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作者：Bernardo López,, Robert P. Ryan,, Carol Moreno,, Albert Sarkis,, Jozef Lazar,, Abraham P. Provoost, Howard J. Jacob,,, and Richard J. Roman,,作者单位：1 Kidney Disease Center, 3 Human and Molecular Genetics Center, and Departments of 2 Physiology, 7 Medicine, 6 Pediatrics, and 4 Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin; and 5 Department of Pediatric Surgery, Erasmus Medical Center, Rotterdam, The Netherlands : n# I8 n* ?5 |6 k' l( C. l
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【摘要】
: T J+ t6 o+ q' i8 z The present study evaluated whether the impairment in autoregulation of renal blood flow (RBF) in the fawn-hooded Hypertensive (FHH) rat colocalizes with the Rf-1 region on chromosome 1 that has been previously linked to the development of proteinuria in this strain. Autoregulation of RBF was measured in FHH and a consomic strain (FHH.1 BN ) in which chromosome 1 from the Brown-Norway (BN) rat was introgressed into the FHH genetic background. The autoregulation indexes (AI) averaged 0.80 ± 0.08 in the FHH and 0.19 ± 0.05 in the FHH.1 BN rats. We next performed a genetic linkage analysis for autoregulation of RBF in 85 F2 rats generated from a backcross of FHH.1 BN consomic and FHH rats. The results revealed a significant quantitative trait locus (QTL) with a peak logarithm of the odds score of 6.3 near marker D1Rat376. To confirm the existence of this QTL, five overlapping congenic strains were created that spanned the region from markers D1Rat234 to D1Mit14. Transfer of a region of BN chromosome 1 from markers D1Mgh13 to D1Rat89 into the FHH genetic background improved autoregulation of RBF (AI = 0.23 ± 0.04) and reduced protein excretion. In contrast, RBF was poorly autoregulated and the rats were not protected from proteinuria in congenic strains in which other regions of chromosome 1 that exclude the D1Rat376 marker were transferred. These results indicate that there is a gene(s) that influences autoregulation of RBF and proteinuria between markers D1Mgh13 and D1Rat89 on chromosome 1 that lies within the confidence interval of the Rf-1 QTL previously linked to the development of proteinuria in FHH rats.
[1 N! e! b k' u$ V. K 【关键词】 renal disease Rf consomic and congenic rats quantitative trait locus
- M1 |0 K" l$ }. B& }0 M END - STAGE RENAL DISEASE (ESRD) is a major health problem in the United States and the incidence of the disease is expected to increase over the next decade ( 35 ). Hypertension and diabetes 67% of the newly diagnosed cases of ESRD ( 34 ). However, not all patients with diabetes or hypertension develop ESRD and little is known about the genes that determine the genetic susceptibility to develop diabetic- or hypertension-induced nephropathy./ K; f: C3 j: a
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The fawn-hooded hypertensive (FHH) rat is a genetic model of hypertension-induced renal disease ( 26, 27 ). These animals gradually develop systolic hypertension, followed by progressive proteinuria, focal glomerulosclerosis, and, eventually, ESRD ( 16, 17 ). Previous genetic studies identified two quantitative trait loci (QTL) on chromosome 1 ( Rf-1 and Rf-2) that are associated with the development of proteinuria ( 3, 32 ). Rf-1 QTL is homologous to a region on human chromosome 10 that has been linked to the development of hypertension- and diabetic-induced glomerulosclerosis in humans ( 12, 13 ). A more recent study has identified an functional mutation in the Rab38 gene in the FHH rat as the likely candidate gene for Rf-2 ( 29 ). This gene influences the intracellular trafficking of proteins. The mutation in Rab38 was postulated to alter the reabsorption of filtered protein in FHH rats ( 29 ). On the other hand, very little is known about the nature of the genes in the Rf-1 locus linked to the development of proteinuria and renal disease.
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' d. [9 R9 l MIn a previous study, our group reported that the myogenic response of preglomerular renal arterioles and autoregulation of glomerular capillary pressure are markedly impaired in FHH rats ( 38 ). We suggested that elevations in glomerular capillary pressure may trigger the development of proteinuria and glomerular disease in FHH rats ( 37 ). However, the chromosomal location of the gene or genes contributing to the impaired autoregulation of renal blood flow (RBF) in FHH rats and the relationship of this defect to the development of ESRD are uncertain. Thus the purpose of the present study was to determine whether the gene mediating the impaired autoregulation of RBF in FHH rats is located on chromosome 1 and whether this gene colocalizes with the Rf-1 region that was previously linked to the development of proteinuria in this strain.( r* w3 T8 [! z9 X$ w! c4 X2 J
, g( S$ R- @! h/ b/ o M/ kMATERIALS AND METHODS. u/ E9 h; X. R1 |) n$ N) N
. I6 }" a) V. t+ YExperiments were performed in FHH/EurMcwi rats, consomic FHH.1 BN /Mcwi rats generated from a cross of FHH and Brown-Norway (BN) rats ( 23 ) and congenic strains generated from a backcross of FHH.1 BN and FHH rats ( 5 ). The rats were maintained in an animal care facility at the Medical College of Wisconsin, which is approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. The breeding colonies were maintained on a Laboratory Autoclavable Rodent Diet 5010 containing 0.28% NaCl purchased from LabDiet (PMI Nutrition International, Brentwood, MO). At weaning, the pups were switched to a purified AIN-76A rodent diet containing 0.4% NaCl (Dyets, Bethlehem, PA) that we have previously reported accelerates the development of renal disease in other strains of rats ( 22 ).
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Protocol 1: comparison of autoregulation of RBF in FHH and FHH.1 BN consomic rats. These experiments were performed to assess whether the locus responsible for the impairment of autoregulation of RBF in the FHH rat lies on chromosome 1. Experiments were performed on 20-wk-old male FHH rats ( n = 12) and FHH.1 BN consomic rats ( n = 12) in which chromosome 1 from the BN rat was introgressed into the FHH genetic background ( 23 ). The rats were anesthetized with ketamine (30 mg/kg im; Ketaject, Phoenix Pharmaceutical, St. Joseph, MO) and thiobutabarbital sodium (50 mg/kg ip; Inactin, Sigma, St. Louis, MO) and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. The trachea was cannulated with PE-240 tubing to facilitate breathing. PE-50 cannulas were placed in the femoral artery and vein to record renal perfusion pressure (RPP) and for intravenous (iv) infusions. The rats received an iv infusion of a 1% solution of BSA in 0.9% NaCl at a rate of 100 µl/min throughout the experiment. A 2.0-mm-diameter flow probe was placed around the left renal artery, and RBF was measured using an ultrasonic flowmeter (Transonic System, Ithaca, NY). A micro-Blalock clamp was positioned on the aorta between the renal and the superior mesenteric arteries, and ligatures were placed around the superior mesenteric and celiac arteries for manipulation of RPP. The kidney was denervated by stripping the visible renal nerves and by coating the renal artery with a 5% solution of phenol in ethanol. Circulating levels of vasopressin (2.4 U·ml -1 ·min -1 ) and norepinephrine (100 ng/min) were fixed by iv infusion ( 30 ).9 O$ k; z2 S& P1 K
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After surgery and a 30-min equilibration period, the rats received a bolus iv infusion of 6 ml of a 2% solution of BSA in 0.9% NaCl to inhibit the tubuloglomerular component of renal autoregulation ( 37 ). After a 15-min equilibration period, RBF was measured as RPP was varied from 140 to 80 mmHg in steps of 10 mmHg. The kidney was perfused at each level of RPP for 3 min. RBF autoregulatory indexes (AI) over the range of pressures from 100 to 140 mmHg were calculated by the method of Semple and De Wardener ( 31 ). According to this analysis, an AI of 0 indicates perfect autoregulation of RBF; an AI of 1 is characteristic of a circulation with a 1 is indicative of a compliant system in which vascular resistance decreases as RPP increases.* X" p* G @5 o, d& B# G
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Protocol 2: genetic linkage analysis for autoregulation of RBF on chromosome 1. These experiments were performed to identify the genomic region of chromosome 1 that participates in the impaired autoregulation of RBF in the FHH rat. An F2 population of rats ( n = 85) was generated by backcrossing male FHH.1 BN consomic rats to female FHH rats and then intercrossing the progeny. The rats were phenotyped for autoregulation of RBF as described above, and a sample of the tail was collected for isolating genomic DNA. The rats were genotyped using 16 polymorphic simple-sequence-length polymorphism markers with an average spacing of 8.7 centimorgans (cM) along chromosome 1. A genetic linkage analysis was performed using MapMaker/Exp and MapMaker/QTL (19-21, 25). Linkage maps were constructed using the Kosambi mapping function for genetic distance calculations. For this linkage analysis, a logarithm of the odds threshold 3.0 was considered to be significant ( 18 ).
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; w5 r9 D# B, F9 C# ~Protocol 3: generation and phenotyping of FHH.1 BN congenic strains for autoregulation of RBF. To confirm the region of chromosome 1 that harbors the loci influencing RBF autoregulation, we developed a series of overlapping FHH.1 BN congenic strains around the region of the QTL identified in the linkage analysis study. The FHH chromosome 1 congenic strains were generated by backcrossing FHH.1 BN consomic rats to FHH rats to generate an F1 population, heterozygous along chromosome 1 ( 5 ). F1 rats were intercrossed to generate an F2 population with random recombinations occurring along chromosome 1 but that is homozygous for FHH alleles on all other chromosomes. The F2 rats were further backcrossed to FHH rats, and the pups were genotyped to further narrow the region of interest. Overlapping congenic strains were then established by intercrossing male and female littermates heterozygous for the region of interest. A portion of the ear of each animal was collected for isolating DNA. Rats were genotyped using fluorescently labeled primers as previously described ( 24 ). A total genome scan was performed using 96 microsatellite markers spaced at a 10- to 20-cM resolution across the genome along with more detailed genotyping on chromosome 1 with 30 markers placed at a density of 1 marker/9 Mbp to map the introgressed region. RBF AI were measured in six to nine male rats for each of the five FHH.1 BN congenic strains as described in protocol 1. K1 b# k( G. l0 x! Z$ {
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Protocol 4: phenotyping of FHH and FHH.1 BN consomic and congenic rats for assessment of protein excretion, degree of renal injury, and arterial pressure. Experiments were performed in 20-wk-old FHH ( n = 9) and FHH.1 BN consomic ( n = 8) rats and FHH.1 BN congenic strains A ( n = 9), B ( n = 11), C ( n = 10), D ( n = 8), and E ( n = 8) ( Fig. 3 ). The rats were placed in metabolic cages for collection of a 24-h urine sample. The concentration of protein in the samples was measured using the Bradford method (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA) ( 2 ) and BSA as a standard. After protein excretion was measured, the FHH, FHH.1 BN consomic rats and rats from the FHH.1 BN congenic strains A, B, C, and E were anesthetized. The kidneys were flushed with 10 ml of saline, bisected along the midsagittal plane, and fixed by immersion in a 10% phosphate-buffered formaldehyde solution for histological analysis. The tissue was embedded in paraffin, cut into 3-µm sections, and stained with Mason?s trichrome stain. Individual glomeruli (30/rat) were graded from 0 (best) to 4 (worst) on the basis of mesangial matrix expansion and loss of glomerular capillaries as described previously ( 6, 14, 22, 28 ).
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Fig. 3. Comparison of the parental FHH and FHH.1 BN consomic and congenic rats. Left : location of genetic markers and previously identified Rf-1 and Rf-2 QTLs on chromosome 1 in FHH rats. The open and filled bars refer to FHH and BN genome, respectively. Right : some of the known genes in the 12.8-Mbp region of interest that influences autoregulation of RBF.3 }# v3 {" Z6 ~; `$ k
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To determine whether the renoprotection seen in the FHH.1 BN consomic or the congenic strains C and E is due to differences in blood pressure, mean arterial pressure (MAP) was measured in conscious 20-wk-old FHH ( n = 5) and FHH.1 BN consomic ( n = 4) rats and in FHH.1 BN congenic strains A ( n = 5), B ( n = 9), C ( n = 5), and E ( n = 7) ( Fig. 3 ). The rats were anesthetized with ketamine, xylazine, and acepromazine (56, 3.2, and 0.8 mg/kg im), and a microrenathane catheter (MRE-025/Tygon) was implanted in the femoral artery. The catheter was routed subcutaneously to the scapular region, exteriorized through a Dacron mesh button (Instech Laboratories, Plymouth Meeting, PA), and protected with a stainless steel spring that was used to tether the rats to a swivel. After surgery, 1 wk was allowed for full recovery. Then, the arterial catheters were connected to solid-state pressure transducers (Argon Medical Technologies, Athens, TX) interfaced with a computerized data-acquisition system, and heart rate, systolic and diastolic blood pressure, and MAP were recorded over a 3-h recording session during the light cycle between 9 AM and 1 PM.
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* a2 l; r! Y' G5 kStatistical analysis. Data are presented as means ± SE. A two-way ANOVA for repeated measures followed by a Holm-Sidak post hoc test was used to determine the differences between and within mean values ( 11 ). P 9 @" o4 a" ]! V+ [2 c3 L
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Protocol 1: comparison of autoregulation of RBF in FHH and FHH.1 BN consomic rats. The relationships between RBF and RPP in FHH and FHH.1 BN consomic rats are presented in Fig. 1. Autoregulation of RBF is impaired in FHH rats. RBF rose significantly from 6.16 ± 0.43 to 7.95 ± 0.50 ml·min -1 ·g -1 as RPP was varied over the normal range of autoregulation, from 100 to 140 mmHg. The AI averaged 0.80 ± 0.08 in FHH rats over this range of RPP. Autoregulation of RBF was greatly improved in FHH.1 BN consomic rats (AI = 0.19 ± 0.05), and RBF remained relatively constant when RPP was varied from 100 to 140 mmHg.
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Fig. 1. Left : relationship between renal perfusion pressure (RPP) and renal blood flow (RBF) in volume-expanded fawn-hooded hypertensive (FHH) and FHH.1 BN consomic rats, in which chromosome 1 from the Brown-Norway (BN) rat was introgressed onto the FHH genetic background. Right : RBF autoregulation indexes measured in the FHH and FHH.1 BN consomic rats. The no. of animals/group is indicated in parentheses. Values are means ± SE. *Significantly different from RBF value at RPP of 140 mmHg within same group ( P
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& _6 z" r# I+ lProtocol 2: linkage analysis for autoregulation of RBF along chromosome 1. A genetic linkage map for autoregulation of RBF in an F2 population generated from a cross of FHH and FHH.1 BN rats is presented in Fig. 2. This analysis reveals the presence of a QTL with a logarithm of the odds score of 6.31 for autoregulation of RBF on the q-end of chromosome 1. This QTL spans a region of 21.8 cM (95% confidence interval), and the peak of the QTL is located near marker D1Rat376. The magnitude of the effect of genotype at marker D1Rat376 on autoregulation of RBF is also depicted in Fig. 2. F2 rats homozygous or heterozygous for the FHH allele at this marker exhibit RBF AI of 0.47 ± 0.06 and 0.40 ± 0.04, respectively. F2 rats homozygous for the BN allele at this marker exhibit normal autoregulation of RBF with an AI = 0.18 ± 0.02. The magnitude of the effect of this locus is quite large, and genotype at the D1Rat376 marker contributes 50% to the overall variance in autoregulation of RBF seen in the F2 population derived from the cross of FHH rats and the consomic strain.
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Fig. 2. Left : genetic linkage map for RBF autoregulation index along chromosome 1. RBF autoregulation index was mapped to the Rf-1 quantitative trait locus (QTL) with a peak logarithm of the odds (LOD) score of 6.31 at marker D1Rat376. No association was found at the locus for the Rf-2 region. Right : effect of genotype at marker D1Rat376 on autoregulation index of RBF in the F2 population generated from the backcross of male FHH.1 BN consomic and female FHH rats. F2 rats were grouped by their genotype at marker D1Rat376 regardless of the genotype on the rest of chromosome 1. The no. of animals/group is indicated in parentheses. Values are means ± SE. Significantly different vs. FHH/FHH rats ( P
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' }7 y8 a+ l5 b6 j1 {: OProtocol 3: generation and phenotyping of FHH.1 BN congenic strains for autoregulation of RBF. The regions of chromosome 1 transferred from the BN rat into the overlapping FHH.1 BN congenic strains are presented in Fig. 3. Congenic line A is designed to be a control strain in which a region of BN chromosome 1 outside of the Rf-1 and Rf-2 regions and the QTL region near marker D1Rat376 are introgressed into the FHH genome. The introgressed region in line B from markers D1Rat183 to D1Rat76 (99.6 Mbp) captures the Rf-2 region but excludes Rf-1 region and the QTL near marker D1Rat376. Line C is homozygous for the BN alleles across the Rf-1 locus from markers D1Rat287 to D1Rat84 (107.5 Mbp). Strains D and E are similar to line B in the Rf-2 region; however, line D is homozygous for the BN alleles from markers D1Rat173F6B to D1Mit14 (23.41 Mbp) across the Rf-1 region and in the QTL region near marker D1Rat376. Finally, line E is identical to line D, except that the introgressed region is narrower (12.85 Mbp) and extends from markers D1Mgh13 to D1Rat89. It includes the QTL region near marker D1Rat376 and excludes most of the remaining portion of the Rf-1 region.
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1 r: c8 T2 W$ h/ A. N5 ~# C' PThe relationships between RBF and RPP ( left ) and AI ( right ) in the FHH.1 BN congenic strains are presented in Fig. 4. Congenic strains A and B, in which regions of chromosome 1 that exclude the QTL region near marker D1Rat376 from BN rats are introgressed into the FHH background, exhibit impaired autoregulation of RBF similar to that seen in the parental FHH strain, regardless of the presence of a BN allele at the Rf-2 locus in line B. The AI in congenic strains A and B average 0.55 ± 0.07 and 0.61 ± 0.06, respectively. Autoregulation of RBF is intact in congenic strains C (AI = 0.20 ± 0.03), D (AI = 0.20 ± 0.06), and E (AI = 0.23 ± 0.04) over the normal range of RPP from 100 to 140 mmHg. RBF per gram of kidney weight is lower in the congenic strains A and B because the kidneys are hypertrophied. Kidney weight averages 1.7 g in these strains vs. 1.4 g in congenic strains C, D, and E, which are protected from renal disease. In aggregate, these results suggest that the gene(s) responsible for impaired autoregulation of RBF in the FHH rats is located between markers D1Mgh13 and D1Rat89 (Chr1: 252.6 to 265.5 Mbp) on chromosome 1.
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# r! m& ?. \" k3 pFig. 4. Left : relationship between RPP and RBF in volume-expanded FHH.1 BN congenic strains A, B, C, D, and E. Right : RBF autoregulation indexes measured in FHH and FHH.1 BN consomic rats and in congenic strains A, B, C, D, and E. The no. of animals/group is indicated in parentheses. Values are means ± SE. *Significantly different from RBF value at RPP of 140 mmHg within same group ( P
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1 C: |2 I, ^: U7 w" I% {" K& Y2 r1 nProtocol 4: phenotyping of FHH and FHH.1 BN consomic and congenic strains for proteinuria and the degree of renal injury. A comparison of protein excretion in FHH and FHH.1 BN consomic rats and FHH.1 BN congenic strains is presented in Fig. 5. Protein excretion in FHH rats and in congenic strains A and B is significantly higher than the levels seen in FHH.1 BN consomic rats and in congenic strains C, D, and E. Interestingly, protein excretion in congenic strain B is significantly reduced compared with that measured in FHH rats, confirming that the Rf-2 region alone influences protein excretion in FHH rats ( 29 ) even though it has no effect on autoregulation of RBF.
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Fig. 5. Comparison of protein excretion rate in FHH and FHH.1 BN consomic rats and in congenic strains A, B, C, D, and E. The no. of animals/group is indicated in parentheses. Values are means ± SE. Significantly different vs. FHH rats ( P 5 Q8 H( g) ^. s9 u: O
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Representative histological sections of kidneys obtained from FHH and FHH.1 BN consomic rats and congenic strains A, B, C, and E are presented in Fig. 6. Glomerular damage with mesangial matrix expansion, increased collagen deposition and fibrosis (blue stain), and collapsed capillaries is found in most of the glomeruli in the kidneys of the FHH rats and FHH.1 BN congenic strains A and B. The degree of glomerular injury is less prominent in FHH.1 BN consomic rats and in congenic strains C and E, in which BN alleles in the D1Rat376 QTL region are introgressed in the FHH genetic background ( Fig. 7 ). The glomerular injury score is significantly greater in FHH rats (2.77 ± 0.15) and in the FHH.1 BN congenic strains A (3.01 ± 0.09) and B (2.85 ± 0.09) than those observed in the FHH.1 BN consomic (1.27 ± 0.09) rats or in the congenic strains C (1.91 ± 0.15) and E (1.26 ± 0.10).. e6 a7 c1 S7 B, O
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Fig. 6. Representative images of glomeruli ( x 40 original magnification) from FHH and FHH.1 BN consomic rats and congenic strains A, B, C, and E. Severe glomerular damage with mesangial matrix expansion, fibrosis (blue stain), and collapsed capillaries is evident in the kidneys of the FHH rats and FHH.1 BN congenic strains A and B. The glomerular damage is less in kidneys obtained from FHH.1 BN consomic rats and congenic strains C and E.9 Y& ~" X) p9 G$ b, N4 ]) `
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Fig. 7. Glomerular injury score in FHH and FHH.1 BN consomic and congenic strains A, B, C, and E, in which different regions of chromosome 1 from the BN rat were introgressed to the FHH genetic background. The no. of animals/group is indicated in parentheses. Values are means ± SE. Significantly different vs. FHH rats ( P
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A comparison of MAP measured in FHH and FHH.1 BN consomic rats and in FHH.1 BN congenic strains A, B, C, and E is presented in Fig. 8. MAP values in FHH rats and FHH.1 BN congenic strains A and B are similar and averaged 150 mmHg. MAP values are slightly but significantly lower ( 8 mmHg) in rats from the FHH. 1 BN consomic strain and in rats from congenic strains C and E compared with the value measured in FHH rats.8 w0 c8 c# m4 s
+ e& s) z: ^& } HFig. 8. Comparison of mean arterial pressure (MAP) measured in conscious 20-wk-old FHH and FHH.1 BN consomic rats and congenic strains A, B, C, and E. The no. of animals/group is indicated in parentheses. Values are means ± SE. Significantly different vs. FHH rats ( P
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9 w2 c. a) ~- b- {3 JThe present study examined whether the impaired autoregulation of RBF in the FHH rat is linked to a gene(s) on rat chromosome 1 and whether this gene(s) colocalizes with the Rf-1 region that has previously been linked to the development of proteinuria in this strain ( 32 ). The major finding was that introgression of chromosome 1 in the FHH.1 BN consomic strain, or a smaller segment of chromosome 1 that includes a 12.8-Mbp region spanning markers D1Mgh13 - D1Rat89 from BN rats into the genetic background of the FHH rat (congenic strains C, D, and E), improves autoregulation of RBF, reduces protein excretion, and diminishes the degree of glomerular injury compared with that seen in FHH rats. The marked degree of renal protection observed in the FHH.1 BN consomic strain and congenic strains C and E cannot be simply explained on the basis of differences in the degree of hypertension because MAP is, at most, only 8 mmHg lower in these strains than that seen in the FHH rats and congenic strains A and B that all developed proteinuria and glomerular disease. Overall, these results indicate that there is a gene(s) for impaired autoregulation of RBF and proteinuria on chromosome 1 of the FHH rat between markers D1Mgh13 and D1Rat89 (Chr1: 252.6-265.5 Mbp). This gene(s) lies within the confidence interval previously reported for the Rf-1 QTL ( 32 ).
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$ M/ e1 @' I6 c. R% U) dTransfer of a 12.8-Mbp segment within the Rf-1 region on chromosome 1, between markers D1Mgh13 and D1Rat89, from the BN rat into the genetic background of the FHH rat restored the ability of the FHH rat to autoregulate RBF and attenuated the degree of proteinuria observed in 20-wk-old rats from congenic strains C, D, and E. The results from the present F2 study further indicate that the impairment of RBF autoregulation in the FHH rat follows a dominant mode of inheritance.. \% Y5 V7 S$ h& _' W h" A9 L1 x: X
( | m2 m0 h0 q" J# M x; f6 e$ MThe mechanism by which a gene(s) in the region from D1Mgh13 to D1Rat89 alters autoregulation of RBF, proteinuria, and glomerular disease in FHH rats remains to be determined. The FHH rat gradually develops glomerulosclerosis and proteinuria after the onset of hypertension ( 26, 27 ). We have previously shown that FHH rats exhibit an impaired myogenic response in small renal arteries ( 37 ), which contributes to diminished autoregulation of RBF ( 38 ) and increased transmission of systemic pressure to the glomerular capillaries ( 37 ). Glomerular hyperfiltration and increased glomerular capillary pressure (P GC ) have been repeatedly implicated as factors determining the susceptibility to the development of glomerulosclerosis in many models of hypertension. For example, autoregulation of RBF is impaired in the DOCA-salt and nephrectomy models of hypertension ( 1, 8 ). P GC is elevated in both of these models, and they rapidly develop severe proteinuria and glomerulosclerosis. These findings suggest that the restoration of autoregulation of RBF in congenic strains C, D, and E may oppose the transmission of elevated systemic pressure to the glomerulus during the development of hypertension in FHH rats and that this likely opposes the development of proteinuria and glomerular disease.& r* y- P c: R6 h% Q5 ~
6 T/ n' M) n+ t% T* _+ r3 _Several QTLs linked to the development of proteinuria and renal disease have been previously identified on rat chromosome 1 and in the homologous region of chromosome 10 in humans. In FHH rats, Shiozawa et al. ( 32 ) identified five QTLs ( Rf-1 - Rf-5 ) that contribute to the development of hypertension-induced proteinuria and glomerular injury in a linkage analysis of a cross of FHH and the August Copenhagen Irish (ACI) rats. Two of these QTLs, Rf-1 and Rf-2, map to chromosome 1. The confidence interval for the Rf-1 QTL includes the region of chromosome 1 between markers D1Mgh13 and D1Rat89 that is the same region that contributes to the impaired autoregulation of RBF, proteinuria, and renal injury identified in the present study. QTLs for elevated albumin excretion have also been identified on rat chromosome 1 in a backcross population generated from a cross of Dahl S and spontaneously hypertensive rats (SHR). These QTLs also overlap with the Rf-1 region identified in FHH rats ( 10 ). The Rf-1 locus on rat chromosome 1 is homologous to a corresponding region on human chromosome 10 ( 9 ) that has been linked to an increase susceptibility to develop ESRD ( 15 ) and diabetic nephropathy in humans ( 13 ). These later findings increase the likelihood that identification of the gene(s) that increase the susceptibility to renal disease in the FHH rat may provide insight concerning candidate genes and pathways that may affect the development of glomerular disease in humans.8 ]: @( O" e( _2 j" s0 f
0 _6 ]1 n, s1 i/ z _8 L+ B! ]Previous studies by St. Lezin et al. ( 33 ) reported that the transfer of a 22-cM segment of chromosome 1, spanning the Rf-2 region between markers D1Mit3 and Igf2, from the normotensive BN rat into the SHR genetic background rendered the kidney more susceptible to develop hypertension-induced damage compared with SHR. This region extends across the Rf-2 region that was introgressed in congenic strain B. In further studies, Churchill et al. ( 4 ) obtained evidence that BN kidneys transplanted into SHR rapidly develop proteinuria and glomerular sclerosis. An impairment of RBF autoregulation in BN rats ( 39 ) relative to that seen in SHR was suggested to explain the greater degree of renal injury in BN kidneys transplanted into a hypertensive background. However, none of these previous studies confirmed that a gene in the Rf-2 region was responsible for the difference in autoregulation of RBF seen in SHR and BN rats. In the present study, the transfer of the Rf-2 region from BN rats (congenic line B) had no effect on autoregulation of RBF in FHH rats. However, we may not have been able to detect whether the transfer of Rf-2 QTL from BN into the FHH rats genetic background promotes renal disease by impairing autoregulation of RBF because autoregulation of RBF is basically absent in the FHH genetic background ( 37 ), unlike the high degree of RBF autoregulation seen in SHR. We did find partial protection against proteinuria when the Rf-2 region from the BN rat was introgressed into the FHH genetic background in congenic line B. Overall, these results are consistent with the view that transfer of the Rf-2 region from the BN rat into the FHH background opposes the development of proteinuria and hypertension-induced renal disease through a mechanism other than altering autoregulation of RBF, perhaps by affecting tubular reabsorption of protein as suggested by Rangel-Filho et al. ( 29 ).$ ?6 k# U9 d. n$ w
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A recent study by Van Dijk et al. ( 36 ) indicated that transfer of the Rf-1 region from the FHH rat into the genetic background of the ACI rat impairs renal autoregulation and increases susceptibility to renal damage in rats treated with L -NAME. These results also suggest that the Rf-1 QTL might harbor one or more genes influencing renal autoregulation. The 24.4-Mbp region of chromosome 1 transferred from FHH to ACI rats (from D1Rat324 to D1rat156; Chr1: 229.4-253.8 Mbp) in this previous study is distinct from the 12.8-Mbp region identified in the present study (from D1Mgh13 to D1Rat89; Chr1: 252.6- 265.5). At most, there is only a small region of overlap of
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% R5 p7 ?' p: FThe myogenic response is inherent to smooth muscle and is associated with depolarization followed by Ca 2 entry through voltage-operated Ca 2 channels ( 7 ). The mechanotransduction in vascular smooth muscle is dependent on cytoskeletal proteins (integrins) that activate mechanosensitive ion channels and a number of intracellular signaling pathways ( 7 ). The signaling events include activation of Ca 2 /calmodulin-myosin light chain kinase, caldesmon, calponin, serine/threonine kinases, PKC, the G protein-phosphatidylinositol-PLC pathway, adenylate cyclase, and cytochrome P -450 (20-HETE) ( 7 ). Other factors that could modulate myogenic tone include Ca 2 -ATPase and Na /Ca 2 pump exchangers and K channels ( 7 ). Changes in genes regulating the expression or activity of any of these proteins would be potential candidates for the impaired autoregulation of RBF in FHH rats. The 13-Mbp QTL region on chromosome 1 between markers D1Mgh13 and D1Rat89 for autoregulation of RBF identified in the present study contains 28 known genes. Some of the candidate genes in this region that potentially could influence the myogenic response include Slk (a member of the serine/threonine kinase family); Add3 (a member of the PKC-, calmodulin-, calcium-binding protein family); Adrb1 (a G protein-mediated, -stimulated adenylyl cyclase activation family protein); and Gfra1 (a transmembrane receptor protein tyrosine kinase signaling pathway family protein). However, the region identified in the present study is still quite large, and further fine mapping through the development of subcongenic lines, as well as differential expression studies and sequencing, is necessary to narrow the list of candidate genes responsible for the lack of RBF autoregulation and the development of renal disease in FHH rats.2 ~* Y* o' A: u7 K1 G, C$ i' N! I9 j7 s
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The present study indicates that there is a gene(s) that influences autoregulation of RBF and proteinuria between markers D1Mgh13 and D1Rat89 on chromosome 1. This gene(s) lies within the confidence of the Rf-1 QTL previously linked to the development of proteinuria in FHH rats and within a homologous region linked to hypertension- and diabetes-induced renal disease in humans ( 9 ). A better understanding of the nature of the gene(s) and the pathways in this region that influence the autoregulation of RBF and the development of proteinuria and glomerular injury in FHH rats may provide insight into differences in the genetic susceptibility to develop ESRD and treatment of hypertension- and diabetic-induced nephropathy in humans.3 H- u& `2 | V# c" ^! {0 v4 A8 K
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u$ W4 c$ |( C/ `# pThis work was supported by National Heart, Lung, and Blood Institute Grants HL-36279, HL-29587, and HL-69321. B. López was supported as a Fulbright Scholar awarded by the Fulbright Commission/Spanish Ministry of Education, Culture and Sports (MECD) award FU2003-0973.
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