SA Gene Expression in the Proximal Tubule of Normotensive and Hypertensive Rats
Abstract Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability.
Essential hypertension is a polygenic, heterogeneous, and multifactorial disease with a high genetic predisposition. Transplantation studies indicate that the kidneys play a central role in the pathogenesis of hypertension in genetically hypertensive rats.1 2 Using the differential hybridization technique, Iwai et al3 identified a previously unknown gene that they called SA that shows a 10-fold higher expression in the kidneys of SHRs than in kidneys from normotensive WKY control rats. In a subsequent study in Dahl salt-sensitive rat kidneys, Harris et al4 described a second isoform of the SA gene that has a 102-bp insert in the 3′ end of the cDNA sequence of Iwai et al (in this article, the original SA gene isoform of Iwai et al is called SA-1 and the second SA isoform of Harris et al, SA-2). The SA gene was mapped to chromosome 1 in the rat5 6 and to chromosome 16 in humans.7 Genetic markers in or close to the SA locus were linked to increased blood pressure in several F2 crosses involving SHRs,8 9 10 stroke-prone SHRs,3 11 and Dahl salt-sensitive rats.4 Moreover, antihypertensive treatment in SHRs could modulate hepatic expression of the SA gene.3 These observations suggest a possible involvement of the SA gene in the pathogenesis of hypertension in genetically hypertensive rodent models. In human studies, a Pst I polymorphism of the SA gene is linked to hypertension,12 but polymorphic microsatellite markers in the human SA locus are not.7
At present, the function of the SA gene product is entirely unknown. With the hope that localization information might provide clues to the possible function of the SA gene, we used RT-PCR methodology to study the localization of the SA gene in microdissected nephron segments and to compare the tubular expression pattern in hypertensive and normotensive animals. We found that renal expression of SA mRNA is virtually exclusively confined to the proximal tubules of both hypertensive and normotensive rats. SHRs showed high levels of expression in both the convoluted and straight portions of the proximal tubule, whereas WKY rats showed selective expression in the PSTs, while in SD rats, SA expression was restricted to the PCTs.
Animals and Dissection Methods
Male SD rats, SHRs, and WKY rats (all about 8 weeks of age) were anesthetized with Inactin (120 mg/kg; Byk-Gulden). After aortic blood flow to the left kidney was interrupted, the kidney was perfused through a cannula inserted into the abdominal aorta below the origin of the renal artery with 30 mL cold saline followed by perfusion with 30 mL culture medium (DMEM; Sigma Chemical Co) containing 1 mg/mL collagenase. The left kidney was then removed, cut into slices, and incubated in the DMEM/collagenase solution for 22 minutes at 37°C. Microdissection was performed with sharpened forceps at 4°C under a stereomicroscope. Lengths of dissected segments were measured. In general, 10 glomeruli or 6 to 10 mm of tubule segments were dissected and pooled to constitute one sample. The following specimens were dissected from all slices: glomeruli, PCTs, PSTs, cortical thick ascending limb, cortical collecting ducts, outer medullary collecting ducts, inner medullary collecting ducts, and arcuate arteries. Lengths of the dissected segments were measured with an eyepiece micrometer. Samples were placed in 100 μL GITC buffer (4 mol/L GITC, 25 mmol/L sodium acetate, pH 6.0, 0.8% β-mercaptoethanol), snap-frozen in liquid nitrogen, and stored at −80°C.
Four male SD rats weighing about 120 to 130 g were anesthetized with sodium pentobarbital. The left renal artery was clipped as previously described.13 The same surgical procedure except for clipping of the renal artery was performed on four rats to prepare sham-operated controls. All rats were maintained on normal rat chow and had free access to tap water. Blood pressure was measured in conscious animals by tail-cuff plethysmography. At 4 weeks after surgery, microdissection was performed as described above, except that both kidneys were perfused and removed for microdissection.
RNA from glomerular and tubular samples was isolated as previously described.14 15 Briefly, glomerular and tubular samples were thawed in an ice slurry and sonicated for 15 seconds. Twenty micrograms of ribosomal RNA from Escherichia coli (Boehringer Mannheim) was added as carrier, and the sample in 100 μL of GITC buffer was layered onto a gradient of cesium chloride (100 μL of 97% and 20 μL of 40% cesium chloride in 25 mmol/L sodium acetate buffer) in a 250-μL polycarbonate ultracentrifuge tube. Samples were centrifuged for 2 hours at 300 000g in a Beckman TLA 100 ultracentrifuge (Beckman Instruments) with a fixed-angle rotor. The RNA pellet was redissolved in 0.3 mol/L sodium acetate and precipitated in ethanol.
Total RNA from different organs, including liver, kidney, stomach, small intestine, lung, spleen, aorta, and brain from 8-week-old SD rats, was isolated with TRI reagent (Molecular Research Center). Briefly, tissue samples were homogenized in TRI reagent solution. After addition of chloroform and centrifugation, the homogenates separated into three phases: aqueous, interphase, and organic. RNA was precipitated from the aqueous phase by addition of isopropanol. Contaminating genomic DNA was removed with RNase-free DNase I (GeneHunter). The purified RNA was redissolved in diethyl pyrocarbonate–treated water containing 20 U RNAsin.
RT was performed in the presence of 100 IU Moloney murine leukemia virus reverse transcriptase (Superscript; BRL), 0.5 μg oligo (dT)12-18 (Pharmacia), 20 IU RNAsin (Promega Biotech), 10 mmol/L dithiothreitol, 0.5 mmol/L dNTP (Pharmacia), and 1% bovine serum albumin (Boehringer Mannheim) in the buffer provided by the manufacturer in a total volume of 20 μL. Before the addition of reverse transcriptase, dNTPs, and bovine serum albumin, the reaction mixture was incubated at 65°C for 5 minutes to allow the primers to anneal to the poly A tail of mRNA. cDNA was synthesized at 42°C for 1 hour and precipitated with 1 μL linear acrylamide, 4 mol/L ammonium acetate, and 100% ethanol. The pellets were redissolved in Tris-EDTA buffer at a dilution adjusted so that each 2 μL of cDNA corresponded to 1 mm of tubule or 1 glomerulus.
Polymerase Chain Reaction
Primers for the SA gene were chosen from the SA-1 sequence.3 The sequence of sense primer was 5′-GACTGTCTGTCAACGGAAGG-3′ (bp 1034 to 1053) and that of the antisense primer was 5′-TGAGAGCACTCTCTACCTCA-3′ (bp 1641 to 1660). The primers flank a 102-bp insertion region in the SA-2 sequence.4 The two predominant products expected to be amplified from SA-1 and SA-2 are predicted to be 630 and 732 bp, respectively.
To control for variations in RNA amount and efficiency of RT, PCR amplification for β-actin was also performed. The primers for β-actin were chosen from the human published sequences.16 The sequences of the β-actin primers were as follows: sense, 5′-AACCGCGAGAAGATGACCCAGATCATGTTT-3′ (bp 384 to 413); and antisense, 5′-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3′ (bp 705 to 734).
PCRs were performed in a total volume of 50 μL in the presence of 5 pmol of each oligonucleotide primer, 200 μmol/L dNTP, 10 mmol/L dithiothreitol, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl, pH 8.3, 0.001% gelatin, 1.25 IU AmpliTaq DNA polymerase (Perkin-Elmer Cetus Corp), and 1.5 μCi [32P]dCTP (Amersham Corp). Mineral oil was layered on the top of each sample to prevent evaporation of the liquid. The samples were first denatured at 94°C for 3.5 minutes. The PCR cycle was programmed as follows: 94°C for 1 minute (melt), 55°C for 1 minute (anneal), and 72°C for 1 minute (extend). PCR was run for 25 cycles, and the last cycle was followed by additional incubation for 8 minutes at 72°C.
Analysis of PCR Products
After amplification, PCR products were subjected to size separation by polyacrylamide gel electrophoresis. The band intensity was determined by phosphoimaging with Phosphor Analyst software on a GS-250 Molecular Imager System (Bio-Rad Inc).
The identity of the PCR products was determined by restriction digestion and sequencing. The single 732-bp PCR products from PCTs of SD rats, SHRs, and WKY rats were gel-purified and digested with HaeIII and HinfI. Digestion reactions were incubated at 37°C for 1 hour in the buffer provided by the manufacturer. The gel-purified products were subcloned to pCR II (Invitrogen) and sequenced by the dideoxynucleotide chain reaction.
Identity of SA Gene PCR Products
Primers chosen were expected to generate PCR products from both isoforms of the SA gene of 630 and 732 bp in length, respectively. However, we consistently detected only the 732-bp product in all tissues from SD rats, SHRs, and WKY rats; the 630-bp product could not be found in any strain. PCR performed in the absence of RT did not yield a recognizable band, indicating that the 732-bp product originated from mRNA, not from genomic DNA (Fig 1⇓). To confirm the identity of the product, the 732-bp band was analyzed by restriction digestion and sequencing. In restriction analysis, we chose HaeIII with a unique cutting site in the 102-bp insertion region of SA-2 and HinfI with a cutting site common to both isoforms. As shown in Fig 1⇓, the 732-bp PCR product could be digested into predicted fragments with both Hinf1 and HaeIII, indicating that the products originated from SA-2. Sequencing of the 732-bp product from PCTs of SHRs and WKY rats demonstrated its complete agreement with the published sequence of the SA-2 gene.
Organ Distribution of SA Gene mRNA in SD Rats
To test the tissue-specific expression of the SA gene, RT-PCR was performed using 1 μg of total RNA isolated from different SD rat tissues. As shown in Fig 2⇓, product could be found only in kidney and liver, and no signal could be detected from other tissues, including heart, brain, lung, spleen, stomach, and small intestine. RT-PCR of β-actin was run to verify homogeneity of mRNA amounts in each sample. A single 351-bp band with similar intensity was detected among all the tissues.
Localization of SA Gene mRNA in Microdissected Renal Structures From SD Rats
RT-PCR for SA mRNA was performed on cDNA prepared from microdissected nephron segments from SD rats. Product abundance was determined by serial dilutions of cDNA (×10, ×100, and ×1000) and normalized for equal levels of β-actin expression. SA gene mRNA was found to be abundant in PCTs. Product was occasionally detected, at approximately 100-fold lower abundance, in cDNA prepared from glomeruli and PSTs, and no signal was detected in the cortical thick ascending limb, cortical collecting ducts, outer medullary collecting ducts, inner medullary collecting ducts, and arcuate artery (Fig 3⇓).
SA mRNA Levels in Proximal Tubules From SHRs and WKY Rats
Studies of the localization of SA gene mRNA in microdissected nephron segments of SHRs and WKY rats showed that, similar to SD rats, SA gene mRNA was expressed predominantly in the proximal tubule. However, whereas SA gene mRNA in SD rats was expressed at high levels in the PCTs, SA PCR products in WKY rats were abundant in the PSTs. In SHRs, SA mRNA was expressed in both PCTs and PSTs, with levels being similar in both parts of the proximal tubule (Fig 4⇓, top and bottom; Table⇓). This result indicates a higher level of SA gene expression in the proximal tubule of hypertensive rats and a strain-dependent distribution of SA gene mRNA along the proximal tubules of normotensive rats.
Nephron Localization in Kidney of Two-Kidney, One Clip Goldblatt SD Rats
Systolic blood pressure was significantly increased in Goldblatt rats compared with the sham-operated rats (165±15 versus 110±5 mm Hg, P<.01, n=4). SA gene mRNA was confined to PCTs, but the expression levels in PCTs showed no significant difference in nonclipped and clipped kidneys of SD rats and in sham-operated controls (Fig 5⇓). The relative levels of SA gene PCR products in PCTs of non–clipped-kidney, clipped-kidney, and sham-operated controls are 1.03±0.08, 1.02±0.28, and 1.24±0.24 (n=4, P=NS), respectively.
Since identification of the SA gene as a sequence expressed at substantially higher levels in hypertensive than normotensive rats,3 support has accrued for its possible involvement in the pathogenesis of hypertension in rodent models.4 8 10 Nevertheless, its function and the explanation for the correlation between high expression levels and high blood pressure remain completely unknown. The present studies were undertaken with the hope that the identification of the intrarenal sites of SA gene expression might provide some clues that could contribute to understanding its function. In the present experiments, we used a combination of microdissection and RT-PCR techniques to study the localization of SA gene mRNA expression in defined nephron segments. In performance of PCR, attention was paid to preventing plateau amplification with the use of low cycle number and serial dilutions of cDNA. RT-PCR of β-actin was used to validate that comparable amounts of template cDNA were used in each sample. These controls allowed us to provide some quantitative assessment of relative expression levels along the nephron.
Our results demonstrate that mRNA expression of the SA gene is virtually exclusively localized to the proximal tubules in all three strains of rats tested in the present study. The demonstration that expression of the SA gene product in the kidney is confined to the proximal tubule suggests its participation in a cellular function with some specificity to this epithelium. Given the importance of proximal tubule resorption in overall fluid homeostasis; the fact that the proximal tubule is the site of action of a number of agonists potentially important in hypertension, such as angiotensin and dopamine17 ; and the fact that the proximal tubule is the site of renal synthesis of angiotensinogen and a number of other gene products with both local and systemic effects,17 18 this nephron localization was not surprising.
Unexpected, however, was the finding that localization within the proximal tubule varied markedly with strain. Whereas in WKY rats, SA gene mRNA was expressed only in the PST segment, it was found all along the proximal tubule in SHRs. This extension of SA gene mRNA expression in SHRs to the entire proximal tubule is consistent with the notion that intrarenal levels of the SA gene product correlate with the hypertensive phenotype. In a second normotensive control strain, the SD rat, however, the expression pattern was the converse of that observed in the WKY rat, restricted to the PCTs and present only at very low levels in PSTs. Furthermore, in the SD rat, the expression pattern was not altered by induction of hypertension by renal artery clamping.
This variability in PCT/PST expression is not consistent with the participation of the SA gene product in a number of transport activities of the proximal tubule. For example, a function related to organic anion transport, a transport activity localized to the PSTs,17 would be difficult to reconcile with its low levels of expression in this segment in SD rats; similarly, a key role in sodium-dependent glucose transport, which occurs along the proximal portions of the PCTs,17 would appear to be inconsistent with the low levels seen in WKY rats in this segment. Similar arguments could be made against its participation in other proximal transport activities for which well-established, species-independent PCT/PST gradients exist, such as bicarbonate transport.18
In addition to its importance for transport, the proximal tubule is also the site of synthesis of a number of bioactive metabolites with both local and systemic effects. An alternative possibility to participation in transport is that the SA gene product participates in synthetic activities of the proximal tubule. This possibility is supported, albeit somewhat indirectly, by the only known homologies of its sequence with gene products of known function. The SA gene product shows its highest overall homology with a family of prokaryotic enzymes considered to be acyl CoA synthetases (12, ie, Gen Bank P27095, P36333, and P27550). The overall homology between the SA gene and members of this family, approximately 25% to 30% nucleotide identity, is not impressive, but there are several short regions of very high homology at the amino acid level, suggesting the possible presence of conserved structural motifs. It is tempting to speculate that the SA gene product might participate in a synthetic process with some similarities to that catalyzed by the acyl CoA synthetases. Obviously, further work is needed to clarify this issue.
Published sequences for the SA gene suggest the existence of at least two different isoforms, the original form (which for convenience we call SA-1) obtained by Iwai et al3 and another isoform reported by Harris et al4 (SA-2) with a 102-bp insertion near the 3′ end. In the present study, we performed RT-PCR with primers that should amplify both isoforms of the SA gene and yield distinctive products. We consistently detected only the product for the SA-2 form in the tissues from all three strains of rats. Restriction digestion and sequencing confirmed that the product was identical to SA-2. The reasons for the absence of an SA-1 product are not clear, but our results would suggest that it is in much lower abundance than the SA-2 isoform.
In summary, the present data show that mRNA expression of the SA gene is restricted to the proximal tubule; in SHRs, it is found all along the proximal tubule, whereas in normotensive control rats, it is more restricted in its expression, with variable proximal tubule expression patterns that shown strain specificity. Further study will be needed to determine whether high expression of SA gene mRNA in the proximal tubules of SHRs is causally related to the development of hypertension.
Selected Abbreviations and Acronyms
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|PCT||=||proximal convoluted tubule|
|PST||=||proximal straight tubule|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
|SHR||=||spontaneously hypertensive rat|
Work performed in this laboratory is funded by National Institutes of Health grants DK-37448, DK-39255, and DK-40042. The authors wish to thank Ann Smart for expert technical assistance in the microsample RNA isolation and cDNA preparation.
Reprint requests to Josie P. Briggs, MD, Department of Internal Medicine, George M. O’Brien Renal Center, 1150 W Medical Center Dr, 1560 MSRB II, Box 0676, University of Michigan, Ann Arbor, MI 48109-0676. E-mail email@example.com.
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