Transfer of chromosome 13 from the Brown Norway (BN) rat onto the Dahl salt-sensitive (SS) genetic background attenuates the development of hypertension, but the genes involved remain to be identified. The purpose of the present study was to confirm by telemetry that a congenic strain [SS.BN-(D13Hmgc37-D13Got22)/Mcwi, line 5], carrying a 13.4-Mb segment of BN chromosome 13 from position 32.4 to 45.8 Mb, is protected from the development of hypertension and then to narrow the region of interest by creating and phenotyping 11 additional subcongenic strains. Mean arterial pressure (MAP) rose from 118 ± 1 to 186 ± 5 mmHg in SS rats fed a high-salt diet (8.0% NaCl) for 3 wk. Protein excretion increased from 56 ± 11 to 365 ± 37 mg/day. In contrast, MAP only increased to 152 ± 9 mmHg in the line 5 congenic strain. Six subcongenic strains carrying segments of BN chromosome 13 from 32.4 and 38.2 Mb and from 39.9 to 45.8 Mb were not protected from the development of hypertension. In contrast, MAP was reduced by ∼30 mmHg in five strains, carrying a 1.9-Mb common segment of BN chromosome 13 from 38.5 to 40.4 Mb. Proteinuria was reduced by ∼50% in these strains. Sequencing studies did not identify any nonsynonymous single nucleotide polymorphisms in the coding region of the genes in this region. RT-PCR studies indicated that 4 of the 13 genes in this region were differentially expressed in the kidney of two subcongenic strains that were partially protected from hypertension vs. those that were not. These results narrow the region of interest on chromosome 13 from 13.4 Mb (159 genes) to a 1.9-Mb segment containing only 13 genes, of which 4 are differentially expressed in strains partially protected from the development of hypertension.
- Dahl S rats
- renal disease
previous studies have shown that introgression of chromosome 13 (Chr 13) from the Brown Norway (BN) rat into the salt-sensitive (SS) genetic background attenuates the development of hypertension in a SS-13BN consomic strain (4), but the genes and pathways involved remain to be identified. Our laboratory recently developed 26 overlapping congenic strains and found that 4 different regions of Chr 13 contribute to the lowering of blood pressure (BP) in the SS-13BN consomic strain (10). BP measured via an arterial catheter after 3 wk on a high-salt diet in one of these congenic strains [SS.BN-(D13Hmgc37-D13Got22)/Mcwi, line 5] carrying a 13.4-Mb segment of BN Chr 13 between 32.4 and 45.8 Mb was reduced by 30 mmHg (10). This region is distinct from the BP quantitative trait loci (QTL) near the renin gene that was originally identified in an F2 cross and congenic strains derived from Dahl SS and salt-resistant rats (7, 14, 20–21). This QTL on rat Chr 13 is homologous with a region on human Chr 2 that has been linked to an elevation in BP in both the Quebec Family Study (15) and the Family Blood Pressure Program (9). The purpose of the present study was to characterize the time course of changes in BP measured by telemetry in SS rats vs. those seen in the line 5 SS.BN13 congenic strain fed a high-salt diet and then to narrow the region of interest by creating and phenotyping 11 additional subcongenic lines.
Experiments were performed on male Dahl SS/JrHsdMcwi (SS) rats and congenic strains derived from a cross between SS-13BN consomic and SS rats. The congenic strains were developed using marker-assisted breeding, as previously described (10). Rats from a previously described congenic strain line 5 [SS.BN-(D13Hmgc37-D13Got22)/Mcwi] that were partially protected from the development of hypertension (10) were crossed with SS rats. Rats in the F1 generation were intercrossed, and the progeny was genotyped with 20 microsatellite markers covering the entire 13.4-Mbp congenic interval (10, 11). Recombinant rats were selected as breeders for the establishment of congenic strains. A total of 11 overlapping subcongenic strains were developed. All the rats were bred and housed in an American Association for Accreditation of Laboratory Animal Care-approved animal care facility at the Medical College of Wisconsin. The rats had free access to food and water throughout the study. All experimental procedures were approved by the Institutional Care and Use Committee of the Medical College of Wisconsin.
Phenotyping of BP and proteinuria.
Experiments were performed on 9-wk-old SS, SS-13BN consomic animals, and 11 overlapping SS-13BN congenic strains. The rats were all fed a defined diet (AIN76 diet, Dyets) containing 0.4% NaCl until they were 8 wk old. The rats were then anesthetized with 2% isoflurane, and a gel-filled catheter attached to a BP telemetry transmitter (Data Sciences) was implanted into the femoral artery for chronic measurement of BP. After a 7-day recovery period, mean arterial pressure (MAP) was measured between 9 AM and 12 PM on 3 consecutive control days. Rats were then placed in metabolic cages without restricting food or water for an overnight urine collection to measure baseline proteinuria. The concentration of protein in the urine samples was measured using the Bradford method using bovine serum albumin as the standard (Bio-Rad Laboratories, Hercules, CA). The rats were then switched to a high-salt diet (8.0% NaCl), and BP was measured on days 3, 7, 14, and 21, and proteinuria was reassessed on days 7, 14, and 21.
The expression of 11 of 13 genes in a common 1.9-Mb candidate region identified by our BP phenotyping studies (Table 1) was compared in samples of the renal cortex obtained from subcongenic strains fed a low (0.4%) NaCl diet vs. groups fed a 4.0% NaCl diet for 3 or 7 days. The rationale for studying the rats after 3 or 7 days on a high-salt diet was to look for differentially expressed genes associated with the development of hypertension rather than to changes that might be secondary to the renal damage that begins to be evident in SS rats fed a high-salt diet for 14–21 days. Real-time PCR analysis was performed in triplicate using cDNAs prepared from pooled RNA samples collected from six rats per strain using the SYBR green chemistry and a Stratagene Mx3000P instrument. The results of this screening analysis were then confirmed by reverse transcribing and reanalyzing the individual RNA samples for those genes found to be differentially expressed in the pooled samples. The sequences of the primers used to amplify the genes studied are presented in Table 2. B-actin was used as the normalizer.
The Primer 3 program (http://frodo.wi.mit.edu/cgi-in/primer3) (16) was used to design overlapping primer pairs to sequence all of the exons and 1,000 bps of the 5′ region for all 13 genes within the narrowed region of interest. BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start; Ref. 8) was used to ensure that the primers were specific for the targeted region. The reactions were performed by using a PCR-based Sanger sequencing method, as previously described (17). The sequencing files were analyzed with the Phred/Phrap/Consed package (5), and single-nucleotide substitutions and insertion/deletions were confirmed in the trace files using Consed. Sequence variants were analyzed with PolyPhen (13) and Sift (12), which predict the possible impact of an amino acid substitution on the structure and/or function of protein.
Data are presented as means ± 1 SE. The significance of differences in mean values between and within groups was evaluated using a repeated-measure ANOVA for the BP or proteinuria data or a two-way ANOVA for the gene expression data, followed by a Holm-Sidak test for preplanned comparisons. A P < 0.05 using a two-tailed test will be considered significant.
Phenotyping for BP and proteinuria.
A map of the introgressed regions in the congenic strains and a summary of the MAP and protein excretion data are presented in Fig. 1. MAP rose from 117 ± 1 to 186 ± 5 mmHg in SS rats fed a high-salt diet for 3 wk. Protein excretion increased from 56 ± 11 to 365 ± 37 mg/day. In contrast, MAP only increased from 110 ± 2 to 146 ± 6 mmHg in SS-13BN consomic rats and from 110 ± 3 to 152 ± 9 mmHg in the line 5 congenic strain carrying the entire 13.4-Mb segment of BN Chr 13 from position 32.4 to 45.8 Mb. Six congenic strains carrying segments of BN Chr 13 in the region from 32.4 to 38.2 (lines 5–8 through 5–10) and from 39.9 to 45.8 Mb (lines 5-5 through 5–7) were not protected from the development of hypertension (Fig. 1). Proteinuria was also not significantly reduced in any of these strains relative to the values seen in SS rats. In contrast, MAP was reduced by ∼30 mmHg in six congenic strains (line 5, lines 5-1 through 5-4, and line 5–11), carrying a common segment of BN Chr 13 spanning a 1.9-Mb segment of Chr 13 from 38.5 to 40.4 Mb. Proteinuria was significantly reduced in subcongenic strains 5-1, 5-2, 5-3, 5-4, and 5–11 relative to the value seen in SS rats.
A comparison of the time course of the development of hypertension and proteinuria in SS and SS-13BN consomic rats and in line 5 and two of the other congenic strains (5-4 and 5–11 are shown) that were partially protected from the development of hypertension vs. the results obtained in the most closely related overlapping congenic strains (lines 5-5 and 5–10) that were not protected are presented in Figs. 2 and 3. Similar time courses were also seen in line 5, 5-1, 5-2, and 5-3 rats that were partially protected from the development of hypertension vs. the results seen in lines 5–6, 5–7, 5–8, and 5–9 that were not. Baseline MAP measured in SS, SS-13BN consomic strain, and the SS-13BN congenic strains were not significantly different. All of the strains exhibited a similar 10- to 15-mmHg increase in BP (salt sensitivity) on days 3 and 7 of the high-salt (8% NaCl) diet. Thereafter, pressure increased by another 50 mmHg in SS rats and in the two subcongenic lines 5-5 and 5–10 that were not protected from the development of hypertension. In contrast, the increase in MAP seen on days 7–21 was significantly less in SS-13BN consomic strain, as well as in the SS-13BN congenic line 5 and the subcongenic lines 5-4 and 5–11, than the changes seen in SS rats or the unprotected congenic strains (5-5 and 5–10).
The rise in MAP in SS rats and SS-13BN subcongenic lines 5-5 and 5–10 was associated with the development of proteinuria (Fig. 3). Protein excretion rose to a lesser extent in the SS-13BN consomic strain and in the SS-13BN congenic lines that were partially protected from the development of hypertension (5-4 and 5–11). Similar significant reductions in proteinuria were also seen in strains 5-1, 5-2, and 5-3 (Fig. 1). The only exception was in line 5, in which proteinuria was not significantly reduced relative to the values seen in SS rats.
To better define the limits of the region of interest, we fine mapped ends of the introgressed region in the two smallest subcongenic strains 5-4 and 5–11 that were partially protected from the development of hypertension vs. those seen in the most closely related overlapping lines 5-5 and 5–10 that are not, by spot sequencing in the confidence interval region. The results defined the limits of the candidate region between positions 38.56 Mbp and 40.436 Mbp on Chr 13. This region contains the 13 known and predicted genes presented in Table 1.
We also sequenced the entire coding region and 1,000 bp of the 5′ regulatory regions for 9 of the 13 genes in samples of DNA obtained from SS and BN rats. We detected sequence variants between SS and BN rats in the coding region of only one gene, Nap5, where we found eight single nucleotide polymorphisms (SNPs). Five of them were synonymous. Variants A1958V and T1001M were predicted to be benign by Polyphen and Sift. Variant R1697G was predicted to potentially alter protein structure, but it was found to be located at position 38,511,189 Mbp, which is outside the confidence interval of the candidate region. We further confirmed this by sequencing samples obtained from subcongenic line 5-4 and 5–11 rats that are partially protected from hypertension and found that these strains carry the SS rather than the BN allele at this site.
The results of experiments to compare the expression of 11 of the 13 genes in samples of RNA extracted from the renal cortex of rats in the two smallest subcongenic lines 5-4 and 5–11 that are partially protected from the development of hypertension vs. that seen in the most closely related overlapping subcongenic strains (5-5 and 5–10) that are not protected are presented in Table 3. The expression of two of the predicted genes (LOC680625 and LOC680642) in the region was not studied, because the expression of these genes was very low in the kidney (>50 cycles). The expression of 6 of 11 of the genes in this region was significantly elevated more than twofold in the pooled samples of renal cortex obtained from rats in subcongenic line 5-4 vs. the corresponding levels seen in line 5-5 at all of the time points studied. The differential expression of these genes, Nap5, LOC680596, LOC68052, Mgat5, Tmem163, and LOC501853, was confirmed in line 5-5 by separately analyzing the expression levels of these genes in the RNA samples prepared from 6 individual rats (Table 4). Significant differences in the expression of four of these genes, NAP5, LOC68052, Tmem163, and LOC501853, were also detected after 7 days on a high-salt diet in the renal cortex samples obtained from rats in subcongenic line 5–11, which is also partially protected from the development of hypertension vs. the levels seen in the overlapping unprotected subcongenic line 5–10.
We further explored the effects of a high-salt intake on the expression of the four genes that were differentially expressed in common in the strains partially protected from the development of hypertension. The renal expression of Nap5, LOC680652, and LO501853 increased significantly following 3 days on a high-salt diet in the “protected” 5-4 and 5–11 strains, but they did not increase significantly in the 5-5 and 5–10 unprotected control strains. The expression of Tmem163 also increased to a greater extent when rats were fed a high-salt diet in the 5–11 vs. the 5–10 strain following 3 days of a high-salt diet, but did not increase in the 5-4 strain, as it was already highly differentially expressed in this strain relative to the 5-5 control strain on a low-salt diet. The expression of all four of these genes returned toward control in the 5-4 and 5–11 congenic strains that were partially protected from the development of hypertension after 7 days on a high-salt diet. Nevertheless, they remained significantly higher than the levels seen in the control strains, because the expression of most of these genes tended to decrease with time in congenic lines 5-5 and 5–11.
Previous studies indicated that transfer of Chr 13 from the BN rat onto the Dahl SS genetic background attenuates the development of hypertension (4), but the mechanism and genes involved remain to be determined. In a recent study of 26 overlapping SS-13BN congenic strains, Moreno et al. (10) reported that there are four different QTL regions on this chromosome that contribute to the reduction in pressure. However, the sizes of the QTLs defined in this study (5–16 Mb) were far too large to begin any meaningful search for genes involved. Moreover, it was also important to first confirm the results of the previous study using telemetry before launching into the time-consuming task of narrowing the congenic region and gene identification, since BP was only measured at a single time point in the previous study after the rats were fed a high-salt diet for 3 wk (10). In the present study, we more fully characterized the time course of the development of hypertension in the line 5 SS.13BN congenic strain in which a 13.4-Mbp segment of BN Chr 13 from position 32.4 to 45.8 Mbp was introgressed using telemetry, as well as in all of the subcongenic strains developed to narrow the region of interest. We found that complete transfer of BN Chr 13 in SS-13BN consomic strain fed a high-salt diet attenuated the rise in pressure by ∼40 mmHg vs. the increase seen in SS rats. Transfer of a 13.4-Mb segment of BN Chr 13 in the line 5 congenic strain was only slightly less effective at attenuating the development of hypertension (152 vs. 142 mmHg) than that seen with complete transfer of Chr 13. This finding confirms that there is a gene or genes in this smaller introgressed region of Chr 13 that plays a major role in the regulation of BP in SS rats.
Phenotypic analysis of the results obtained in the subcongenic strains indicated that transfer of either a 5.8-Mb region from 32.4 to 38.2 Mb, or a 5.9-Mb region from 39.9 to 45.8 Mb did nothing to prevent the development of hypertension or proteinuria. This result eliminates the genes in 11.5 of the 13.4 Mb of the introgressed region in the line 5 congenic strain from further consideration as causal. In contrast, BP was reduced in all of the SS-13BN congenic strains that included a common 1.9-Mb segment of the BN genome in the region from 38.5 to 40.4 Mb. It is also interesting to note that introgression of this region did not lower baseline pressure or salt-sensitivity of BP, since there was no difference in the rise in pressure in SS rats and the line 5 congenic and subcongenic strains over the first 7 days of exposure to a high-salt diet. Rather, this region influenced the secondary rise in MAP seen on days 7–21 of the high-salt diet that was associated with the development of proteinuria, which is a marker for glomerular injury.
The region of interest contains just 13 known and predicted genes. We failed to identify any differences in the coding region of nine of the genes we sequenced in SS and BN rats that would be predicted to alter the structure or function of any of these proteins. We also failed to detect any sequence difference in the first 1,000 bp of the 5′ regulatory region of these genes. This finding was quite unexpected, since, on average, one SNP is found for every 1,000 bp of sequence across the genome. Thus it appears that the genes in this region are very highly conserved in SS and BN rats.
In further RT-PCR studies, we found that six of the genes in this region were differentially expressed in the renal cortex of the subcongenic strain 5-4 with the smallest BN insert that was partially protected from hypertension vs. the levels seen in the overlapping 5-5 strain that was not protected. Similar results were obtained for four of six of these genes (Nap5, LOC680652, Tmem163, and LOC501853) in the renal cortex of the other narrow subcongenic strain (5–11) that was protected from hypertension vs. the levels seen in the most closely related 5–10 strain that was not. The expression of three of four of these genes increased in both strains in response to 3 days of high-salt diet in the “protected” strains 5-4 and 5–11. These genes all remained differentially expressed in the protected strains following 7 days on a high-salt diet, because the expression of these genes tended to decrease to a greater extent in the unprotected control strains than in the strains that were partially protected from the development of hypertension, but did not increase in the control strains. Overall, the expression analysis suggests that Nap5, LOC680652, Tmem163, and LOC501853 are differentially expressed in common in the kidney of the two smallest subcongenic strains that are protected from the development of hypertension. Thus these genes are the prime candidate genes for more detailed studies to identify the causal sequence variant and to validate one or more of them using recent advances in rat transgenic technology to knockout or overexpress genes of interest using Sleeping Beauty tranponsons and Zn finger nucleases (3, 6).
It should be noted that our sequencing studies failed to identify any variants in the first 1,000 bp of the promoters of these four genes that can explain the differences in expression between the congenic strains. However, many genes are regulated by motifs located thousands of base pairs from them, with some of them regulating the function of a cluster of genes. The fact that we found 4–6 out of 13 genes that were differentially expressed in the kidney between congenic strains within a rather small 1.9-Mbp region suggests that there might be a common regulatory region in the region that is affecting gene expression in SS than in BN rats. Further studies will be needed to sequence the entire 1.9-Mbp region of interest to identify all of the sequence variants between congenic strains, identify the ones located in highly conserved regions that may affect gene expression, and then prioritize the potential candidate causative variants.
None of the differentially expressed genes identified in the present study have any known association with BP or cardiovascular disease. For example, the function of Nap5 is unknown, although it has been linked to bipolar disorders in human Genome-Wide Association Studies (19). Other Nck-associated proteins have been found in vascular smooth muscle cells and are thought to mediate some of the actions of angiotensin II (18). Tmem163 is also known as SV31. It is a synaptic vesicle membrane protein of 31 kDa that appears to be related to GABA and glutaminergic neurons in the brain (2). A homolog of this gene, actin-related protein 3, has been associated with the development of proteinuria in BUF/Mna rats and abnormalities in actin assembly in podocytes (1). Thus it is possible that changes in the expression of this gene could also play a role in the development of hypertension-induced renal injury in SS rats. Finally, LOC6806252 resembles the structure of 40S ribosomal protein S2, but there is little information available about the function of this protein or that encoded by LOC501853.
Previous studies have indicated transfer of BN Chr 13 protects from the development of hypertension, but the mechanism and genes involved remain to be identified. The present study identified a 1.9-Mb region on BN Chr 13 that contains just 13 genes that capture most of the antihypertensive effect seen with transferring the whole chromosome. Four of the genes in this region are differentially expressed in common in the kidney of congenic strains that exhibit partial protection from the development of hypertension vs. results obtained in closely related congenic strains that are not protected. Further detailed sequencing studies are needed to identify the causal sequence variants that contribute to the differential expression of these genes. Moreover, further gene knockout and transgenic overexpression studies will be required to ultimately determine which of these candidate genes is mechanistically related to development of hypertension in SS rats. Identification of the gene involved may lead to new insights about the pathogenesis of SS hypertension, since none of the genes in the region have any known relation to the regulation of BP.
This study was supported by National Heart, Lung, and Blood Institute Grants HL-082798 and HL-036279.
No conflicts of interest, financial or otherwise, are declared by the author(s).
The authors thank Nadia Barreto, Mathew Hoffman, and Paul Graf for expert technical assistance.
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