Ren1c Homozygous Null Mice Are Hypotensive and Polyuric, but Heterozygotes Are Indistinguishable from Wild-Type
Nobuyuki Takahashi*,,
Maria Luisa S. Sequeira Lopez,
John E. Cowhig, Jr.*,
Melissa A. Taylor*,
Tomoko Hatada*,
Emily Riggs*,
Gene Lee*,,
R. Ariel Gomez,
Hyung-Suk Kim* and
Oliver Smithies*
Departments of * Pathology and Laboratory Medicine and Cell and Molecular Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Pediatrics, University of Virginia, Charlottesville, Virginia; and Department of Oral Biochemistry College of Dentistry, Seoul National University, Seoul, Korea
Address correspondence to: Dr. Nobuyuki Takahashi, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 701 Brinkhous-Bullitt Building, Chapel Hill, NC 27599-7525. Phone: 919-966-6912; Fax: 919-966-8800; E-mail: ntakaha{at}med.unc.edu
Mice lacking Ren1c were generated using C57BL/6-derived embryonicstem cells. Mice homozygous for Ren1c disruption (Ren1c–/–)are born at the expected ratio, but approximately 80% die ofdehydration within a few days. The surviving Ren1c–/–mice have no renin mRNA expression in the kidney, hydronephrosis,thickening of renal arterial walls, and fibrosis in the kidney.Plasma renin and angiotensins I and II are undetectable. Urinaryaldosterone is 6% wild-type. They have low tail-cuff BP (84± 4 versus 116 ± 5 mmHg in +/+) and excrete largeamounts of urine (5.2 ± 0.8 ml/d, 725 ± 34 mOsmversus 1.1 ± 0.1 ml/d, 2460 ± 170 mOsm in +/+).After 5 d of drinking 5% dextrose, desmopressin does not increasethe osmolality of the urine in –/– mice (624 ±19 to 656 ± 25 mOsm), whereas in +/+, it increases severalfold(583 ± 44 to 2630 ± 174 mOsm). Minipump infusionof angiotensin II to Ren1c–/– mice restores BP towild-type level, but preexisting damage to the medulla preventscomplete restoration of the ability of the kidney to concentrateurine. Heterozygous Ren1c+/– mice, in contrast, are indistinguishablefrom +/+ in BP, urine volume, and osmolality. Kidney renin mRNA,the number of kidney cells producing renin, and plasma reninconcentration in the Ren1c+/– mice are also indistinguishablefrom +/+. These results demonstrate that renin is the only enzymecapable of maintaining plasma angiotensins and that renin expressionin the kidney is very tightly regulated at the mRNA level.
The renin angiotensin system (RAS) is important for regulatingBP and cardiovascular homeostasis (1). Renin secreted from thekidney cleaves angiotensinogen (AGT) to angiotensin I, whichis converted to angiotensin II by the angiotensin-convertingenzyme (ACE). Others and we have been investigating the effectsof changes in the expression of genes of the RAS using mice.Most mouse models have been generated by gene targeting usingembryonic stem (ES) cells of strain 129 (2). This strain differsfrom humans in having two renin genes in tandem on the samechromosome (Ren2 and Ren1d) that differ in their expressionand regulation in ways that complicate interpreting experimentaldata (3). For example, Ren2 can compensate for the effects ofthe absence of Ren1d on BP and kidney development (4). In contrast,strain C57BL/6 mice, like humans, have only one renin gene,Ren1c, which makes them more suitable for research involvingchanges in renin expression (3). Yanai et al. (5) approachedthis problem by generating mice with the Ren1c gene disruptedusing TT2 ES cells derived from an F1 hybrid between C57BL/6and CBA. Although this approach provides a satisfactory modelfor studying the consequences of complete absence of renin,it is less satisfactory for studying the effects of mild quantitativedifference in the expression of Ren1c because the resultingmice have a heterogeneous genetic background. This problem canbe overcome by using mice that have a pure genetic backgroundso that small differences in the phenotype can be detected withoutbeing obscured by the unfavorable heterogeneity of genetic backgroundthat occurs when two strains are combined. Accordingly, we havegenerated mice lacking Ren1c using C57BL/6-derived ES cellsand have maintained the C57BL/6 background in their progeny.Using these coisogenic mice, we show that mice completely lackingRen1c show hydronephrosis and are unable to concentrate urine.It is surprising that the Ren1c+/– heterozygotes haveplasma renin and kidney renin mRNA indistinguishable from wild-type(WT), showing that kidney renin is very tightly regulated atthe mRNA level.
Generation of Ren1c-Deficient Mice
The Ren1c gene was disrupted by conventional gene targeting(6) using C57BL/6 ES cells (Specialty Media, Phillipsburg, NJ).After electroporation of the targeting plasmid (Figure 1A) andG418/ganciclovir selection, the ES cells were screened by PCRusing primers 5'-AGAGCGGTCTCATCTTTCCATAG-3' and 5'-GAGAGGCTTTTTGCTTCCTCTT-3'.Clones that give a 1.1-kb PCR band were expanded, and targetingwas confirmed by Southern blot analysis of their genomic DNAdigested with BamH I and hybridized to the probe c. Targetedclones have a 3.4-kb band in addition to a 2.7-kb endogenousband (Figure 1B). The DNA was also digested with Bgl II andhybridized to the probe d; targeted clones have a 13.5-kb bandin addition to an 11.2-kb endogenous band (data not shown).Male chimeras that carry the disrupted allele were mated withC57BL/6 female mice to obtain animals that retained the geneticallyuniform C57BL/6 background. Genotypes were determined by PCRwith primers 5'-ACGCGTCACCTTAATATGCG-3', 5'-TGACTCCCAAGCCTTACATG-3',and 5'-GGCATCTTGGATCATAGGAC-3'. The presence of a 595-bp fragment,a 389-bp fragment, or both fragments identifies animals withthe +/+, –/–, and +/– genotypes, respectively.Mice were housed in standard cages, on a 12-h light/dark cycle,and allowed free access to normal diet that contained 0.26%sodium and water except where indicated and were handled inaccordance with the National Institutes of Health guidelinesfor the use and care of experimental animals, as approved bythe IACUC of UNC-CH.
Figure 1. Disruption of the Ren1c gene. (A) Targeted disruption of Ren1c gene in embryonic stem (ES) cells from C57BL/6 mouse strain. (top) Target gene. (middle) Targeting construct. (bottom) Targeted allele. a and b, PCR primers; c and d, probes for Southern blot. (B) Southern blot analysis of wild-type (WT) and targeted ES cell genomic DNA.
Analyses of Blood, Plasma Renin Concentration, Angiotensin I, Angiotensin II, and BP Measurement
Electrolytes were analyzed with a VT250 Chemical Analyzer (Johnson& Johnson, New Brunswick, NJ). Plasma renin concentration(PRC) and plasma angiotensins I and II were analyzed as describedpreviously (6,7). Mice were exposed to an atmosphere of CO2,and blood then was rapidly withdrawn from the descending aortaof the unconscious mice into ice-cold microcentrifuge tubesthat contained EDTA (<1 min from loss of consciousness tothe end of collection) and immediately centrifuged to isolateplasma. PRC was determined in the presence of sufficient exogenoushigh AGT rat plasma to maximally stimulate production of angiotensinI using an angiotensin I [125I] RIA kit NEA104 (NEN Life ScienceProducts Inc., Boston, MA). Angiotensin II was measured usingRIA kit S-2012 (Peninsula Laboratories, Inc., San Carlos, CA).BP was measured using a computerized tail-cuff method (8).
Quantification of mRNA Expression
Gene expression was quantified with the ABI Prism 7700 SequenceDetection System (Applied Biosystems, Foster City, CA) usingprimers and probes designed using Primer Express software (AppliedBiosystems) (9,10). Real-time TaqMan PCR reactions were performed,and the log10 RNA concentration versus the threshold cycle number(Ct) was plotted. The primers and probe for Ren1c quantificationwere described previously (9) and yield a linear plot (R2 =0.96) over a range from 0.01 to 1 µg of total kidney RNA.For kidney renin mRNA assay in our animals, we used 0.1 µgof total RNA. Changes in expression were determined by the Ctmethod, which normalized expression to -actin as an internalcontrol by subtracting the Ct value for the gene of interestfrom the Ct value for -actin. Because Ct of 1 corresponds toa twofold difference in expression, expression as a percentageof WT is 100 x 2Ct Ren1c–/– or +/– dividedby the mean of 2Ct +/+. Experiments were carried out it duplicate,and the means of each duplicate were used to calculate expressionas percentage of +/+. For aldosterone synthase, the primersare 5'-AGA GAA CTC CGT GGC CTG A-3' and 5'-CCG CAG TCG GTT GAGACG-3', and the probe is 5'-FAM-CGT GGT GTG TTC TTG CTA AATGGG CC-TAMRA-3'.
Analyses of Urine and Kidney Function
Body weight, water and food intake, and urine excretion weremeasured every 24 h for 3 d (6). Electrolytes and albumin weremeasured using the VT250 Chemical Analyzer and Albuwell (Exocell,Philadelphia, PA), respectively, and normalized to 20 g bodyweight. Aldosterone was measured using aldosterone DA RIA kit(ICN Pharmaceuticals, Inc., Orangeburg, NY). The effect of vasopressinon urine concentrating ability was tested by measuring urineosmolality before and 1 and 2 h after injecting 1 ng/g bodyweight desmopressin (Sigma, St. Louis, MO) intraperitoneally.
Effect of Angiotensin II on Kidney Function
Mice were continuously infused subcutaneously with 4 ng/kg permin angiotensin II (Sigma) using an osmotic minipump, model1002 (Alzet, Cupertino, CA). This dose of angiotensin II givesnormal to slightly higher-than-normal but still subpressor levelsof plasma angiotensin II (11). Before and 1 wk after minipumpimplantation, daily urine excretion, urine osmolality, and BPwere measured.
Low-Salt and Enalapril Studies
A low-salt diet (0.01% sodium TD90228; Harlan-Teklad, Madison,WI) was fed for 3 wk, or enalapril maleate (Sigma) was administeredin the drinking water at a concentration of 0.2 mg/ml for 5d (12) before assessment of PRC, renin mRNA, and renin immunohistochemistry.
Renin Immunohistochemistry and Morphometric Analysis
The distribution and the number of renin-producing cells weredetermined as described previously (7,12,13).
Statistical Analyses
All values are expressed as mean ± SEM. The t test wasused for statistical evaluations.
Generation of Mice with the Ren1c Gene Disrupted
Mice that carry a null allele of the Ren1c gene were generatedfrom C57BL/6 ES cells by deleting the promoter and downstreamsequences through exon 6 that include the catalytic site (Figure 1).Matings between Ren1c+/– heterozygotes produced +/+,+/–, and –/– pups at the expected Mendelianratios at birth (15 +/+, 30 +/–, and 14 –/–).There were no obvious differences in appearance or body weightamong the three genotypes at birth, but approximately 80% ofthe –/– mice died within a few days (56 +/+, 100+/–, 12 –/– at weaning). Plasma Na+ concentrationat 7 d of age does not differ between –/– and +/+(WT, 132.2 ± 1.7 mEq/L; –/–, 134.2 ±1.6; n = 6 each; P = 0.44). However, injection of saline (50µl/g body weight) from day 1 for 2 wk rescued approximately50% of the –/– mice. The life span of the –/–mice that survived to weaning was close to normal, althoughtheir body weights were approximately 20% lower than WT at 4mo of age (male WT, 30.6 ± 1.5 g; male –/–,25.6 ± 0.6*; female WT, 26.4 ± 0.5; female –/–,21.8 ± 0.6**, n > 6 each, *P < 0.005, **P <0.0005 compared with WT). Mating –/– female with–/– male mice produced litters of normal size. Thenewborn –/– pups from this mating did not have anyobvious pathologic changes.
BP, Plasma, and the Renin Angiotensin Aldosterone System in Ren1c–/–
The BP of –/– mice is approximately 30 mmHg lowerthan WT counterparts at 4 mo of age (Table 1). They showed metabolicacidosis and high plasma creatinine and blood urea nitrogen,indicating a substantial reduction in GFR. Renin and angiotensinsI and II in plasma are undetectable. Daily urinary aldosteroneexcretion is <10% of WT. Aldosterone synthase mRNA expressionin adrenal glands is significantly lower in the –/–mice (21.3 ± 10.2 versus 100.0 ± 14.7% in WT;n = 6 each; P < 0.001).
Table 1. BP, plasma, and renin angiotensin system of Ren1c–/– micea
Kidney Function and Response to Desmopressin and Angiotensin II
The –/– mice excrete five times more urine dailythan WT (Table 1). Daily urinary Na+, K+, Cl–, and creatinineexcretion is indistinguishable from WT (data not shown). Dailyurinary albumin excretion in the –/– tends to behigher than WT but does not reach statistical significance (91.6± 17.6 µg/d in Ren1c –/–, n = 9; 53.5± 6.8 µg/d in WT, n = 6; P = 0.11). For determiningwhether the –/– mice can concentrate urine in responseto dehydration, they were deprived of water for 12 h. Althoughthis deprivation induces a loss of >20% of body weight, the–/– mice do not increase the concentration of theirurine. This could be due to a structural kidney defect and/orto unresponsiveness to vasopressin. To test the latter possibility,we administered an antidiuretic hormone analogue, desmopressin(1 ng/g intraperitoneally). Figure 2 shows that this treatmentdid not increase the urine osmolality of the –/–mice (untreated, 624 ± 19 mOsm; treated, 656 ±25 mOsm; n = 10; P = 0.67), although in WT, osmolality increasedfive times (583 ± 44 to 2630 ± 174; n = 12; P< 0.0001). We next tested the possibility that angiotensinII is required for the kidney to respond to antidiuretic signalsby administering angiotensin II subcutaneously using an osmoticminipump. Angiotensin II (4 ng/kg per min) restored the BP ofthe –/– mice to levels not different from WT (withoutangiotensin II, 86.4 ± 2.8 mmHg; with angiotensin II,107.4 ± 2.8 mmHg; n = 6; P < 0.0005) and tended todecrease the daily urine volume and increase urine osmolality(without angiotensin II, 5.6 ± 0.6 ml/24 h per 20 g bodyweight, 772 ± 31 mOsm; with angiotensin II, 5.0 ±0.5 ml/24 h per 20 g body weight; P = 0.5, 854 ± 52 mOsm,P = 0.2, n = 6), although they did not reach statistical significance.
Figure 2. Response to desmopressin. Mice were given water that contained 5% dextrose for 5 d before the experiment to stimulate production of relatively dilute urine. On the experimental day, urine was collected, and 1 ng/g body weight of desmopressin was injected intraperitoneally. Urine osmolality was measured 1 and 2 h after the injection (10). WT mice respond to desmopressin, whereas Ren1c–/– mice, shown in interrupted lines, do not.
Effect of Ren1c Deletion on Kidney Morphology
The kidneys of the –/– mice seem normal at birth(data not shown) but subsequently show hydronephrosis of varyingdegrees (Figure 3, A through C). The severely affected kidneyshave a thin medulla with an atrophic/hypoplastic papilla anda dilated renal pelvis. The surface of these –/–kidneys looks granular. The kidneys of the –/– miceare smaller than normal (142.5 ± 11.6 versus 194.8 ±6.9 mg in WT; n = 6 each; P = 0.006), although the kidney/bodyweight ratio does not differ (WT, 6.4 ± 0.4 mg/g; –/–,5.5 ± 0.4 mg/g; n = 6 each; P = 0.15). The –/–kidneys also show interstitial fibrosis, focal glomerulosclerosis,and perivascular infiltration of mononuclear cells (Figure 3,D through F). The walls of the interlobular arteries and afferentarterioles are thickened as a result of an increased numberof cells (Figure 3E). These vascular abnormalities are confinedto the kidney. Other tissues, including the hearts, adrenalglands, and lungs, seem histologically normal. In particular,there is no evidence of arterial or arteriolar thickening atthese sites.
Figure 3. Histology of kidneys from WT and Ren1c–/– mouse. (A through C) Low-power view of kidneys from WT (A) and Ren1c–/– (B and C). Kidneys show two levels of severity of hydronephrosis. (D through F) High-power view of kidneys from Ren1c–/–. Cortical atrophy with shrinkage of tubules and interstitial fibrosis in a –/– mouse (D). Intrarenal arteries from –/– mice showing a marked mural hypercellularity and thickening with narrowing of the lumen (E and F), glomerular sclerosis (E), and chronic inflammatory cell infiltration surrounding the hypertrophied artery (F). The sections shown were stained with hematoxylin and eosin except D, which was stained with Masson trichrome.
BP, Kidney Function, and Histology in Heterozygous Ren1c+/–
Because of the importance of renin in the regulation of BP,we next asked whether loss of one copy of the Ren1c gene inthe +/– heterozygotes affects BP and renal function. Table 2presents the surprising result that BP and PRC of the +/–mice are not different from WT. Body weight and kidney weight/bodyweight also are not different. Likewise, daily urine volume,Na+, K+, Cl–, creatinine excretion, and urine osmolalityof the +/– are not different from WT (data not shown).
Renin Expression of Heterozygous Ren1c+/–
Past experience with heterozygous null mice showed that theyhave levels of mRNA that are 50% of WT. We therefore expectedthe Ren1c+/– mice to express 50% renin mRNA of WT. However,Figure 4 shows that renin mRNA expression in the kidney of Ren1c+/–mice (90 ± 5% of WT) does not differ significantly (P= 0.15) from WT (100 ± 5%), but the heterozygous expressionis significantly different (P < 0.0001) from the expected50%. This suggests the possibility that the number of renin-producingcells in the kidneys of Ren1c+/– mice is twice WT, butwe found that the numbers of renin-producing cells are not differentbetween WT and heterozygous animals on normal or on low-saltdiets. However, because the number of renin-producing cellsis small, we thought that the differences between the two genotypesmight be difficult to detect. We therefore stimulated reninproduction by treating the mice with enalapril. This treatmentleads to the generation of hypertrophic renin-producing cellsalong the preglomerular arterioles (Figure 5). The number ofrenin-producing cells again was not different between WT andheterozygous animals that were treated with enalapril (WT 1202± 90; +/– 1078 ± 79; P = 0.24).
Figure 4. Renin mRNA expression in kidneys of Ren1c+/– and WT mice. Renin mRNA was quantified using real-time reverse transcriptase–PCR with -actin mRNA as an internal control. Expression levels are expressed as percentage of the mean of WT calculated as described in the Materials and Methods section. Although the Ren1c+/– mice have only one functional copy of the renin gene compared with two copies in the WT mice, they have 90 ± 5% of WT expression of renin mRNA that does not differ significantly from WT (P = 0.15). Number of animals in parentheses.
Figure 5. Renin immunolocalization in the Ren1c+/– kidneys after enalapril treatment for 5 d. (A and B) WT. (C and D) Ren1c+/–. Animals were treated with enalapril for 5 d before they were killed for the study. Renin staining is brown. There are no obvious differences in the number of renin-producing cells or in the number of renin-positive glomeruli between WT and Ren1c+/–.
We have generated mice that lack renin by disrupting the Ren1cgene using ES cells derived from C57BL/6 mice, a strain thathas only a single renin gene. The Ren1c–/– miceare born in expected numbers but have reduced survival (approximately20%), low BP, impaired ability to concentrate urine, and abnormalkidney structure, a phenotype virtually identical to that ofhomozygous Agt–/– (14–17) or Ace–/–(18,19) mice or doubly homozygous Agtr1a–/–/Agtr1b–/–mice (20,21). The survival disadvantage of the Ren1c–/–pups is probably due to their reduced ability to compete asa result of their decreased BP and inability to conserve saltand water normally. The absence of a difference in the plasmaNa+ concentration between Ren1c–/– and WT and therescue of some pups by saline injection suggest that the Ren1c–/–pups die of fluid volume depletion consequent to NaCl lossesas also seen in mineralocorticoid receptor –/– pups(22). This contrasts with the dehydration as a result of waterloss seen in kidney-specific NaK2Cl cotransporter (NKCC2) –/–pups (6). Although renin is normally expressed in the reproductiveorgans of both male and female mice, Ren1c–/– maleand female mice with no renin expression are fertile, indicatingthat renin is dispensable for fertility and that fertility isnot dependent on angiotensin production.
The low BP of the Ren1c–/– mice and the absenceof renin and plasma angiotensins indicate that renin is theonly enzyme capable of maintaining plasma angiotensin levels.The daily urinary aldosterone excretion of the Ren1c–/–mice is 10 times less than WT, indicating that angiotensin IIis required for keeping normal aldosterone levels. In contrast,Agtr1a–/– mice have WT levels of aldosterone (23,24),probably because Agtr1b in adrenal glomerulosa cells (25) issufficient for normal aldosterone production.
The kidneys of the Ren1c–/– mice develop varyingdegrees of hydronephrosis. Previous investigations have shownthat loss of angiotensin II receptors leads to the absence ofureteral peristalsis and to a higher baseline intrapelvic pressure(26). Thus, it is likely that in our Ren1c–/– mice,the lack of angiotensin II also causes malfunction of the ureterand may account for the subsequent development of the medullarydamage and hydronephrosis. The development of hydronephrosiscould also be predicted by their higher urine output comparedwith WT combined with the dramatic increase in urine productionthat normally occurs shortly after birth. These factors mayexceed the maximum capability of the ureter to remove urine,causing an increase in backward pressure in the renal pelvisand leading to hydronephrosis, inflammation, fibrosis, and atrophyof the papilla and enlarged calyx (26).
The Ren1c–/– mice drink several times more waterthan WT and excrete a corresponding amount of urine. Becausewater deprivation does not increase the osmolality of theirurine, they excrete more urine not because they drink more butbecause they are unable to concentrate urine. Polyuria and polydipsiacan be due either to a decrease in vasopressin from the brainor to failure of the kidney to respond to vasopressin. Becausethe Ren1c–/– mice do not respond to desmopressin,their polyuria is nephrogenic. This is largely due to the medullarydamage that disables generation of axial osmotic gradient necessaryto concentrate urine. In addition, angiotensin II may be requiredfor vasopressin to concentrate urine, because Agtr1a–/–mice also show an impaired ability to concentrate urine eventhough they have no hydronephrosis (27). Agtr1a–/–mice have decreased expression of the thiazide-sensitive NaClco-transporter and the subunit of the amiloride-sensitive epithelialtype of Na+ channel (24). Both are important for salt reabsorptionin the renal tubules, and their expression and function arenormally stimulated by angiotensin II. However, we found thatangiotensin II infusion to the Ren1c–/– mice didnot significantly increase the osmolality of their urine.
Another phenotype observed in the Ren1c–/– miceis medial thickening of the small arteries in the kidney butnot of the vessels outside the kidney. This phenotype has beendescribed by others in renin-deficient mice (5) as well as inmice deficient of Agt (15,17,28), Ace (18,19), Agtr1a (29),and Agtr1a/1b (20,21). Mononuclear cell infiltration surroundingthe thickened arteries is evident, suggesting that some commonpathogenic mechanism causes vascular thickening and perivascularinflammation. Because both are seen in the renin-deficient mice,it is clear that neither renin nor the angiotensins are necessaryfor this phenotype. Mice in which all cells that express orhave previously expressed renin have been ablated do not showthis phenotype (30). It therefore is likely that the hypertrophythat we see in the renal vessels of the Ren1c–/–mice involves cells that during development would normally synthesizerenin. This suggests that renin-producing cells per se may contributeto the vessel thickening possibly by excreting some factor(s)other than renin that is coordinately regulated by the expressionlevel of renin.
Because our Ren1c+/– mice are derived from C57BL/6 EScells and are maintained on the pure C57BL/6 genetic background,we have been able to make and interpret quantitative measurementsin the Ren1c+/– mice that are not possible with heterozygousRen1c+/– mice that have a mixed genetic background. Theheterozygous gene disruption of other genes in the renin angiotensinsystem, such as Agt and Agtr1a, shows lower BP than WT (15,31).In contrast, the most striking finding in Ren1c disruption isthat there is no difference between the Ren1c+/– and WTin BP, kidney function, or PRC. This is surprising because,in general, the expression of a gene product is proportionalto the number of copies of the corresponding gene (32,33). Thus,many previous observations have shown that the expression ofa protein is usually close to 50% of normal in individuals whoare heterozygous for a loss of function mutation (32). However,the amount of renin mRNA in the Ren1c+/– kidney is almostas high as WT and does not differ significantly from WT, butit is significantly different from 50% WT. Therefore, some feedbackregulation must have restored the kidney renin mRNA level backto the WT level. Because in earlier studies we had observedthat the number of renin-producing cells is severalfold greaterin Agt+/– mouse kidney compared with WT (7), we lookedto see whether the number of renin-producing cells in the kidneyof Ren1c+/– kidney is two times that in WT. It is surprisingthat it is indistinguishable from WT, and this equality is stillmaintained even when renin synthesis is stimulated by low saltor enalapril. We conclude, therefore, that the production ofrenin mRNA and renin protein by individual renin-producing cellsin the kidney is the same in Ren1c+/– and WT mice. Possibleexplanations for this regulation at the cellular level in thekidney include an increase in the transcription rate of reninmRNA from a single renin gene in the Ren1c+/– and/or adecrease in the rate of degradation of renin mRNA in the Ren1c+/–animals. Further studies are needed to determine how these orother changes are executed in the renin-producing cells of theRen1c+/– mice. However, regardless of the details, theconclusion is clear: Kidney expression of renin mRNA is verytightly regulated. We are not aware of other examples in whichheterozygous mice have WT levels of protein except in the caseof NKCC2+/– mice (10). This is partly because heterozygoteswithout phenotype often are not studied carefully. Heterozygotescan have WT levels of protein at least three ways: By upregulationof mRNA production or stability in each cell, an increase inthe number of cells making the protein (7), or posttranscriptionalregulation (10). There is a need for more studies of heterozygotesto determine how often these types of compensation occur.
In summary, we have generated a mouse disrupted for Ren1c usingC57BL/6 ES cells. Ren1c–/– mice have hypotensionand impaired ability to concentrate urine. Ren1c+/– micehave the same BP, kidney renin mRNA level, and the number ofrenin-producing cells in the kidney as WT. These Ren1c-deficientmice will be useful for studies of regulation of BP and reninexpression.
Acknowledgments
This work is supported by grants from the National Institutesof Health (HL49277) to O.S., and from the Burroughs WellcomeFund (Innovation Awards in Functional Genomics 1001254) andthe American Heart Association (0265464U) to N.T. M.L.S.S.L.is a HHMI postdoctoral fellow.
Portions of this work have been published as an abstract (JAm Soc Nephrol 14, 29A, 2003) and presented at the Annual Meetingof the American Society of Nephrology, November 15, 2003, SanDiego, California.
We thank John Hagaman, Sylvia Hiller, and Lonquan Xu for excellentassistance.
Takahashi N, Hagaman JR, Kim H-S, Smithies O: Computer simulations of blood pressure regulation. Endocrinology 144: 2184–2190, 2003[Abstract/Free Full Text]
Takahashi N, Smithies O: Human genetics, animal models and computer simulations for studying hypertension. Trends Genet 20: 136–145, 2004[CrossRef][Medline]
Sigmund CD, Gross KW: Structure, expression, and regulation of the murine renin genes. Hypertension 18: 446–457, 1991[Abstract/Free Full Text]
Pentz ES, Lopez ML, Kim HS, Carretero O, Smithies O, Gomez RA: Ren1d and Ren2 cooperate to preserve homeostasis: Evidence from mice expressing GFP in place of Ren1d. Physiol Genomics 6: 45–55, 2001[Abstract/Free Full Text]
Yanai K, Saito T, Kakinuma Y, Kon Y, Hirota K, Taniguchi-Yanai K, Nishijo N, Shigematsu Y, Horiguchi H, Kasuya Y, Sugiyama F, Yagami K, Murakami K, Fukamizu A: Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice. J Biol Chem 275: 5–8, 2000[Abstract/Free Full Text]
Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, Smithies O: Uncompensated polyuria in a mouse model of Bartter’s syndrome. Proc Natl Acad Sci U S A 97: 5434–5439, 2000[Abstract/Free Full Text]
Kim HS, Maeda N, Oh GT, Fernandez LG, Gomez RA, Smithies O: Homeostasis in mice with genetically decreased angiotensinogen is primarily by an increased number of renin-producing cells. J Biol Chem 274: 14210–14217, 1999[Abstract/Free Full Text]
Krege JH, Hodgin JB, Hagaman JR, Smithies O: A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25: 1111–1115, 1995[Abstract/Free Full Text]
Kim HS, Lee G, John SW, Maeda N, Smithies O: Molecular phenotyping for analyzing subtle genetic effects in mice: Application to an angiotensinogen gene titration. Proc Natl Acad Sci U S A 99: 4602–4607, 2002[Abstract/Free Full Text]
Takahashi N, Brooks HL, Wade JB, Liu W, Kondo Y, Ito S, Knepper MA, Smithies O: Posttranscriptional compensation for heterozygous disruption of the kidney-specific NaK2Cl cotransporter gene. J Am Soc Nephrol 13: 604–610, 2002[Abstract/Free Full Text]
Siragy HM, Inagami T, Ichiki T, Carey RM: Protective role of the angiotensin AT2 receptor in a renal wrap hypertension model. Proc Natl Acad Sci U S A 96: 6506–6510, 1999[Abstract/Free Full Text]
Gomez RA, Lynch KR, Chevalier RL, Everett AD, Johns DW, Wilfong N, Peach MJ, Carey RM: Renin and angiotensinogen gene expression and intrarenal renin distribution during ACE inhibition. Am J Physiol 254: F900–F906, 1988
Sequeira Lopez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA: Renin-expressing cells are precursors for multiple cell types capable of switching to the renin phenotype when homeostasis is threatened. Dev Cell 6: 719–728, 2004[CrossRef][Medline]
Tanimoto K, Sugiyama F, Goto Y, Ishida J, Takimoto E, Yagami K, Fukamizu A, Murakami K: Angiotensinogen-deficient mice with hypotension. J Biol Chem 269: 31334–31337, 1994[Abstract/Free Full Text]
Kim HS, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda N, Smithies O: Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci U S A 92: 2735–2739, 1995[Abstract/Free Full Text]
Okubo S, Niimura F, Matsusaka T, Fogo A, Hogan BL, Ichikawa I: Angiotensinogen gene null-mutant mice lack homeostatic regulation of glomerular filtration and tubular reabsorption. Kidney Int 53: 617–625, 1998[CrossRef][Medline]
Kihara M, Umemura S, Sumida Y, Yokoyama N, Yabana M, Nyui N, Tamura K, Murakami K, Fukamizu A, Ishii M: Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice. Kidney Int 53: 548–555, 1998[CrossRef][Medline]
Krege JH, John SW, Langenbach LL, Hodgin JB, Hagaman JR, Bachman ES, Jennette JC, O’Brien DA, Smithies O: Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature 375: 146–148, 1995[CrossRef][Medline]
Esther CR Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, Bernstein KE: Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74: 953–965, 1996[Medline]
Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM: Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci U S A 95: 15496–15501, 1998[Abstract/Free Full Text]
Tsuchida S, Matsusaka T, Chen X, Okubo S, Niimura F, Nishimura H, Fogo A, Utsunomiya H, Inagami T, Ichikawa I: Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes. J Clin Invest 101: 755–760, 1998[Medline]
Berger S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe H, Kriz W, Warth R, Greger R, Schutz G: Mineralocorticoid receptor knockout mice: Pathophysiology of Na+ metabolism. Proc Natl Acad Sci U S A 95: 9424–9429, 1998[Abstract/Free Full Text]
Oliverio MI, Best CF, Smithies O, Coffman TM: Regulation of sodium balance and blood pressure by the AT(1A) receptor for angiotensin II. Hypertension 35: 550–554, 2000[Abstract/Free Full Text]
Brooks HL, Allred AJ, Beutler KT, Coffman TM, Knepper MA: Targeted proteomic profiling of renal Na(+) transporter and channel abundances in angiotensin II type 1a receptor knockout mice. Hypertension 39: 470–473, 2002[Abstract/Free Full Text]
de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T: International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52: 415–472, 2000[Abstract/Free Full Text]
Matsusaka T, Miyazaki Y, Ichikawa I: The renin angiotensin system and kidney development. Annu Rev Physiol 64: 551–561, 2002[CrossRef][Medline]
Oliverio MI, Delnomdedieu M, Best CF, Li P, Morris M, Callahan MF, Johnson GA, Smithies O, Coffman TM: Abnormal water metabolism in mice lacking the type 1A receptor for ANG II. Am J Physiol Renal Physiol 278: F75–F82, 2000[Abstract/Free Full Text]
Niimura F, Labosky PA, Kakuchi J, Okubo S, Yoshida H, Oikawa T, Ichiki T, Naftilan AJ, Fogo A, Inagami T: Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation. J Clin Invest 96: 2947–2954, 1995
Inokuchi S, Kimura K, Sugaya T, Inokuchi K, Murakami K, Sakai T: Hyperplastic vascular smooth muscle cells of the intrarenal arteries in angiotensin II type 1a receptor null mice. Kidney Int 60: 722–731, 2001[CrossRef][Medline]
Pentz ES: Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype. Am J Physiol 286: R474–R483, 2004
Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM: Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci U S A 92: 3521–3525, 1995[Abstract/Free Full Text]
Harris H: The Principles of Human Biochemical Genetics, New York, American Elsevier Publishing Company, 1970
Epstein CJ: Down syndrome (trisomy 21). In: The Metabolic Basis of Inherited Disease, Vol. 1, 6th Ed., edited by Scriver CR, New York, McGraw-Hill, 1989, pp 291–326
Received for publication June 21, 2004.
Accepted for publication October 6, 2004.
This article has been cited by other articles:
X. Zhou, E. T. Weatherford, X. Liu, E. Born, H. L. Keen, and C. D. Sigmund Dysregulated human renin expression in transgenic mice carrying truncated genomic constructs: evidence supporting the presence of insulators at the renin locus
Am J Physiol Renal Physiol,
September 1, 2008;
295(3):
F642 - F653.
[Abstract][Full Text][PDF]
R. A. Fenton and M. A. Knepper Mouse Models and the Urinary Concentrating Mechanism in the New Millennium
Physiol Rev,
October 1, 2007;
87(4):
1083 - 1112.
[Abstract][Full Text][PDF]
J. Stubbe, K. Madsen, F. T. Nielsen, R. K. Bonde, O. Skott, and B. L. Jensen Postnatal adrenalectomy impairs urinary concentrating ability by increased COX-2 and leads to renal medullary injury
Am J Physiol Renal Physiol,
September 1, 2007;
293(3):
F780 - F789.
[Abstract][Full Text][PDF]
H. A. Itani, X. Liu, J. H. Pratt, and C. D. Sigmund Functional Characterization of Polymorphisms in the Kidney Enhancer of the Human Renin Gene
Endocrinology,
March 1, 2007;
148(3):
1424 - 1430.
[Abstract][Full Text][PDF]
X. Zhoux, D. R. Davis, and C. D. Sigmund The Human Renin Kidney Enhancer Is Required to Maintain Base-line Renin Expression but Is Dispensable for Tissue-specific, Cell-specific, and Regulated Expression
J. Biol. Chem.,
November 17, 2006;
281(46):
35296 - 35304.
[Abstract][Full Text][PDF]
M. Lacoste, Y. Cai, L. Guicharnaud, F. Mounier, Y. Dumez, R. Bouvier, F. Dijoud, M. Gonzales, J. Chatten, A.-L. Delezoide, et al. Renal Tubular Dysgenesis, a Not Uncommon Autosomal Recessive Disorder Leading to Oligohydramnios: Role of the Renin-Angiotensin System
J. Am. Soc. Nephrol.,
August 1, 2006;
17(8):
2253 - 2263.
[Abstract][Full Text][PDF]
L. J. Mullins, M. A. Bailey, and J. J. Mullins Hypertension, Kidney, and Transgenics: A Fresh Perspective
Physiol Rev,
April 1, 2006;
86(2):
709 - 746.
[Abstract][Full Text][PDF]
I. V. Yosypiv, M. Schroeder, and S. S. El-Dahr Angiotensin II Type 1 Receptor-EGF Receptor Cross-Talk Regulates Ureteric Bud Branching Morphogenesis
J. Am. Soc. Nephrol.,
April 1, 2006;
17(4):
1005 - 1014.
[Abstract][Full Text][PDF]
N. Phadnis and J. D. Fry Widespread Correlations Between Dominance and Homozygous Effects of Mutations: Implications for Theories of Dominance
Genetics,
September 1, 2005;
171(1):
385 - 392.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
Ct method, which normalized expression to 
-actin as an internal control by subtracting the Ct value for the gene of interest from the Ct value for 
subunit of the amiloride-sensitive epithelial type of Na+ channel (24). Both are important for salt reabsorption in the renal tubules, and their expression and function are normally stimulated by angiotensin II. However, we found that angiotensin II infusion to the Ren1c–/– mice did not significantly increase the osmolality of their urine. 
