeed in lowering this particular proteinuria. Detection of CUBN pathogenic variants is crucial in clinical nephrology because it prevents unnecessary kidney biopsies as well as the use of RAS blockers and their potential side effects, such as symptomatic hypotension or, rarely, angioneurotic edema. The benign course of this disease needs to be confirmed by longer-term follow-up.