Clinical and molecular characterization of a family with a dominant renin gene mutation and response to treatment with fludrocortisone

Abstract

Background: A family was identified with autosomal dominant inheritance of anemia, polyuria, hyperuricemia, and chronic kidney disease. Mutational analysis revealed a novel heterozygous mutation c.58T > C resulting in the amino acid substitution of cysteine for arginine in the preprorenin signal sequence (p.cys20Arg) occurring in all affected members.

Methods: Effects of the identified mutation were characterized using in vitro and in vivo studies. Affected individuals were clinically characterized before and after administration of fludrocortisone.

Results: The mutation affects endoplasmic reticulum co-translational translocation and posttranslational processing, resulting in massive accumulation of non-glycosylated preprorenin in the cytoplasm. This affects expression of intra-renal RAS components and leads to ultrastructural damage of the kidney. Affected individuals suffered from anemia, hyperuricemia, decreased urinary concentrating ability, and progressive chronic kidney disease. Treatment with fludrocortisone in an affected 10-year-old child resulted in an increase in blood pressure and estimated glomerular filtration rate.

Conclusions: A novel REN gene mutation resulted in an alteration in the amino acid sequence of the renin signal sequence and caused childhood anemia, polyuria, and kidney disease. Treatment with fludrocortisone improved renal function in an affected child. Nephrologists should consider REN mutational analysis in families with autosomal dominant inheritance of chronic kidney disease, especially if they suffer from anemia, hyperuricemia, and polyuria in childhood.

Figure 1

Pedigree and DNA analysis. A: Family pedigree. Black symbols denote affected individuals, open symbols denote unaffected individuals. Analyzed STR (short tandem repeats) markers, corresponding genotypes and reconstructed haplotypes are provided for each individual. B: Chromatograms showing genomic DNA sequence of the REN exon 1 in control and a heterozygous transition c.58T > C in patient AIII2.

Figure 2

Estimated glomerular filtration rate vs. age in affected family members.

Figure 3

Renal biopsy (Hematoxylin and eosin stain). Kidney biopsy of patient AII6 reveals patchy interstitial fibrosis accompanied with focal chronic inflammatory infiltrate, and focal tubular atrophy with markedly thickened and duplicated basement membranes (insert, periodic acid-Schiff). Focal proximal tubules showed hypertrophy. Only one glomerulus was present and showed periglomerular fibrosis but no other alterations by light microscopy.

Figure 4

Ultrastructural examination of kidney biopsy specimen from patient AII6. A: Glomerulus with segmentally thickened and homogenous basement membranes. Segmental podocyte foot process effacement was present. B: Epithelial cells of proximal tubule demonstrating distention of basal membrane invaginations and thickening of tubular basement membrane. C: Distal tubule with marked distention of the intercellular spaces between epithelial cells and thickening of tubular basement membrane. D: Proximal tubule showing epithelial cell atrophy without any significant reduction of the brush border. Thickening of tubular basement membrane and distention of basal membrane invaginations are also present.

Figure 5

Bioinformatic and in vitro characterization of mutant renin. A: Amino acid conservation of renin signal sequence across higher mammals. B: Hydrophobicity scores for the wild type and mutated preprorenin signal peptides. C: Nascent WT^REN and C20R^REN proteins translated in vitro from corresponding mRNAs in nuclease-treated rabbit reticulocyte lysate in the absence (–) or presence (+) of rough endoplasmic reticulum microsomes (RM) and tripeptide glycosylation acceptor (AP). Without RM and AP only nascent preprorenin (PreProREN) is formed (lanes 1 and 4). With RM and in the absence of AP most of the ER-translocated WT^REN is converted into fully glycosylated prorenin (ProREN); (lane 2) whereas C20R^REN produces only minute amounts of ProREN (lane 5). With RM and AP, the glycosylation of the ER-translocated WT^REN is inhibited and partially glycosylated intermediates are present (lane 3); C20R^REN, which is not ER-translocated, is present as non-glycosylated preprorenin.

Figure 6

Transient expression of WT^REN and C20R^REN preprorenin in HEK-293 cells. A: Western blot analysis of the protein products in cell lysates and medium showing that C20R^REN produces only minute amounts of normally glycosylated, proteolytically processed and secretory competent prorenin (ProREN). To distinguish proteolytic processing and glycosylation status, the expressed proteins were analyzed before (–) and after (+) deglycosylation with PNGase. B: Fluorescence-activated cell sorter analysis showing inability of the C20R^REN to form cytoplasmic granules. The values represent means ± s.d. of the three transfection experiments carried out in triplicates. The differences between the number of granular cells expressing the WT^REN and cells expressing either the C20R^REN or empty vector were statistically significant (p < 0,001). C,D: Cellular localization of preprorenin, prorenin and renin in HEK293 cells detected using antibodies recognizing amino acid residues 288 – 317 of the preprorenin (Yanaihara, Shizuoka, Japan). C: WT^REN is present in a form of fine cytoplasmic granules. D C20R^REN protein is retained intracellularly and shows intense diffuse cytoplasmic staining.

Figure 7

Immunohistochemical analysis of selected RAS components and UMOD performed in kidney biopsy from Patient AII6. Comparison with age matched control. A: Angiotensin converting enzyme 1 (ACE 1) expression at the apical pole of proximal tubules in a control kidney. B: ACE 1 positivity almost comparable with the control in partly atrophic proximal tubules in patient’s kidney. C: Angiotensin II (ANG II) positivity at the apical pole of proximal tubules and in tubular fluid in a control kidney. D: Slightly decreased ANG II staining intensity in patient’s kidney revealing also tubular atrophy and interstitial fibrosis. E: Control kidney characterized by strong uromodulin (UMOD) expression in TALH, with some variability of staining intensity. F: Markedly decreased UMOD positivity in areas of pronounced tubulointerstitial changes in patient’s kidney. Maximal UMOD expression was seen at the apical pole of TALH in both, patient and control.