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. 2009 Aug;85(2):204-13.
doi: 10.1016/j.ajhg.2009.07.010. Epub 2009 Aug 6.

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure

Martina Zivná  1 , Helena Hůlková, Marie Matignon, Katerina Hodanová, Petr Vylet'al, Marie Kalbácová, Veronika Baresová, Jakub Sikora, Hana Blazková, Jan Zivný, Robert Ivánek, Viktor Stránecký, Jana Sovová, Kathleen Claes, Evelyne Lerut, Jean-Pierre Fryns, P Suzanne Hart, Thomas C Hart, Jeremy N Adams, Audrey Pawtowski, Maud Clemessy, Jean-Marie Gasc, Marie-Claire Gübler, Corinne Antignac, Milan Elleder, Katja Kapp, Philippe Grimbert, Anthony J Bleyer, Stanislav Kmoch
Affiliations

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure

Martina Zivná et al. Am J Hum Genet. 2009 Aug.

Abstract

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

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Figures

Figure 1
Figure 1
Pedigrees, Linkage and DNA Analysis (A) Pedigrees of the investigated families. Black symbols denote affected individuals; open symbols denote unaffected individuals; and gray symbols denote individuals in whom clinical, biochemical, and genetic investigations were not yet performed. (B) A whole-genome parametric linkage analysis showing a single statistically significant region on chromosome 1q41 detected in family A. (C) Chromatograms showing genomic DNA sequence of the REN exon 1 in control and a heterozygous deletion c.45_47 delGCT in patient from family A. (D) Chromatograms showing genomic DNA sequence of the REN exon 1 in control and a heterozygous mutation c.47T>G in patient from family C.
Figure 2
Figure 2
Renin Immunohistochemistry and Nephropathology Family A, patients DIV3, DIV7, and DII1. (A–C) Renin expression in control kidney. (A) Control aged 7 years; renin expression in JG cells and individual cells of collecting ducts. (B and C) Adult control; renin staining (B) in JG cells and (C) in renal cortical tubules where the signal is restricted to individual cells of the collecting ducts. (D and E) Kidney biopsies in an early disease stage. Patient DIV3 (D) and patient DIV7 (E) shown. The common denominator is a strong reduction of renin signal in the JG apparatus (marked by arrows) and its absence in the surrounding tubules. Insert in (E) shows glomerulus in detail. (F) Patient in advanced stage of kidney disease (DII1). Renin staining is absent in both JG apparatus and tubular epithelium even in relatively well-preserved regions. (G–I) Renin and prorenin expression inside the wall of small size renal vessels, probably in a sub/endothelial localization, more prominent in adult patient DII1 (the main pictures) than in patients in an early stage of the disease (inserts). Preprorenin antibody detecting prorenin and renin (G), monoclonal antibody detecting active renin (H), and polyclonal antibody detecting prorenin (I). Insert in (G) demonstrates this phenomenon in patient DIV7, inserts in (H) and (I) in patient DIV3. (J–L) Nephropathology in early stage of the disease (HE staining). (J) Morphology in patient DIV3 was dominated by irregular dystrophic changes in tubular epithelium mainly in proximal tubules (coarsely vacuolated or granular cytoplasm) and focal segmental sclerosis of glomerular tufts with adhesions to Bowman's capsule. Tubular atrophy and interstitial fibrosis was less expressed (sampling?). (K and L) Kidney biopsy from patient DIV7 demonstrated more pronounced glomerular sclerosis and hyalinosis (1 out of 8 glomeruli was totally sclerosed, not shown) and focal tubular atrophy accompanied with moderate interstitial fibrosis. Glomeruli, marked as Gs, in various degrees of sclerosis and areas of tubulointerstitial fibrosis are shown. In advanced stage of the disease (patient DII1, not shown), morphology of progressive nephropathy was modified by haemodialysis lasting for 1 year.
Figure 3
Figure 3
Bioinformatic Analysis of the Preprorenin (A) Diagram of the preprorenin sequence showing the locations of the identified mutations and epitopes recognized by prorenin (amino acid residues 21–64) and preprorenin (amino acid residues 288–317) antibodies used in this study. (B) Homology of the mutant and wild-type human preprorenin signal sequences with those of higher mammals. (C) Hydrophobicity plot of the WTREN, ΔL16REN, and L16RREN signal sequences calculated via the Kyte and Doolittle method and scale.
Figure 4
Figure 4
Functional Studies (A) Western blot analysis of WTREN, ΔL16REN, and L16RREN proteins transiently expressed in HEK293 cells. Products of biosynthesis—preprorenin (PreProREN) and prorenin (ProREN)—were analyzed in cell lysates and cell culturing medium. To distinguish proteolytic processing and glycosylation status, the proteins were always analyzed before (−) and after (+) deglycosylation with PNGase. (B and C) In vitro translation and translocation. (B) Nascent WTREN, ΔL16REN, and L16RREN proteins translated from corresponding mRNAs in nuclease-treated rabbit reticulocyte lysate in the absence (−) or presence (+) of rough endoplasmic reticulum microsomes (RM). Without RM, only nascent preprorenin (PreProREN) is formed. With RM, the translocated preprorenin is converted into prorenin (ProREN). In comparing lane 2 to lane 4 (as well as lanes 2 and 4 to lanes 6 and 8 in C), one can see that significantly more WTREN is translocated into the RM and converted to prorenin than with the ΔL16REN mutant. The difference in translocation efficiency between WTREN and ΔL16REN was assessed by densitometry. The L16R mutation completely prevents translocation and L16RREN protein is present as preprorenin. (C) Translation/translocation assay performed in the presence of RM and in the absence (−) or presence (+) of the signal peptide peptidase inhibitor (ZZ-L)2ketone. Upon centrifugation, prorenin as well as out cleaved signal peptide were present in RM pellet fractions (p), whereas preprorenin that does not translocate is found in supernatant (s). The inhibitor only slightly affected preprorenin signal peptide processing (lanes 4 and 8). Compared to ΔL16REN, the WTREN signal peptide is evidently more stable and only slightly affected by signal peptide peptidase-mediated processing. (D and E) Prorenin and renin produced by stably transfected HEK293 cells. Active renin (REN) and trypsin activated total renin + prorenin (ProREN+REN) amounts measured in (D) lysates and (E) medium of the corresponding cell lines. The values represent means ± SD of the measurements performed in two independent clones for each of the constructs. The individual measurements were carried out in triplicate. The statistical significance of the differences between WTREN and ΔL16REN protein amounts was tested by t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. 0REN is an antibiotic-selected cell line originally transfected with WTREN construct, but showing later no renin expression by RT-PCR and western blot analysis. (F) Renin secretion from living stably transfected HEK293 cell lines. The fluorescent signal is released from 5-FAM and QXL520 conjugated renin substrate and corresponds to the activity of renin secreted in the medium. (G and H) Reduced growth rate (G) and activated XBP1 splicing (H) indicating ER stress in ΔL16REN-expressing cells. (I and J) Electron microscopy of stably transfected HEK293 cell lines showing (I) overview of the WTREN -expressing cell with numerous electron-dense granules (arrows) and (J) considerable distensions of ER cisternae (asterisk) observed frequently in ΔL16REN cells. Detail of one of the autophagosomal structures is shown in the insert. These structures were not present in 0REN cells (data not shown).
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