Dissection of a genetic locus influencing renal function in the rat and its concordance with kidney disease loci on human chromosome 1q21

Abstract

Previously, we conducted a genome scan on a population derived from the Dahl salt-sensitive hypertensive (S) and the spontaneously hypertensive rat (SHR) using urinary albumin excretion (UAE) as our primary measure of renal function. We identified 10 quantitative trait loci (QTL) linked to several renal and/or cardiovascular traits. In particular, linkage and subsequent congenic strain analysis demonstrated that the loci on chromosome 2 had a large and significant effect on UAE compared with the S rat. The present work sought to characterize the chromosome 2 congenic strain [S.SHR] by conducting a time-course analysis (week 4-20), including evaluating additional renal parameters, histology, electron microscopy, and gene expression/ pathway analysis. Throughout the time course the congenic strain consistently maintained a threefold reduction in UAE compared with S rats and was supported by the histological findings of significantly reduced glomerular, tubular and interstitial changes. Gene expression/pathway analysis performed at week 4, 12, and 20 revealed that pathways involved in cellular assembly and organization, cellular movement, and immune response were controlled differently between the S and congenic. When all the data are considered, the chromosome 2 congenic appears to attenuate renal damage primarily through an altered fibrotic response. Recombinant progeny testing was employed to reduce the QTL to approximately 1.5 cM containing several interesting candidate genes. The concordance of this rat QTL with renal disease loci on human chromosome 1q21 demonstrate that elucidating the causative gene and mechanism of the rat QTL may be of particular importance for understanding kidney disease in humans.

Fig. 1

Time course for urinary protein excretion (UPE, A); urinary albumin excretion (UAE, B); and systolic blood pressure (SBP, C) in male Dahl salt-sensitive (S), chromosome 2 congenic strain [S.SHR(2)], spontaneously hypertensive (SHR) rats. A schematic of the congenic strain is shown in A, inset. The black bar designates the extent of introgressed SHR alleles on the S background. The open region on each end of the congenic segment represents the recombination interval. In A, the dashed line represents the threshold for “proteinuria” (>20 mg/24 h). Rats were maintained on low-salt diet (0.3% NaCl) for entire course of the experiment. The number of animals at each time varied from n = 50 per group (week 4) to n = 12 per group (week 20). SBP was measured by telemetry (n = 8 per group). Mean values ± SE.

Fig. 2

Time-course histological examination of male S, S.SHR(2), and SHR rats. Percent glomerular injury (A), percent interstitial injury (B), tubular injury score (C), vascular injury score (D). Bar graphs show the effect of the S.SHR(2) congenic (C) strain compared with both parental strains, S and SHR. Glomerular and interstitial injury were evaluated quantitatively (see MATERIALS AND METHODS). Tubular and vascular injury was graded on a semiquantitative scale from 0 (normal) to 4 (severe). Kidneys from 6 animals were evaluated at each time point. ND, not detectable. a, Significantly different from S.SHR(2) and SHR at P < 0.05; b, significantly different from S and S.SHR(2) at P < 0.05. P values are from a 1-way analysis of variance followed by post hoc multiple comparisons using Tukey’s test. Error bars are SE.

Fig. 3

Representative light [×40 hematoxylin & eosin (H&E), ×20 Masson’s trichrome] and electron microscopy (EM, ×3,000) images of week 20 low-salt kidney samples from S, S.SHR(2), and SHR. S rats exhibit glomerulosclerosis and mesangial expansion (thin arrow), arterial wall thickening (open arrow), protein casts (PC), and significant fibrosis (thick arrow) compared with S.SHR(2) and SHR. Kidneys from SHR are essentially normal. *Normal arterial vessel. EM of S kidney illustrated significant glomerular disease including evidence of effacement of foot processes, focal mesangial matrix increase, and diffuse mesangial electron-dense deposits. The S.SHR(2) congenic demonstrated significantly less pathology compared with the S. The SHR appeared essentially normal, although there was some evidence of minimal effacement of foot processes (considering all data).

Fig. 4

Fine mapping of renal function quantitative trait loci (QTL) using recombinant progeny testing (RPT). The physical map of rat chromosome 2 is shown on the left. The solid bars to the right of the physical map indicate the extent of the SHR-donor regions for each recombinant family. The open region represents the recombination interval. UPE was measured at week 6. The solid black bar denotes the location of the QTL based on the RPT. The 95% confidence interval (CI) obtained from the original linkage analysis (20) and the present study is shown to the right of the figure. Red bars denote that recombinant animals (congenic-like) had significantly (P < 0.05) lower UPE compared with nonrecombinant (S-like) littermates. Yellow bars denote there was not a significant difference in UPE between recombinant and nonrecombinant littermates. The number of animals tested from each recombinant family ranged from 16 to 29.

Fig. 5

Enlargement of the QTL region and the 4 recombinant families important in delimiting the QTL region. The physical map of rat chromosome 2 is shown on the left, with an enlargement of the physical map of the QTL region to the right. Map distances are in base pairs (www.ensembl.org, Ensembl v38, Apr 2006). UPE was measured at week 6 and week 12. Data for “UPE Effect” below each bar are UPE of recombinant rats (congenic-like) minus the UPE of nonrecombinant (S-like) littermates. A negative value indicates that recombinant rats had lower UPE than nonrecombinant rats, and when this is significant this indicates that the QTL is in the congenic interval. The numbers of animals tested are shown below the effect. P values are from an independent t-test. *P < 0.0001; NS is not significant. Data are means ± SE.

Fig. 6

Comparative map showing overlap of renal susceptibility loci between rat and human. The physical map of the rat QTL is shown on the left. The region in human that is homologous to the rat QTL lies on both human chromosome 1 and 4. Map distances are in base pairs (www.ensembl.org, Ensembl v38, Apr 2006). Adult onset nephropathy and hypertension (10); MCKD1, medullary cystic kidney disease (30, 56, 58); and creatinine clearance (HyperGen study) (14).