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.

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.

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 WT^REN, ΔL16^REN, and L16R^REN signal sequences calculated via the Kyte and Doolittle method and scale.

Functional Studies
(A) Western blot analysis of WT^REN, ΔL16^REN, and L16R^REN 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 WT^REN, ΔL16^REN, and L16R^REN 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 WT^REN is translocated into the RM and converted to prorenin than with the ΔL16^REN mutant. The difference in translocation efficiency between WT^REN and ΔL16^REN was assessed by densitometry. The L16R mutation completely prevents translocation and L16R^REN 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 ΔL16^REN, the WT^REN 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 WT^REN and ΔL16^REN protein amounts was tested by t test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. 0^REN is an antibiotic-selected cell line originally transfected with WT^REN 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 ΔL16^REN-expressing cells.
(I and J) Electron microscopy of stably transfected HEK293 cell lines showing (I) overview of the WT^REN -expressing cell with numerous electron-dense granules (arrows) and (J) considerable distensions of ER cisternae (asterisk) observed frequently in ΔL16^REN cells. Detail of one of the autophagosomal structures is shown in the insert. These structures were not present in 0^REN cells (data not shown).