Abstract
Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c−/−) are born at the expected ratio, but approximately 80% die of dehydration 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. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 ± 4 versus 116 ± 5 mmHg in +/+) and excrete large amounts of urine (5.2 ± 0.8 ml/d, 725 ± 34 mOsm versus 1.1 ± 0.1 ml/d, 2460 ± 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in −/− mice (624 ± 19 to 656 ± 25 mOsm), whereas in +/+, it increases severalfold (583 ± 44 to 2630 ± 174 mOsm). Minipump infusion of angiotensin II to Ren1c−/− mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/− mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/− mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.
The renin angiotensin system (RAS) is important for regulating BP and cardiovascular homeostasis (1). Renin secreted from the kidney cleaves angiotensinogen (AGT) to angiotensin I, which is converted to angiotensin II by the angiotensin-converting enzyme (ACE). Others and we have been investigating the effects of changes in the expression of genes of the RAS using mice. Most mouse models have been generated by gene targeting using embryonic stem (ES) cells of strain 129 (2). This strain differs from humans in having two renin genes in tandem on the same chromosome (Ren2 and Ren1d) that differ in their expression and regulation in ways that complicate interpreting experimental data (3). For example, Ren2 can compensate for the effects of the 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 involving changes in renin expression (3). Yanai et al. (5) approached this problem by generating mice with the Ren1c gene disrupted using TT2 ES cells derived from an F1 hybrid between C57BL/6 and CBA. Although this approach provides a satisfactory model for studying the consequences of complete absence of renin, it is less satisfactory for studying the effects of mild quantitative difference in the expression of Ren1c because the resulting mice have a heterogeneous genetic background. This problem can be overcome by using mice that have a pure genetic background so that small differences in the phenotype can be detected without being obscured by the unfavorable heterogeneity of genetic background that occurs when two strains are combined. Accordingly, we have generated mice lacking Ren1c using C57BL/6-derived ES cells and have maintained the C57BL/6 background in their progeny. Using these coisogenic mice, we show that mice completely lacking Ren1c show hydronephrosis and are unable to concentrate urine. It is surprising that the Ren1c+/− heterozygotes have plasma renin and kidney renin mRNA indistinguishable from wild-type (WT), showing that kidney renin is very tightly regulated at the mRNA level.
Materials and Methods
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) and G418/ganciclovir selection, the ES cells were screened by PCR using primers 5′-AGAGCGGTCTCATCTTTCCATAG-3′ and 5′-GAGAGGCTTTTTGCTTCCTCTT-3′. Clones that give a 1.1-kb PCR band were expanded, and targeting was confirmed by Southern blot analysis of their genomic DNA digested with BamH I and hybridized to the probe c. Targeted clones have a 3.4-kb band in addition to a 2.7-kb endogenous band (Figure 1B). The DNA was also digested with Bgl II and hybridized to the probe d; targeted clones have a 13.5-kb band in addition to an 11.2-kb endogenous band (data not shown). Male chimeras that carry the disrupted allele were mated with C57BL/6 female mice to obtain animals that retained the genetically uniform C57BL/6 background. Genotypes were determined by PCR with 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 with the +/+, −/−, 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 in accordance with the National Institutes of Health guidelines for the use and care of experimental animals, as approved by the IACUC of UNC-CH.
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 described previously (6,7). Mice were exposed to an atmosphere of CO2, and blood then was rapidly withdrawn from the descending aorta of the unconscious mice into ice-cold microcentrifuge tubes that contained EDTA (<1 min from loss of consciousness to the end of collection) and immediately centrifuged to isolate plasma. PRC was determined in the presence of sufficient exogenous high AGT rat plasma to maximally stimulate production of angiotensin I using an angiotensin I [125I] RIA kit NEA104 (NEN Life Science Products Inc., Boston, MA). Angiotensin II was measured using RIA 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 Sequence Detection System (Applied Biosystems, Foster City, CA) using primers and probes designed using Primer Express software (Applied Biosystems) (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 quantification were 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 μg of total RNA. Changes in expression were determined by the ΔCt method, which normalized expression to β-actin as an internal control by subtracting the Ct value for the gene of interest from the Ct value for β-actin. Because ΔCt of 1 corresponds to a twofold difference in expression, expression as a percentage of WT is 100 × 2ΔCt Ren1c−/− or +/− divided by the mean of 2ΔCt +/+. Experiments were carried out it duplicate, and the means of each duplicate were used to calculate expression as percentage of +/+. For aldosterone synthase, the primers are 5′-AGA GAA CTC CGT GGC CTG A-3′ and 5′-CCG CAG TCG GTT GAG ACG-3′, and the probe is 5′-FAM-CGT GGT GTG TTC TTG CTA AAT GGG CC-TAMRA-3′.
Analyses of Urine and Kidney Function
Body weight, water and food intake, and urine excretion were measured every 24 h for 3 d (6). Electrolytes and albumin were measured using the VT250 Chemical Analyzer and Albuwell (Exocell, Philadelphia, PA), respectively, and normalized to 20 g body weight. Aldosterone was measured using aldosterone DA RIA kit (ICN Pharmaceuticals, Inc., Orangeburg, NY). The effect of vasopressin on urine concentrating ability was tested by measuring urine osmolality before and 1 and 2 h after injecting 1 ng/g body weight desmopressin (Sigma, St. Louis, MO) intraperitoneally.
Effect of Angiotensin II on Kidney Function
Mice were continuously infused subcutaneously with 4 ng/kg per min angiotensin II (Sigma) using an osmotic minipump, model 1002 (Alzet, Cupertino, CA). This dose of angiotensin II gives normal to slightly higher-than-normal but still subpressor levels of plasma angiotensin II (11). Before and 1 wk after minipump implantation, daily urine excretion, urine osmolality, and BP were 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 administered in the drinking water at a concentration of 0.2 mg/ml for 5 d (12) before assessment of PRC, renin mRNA, and renin immunohistochemistry.
Renin Immunohistochemistry and Morphometric Analysis
The distribution and the number of renin-producing cells were determined as described previously (7,12,13).
Statistical Analyses
All values are expressed as mean ± SEM. The t test was used for statistical evaluations.
Results
Generation of Mice with the Ren1c Gene Disrupted
Mice that carry a null allele of the Ren1c gene were generated from C57BL/6 ES cells by deleting the promoter and downstream sequences through exon 6 that include the catalytic site (Figure 1). Matings between Ren1c+/− heterozygotes produced +/+, +/−, and −/− pups at the expected Mendelian ratios at birth (15 +/+, 30 +/−, and 14 −/−). There were no obvious differences in appearance or body weight among the three genotypes at birth, but approximately 80% of the −/− mice died within a few days (56 +/+, 100 +/−, 12 −/− at weaning). Plasma Na+ concentration at 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 (50 μl/g body weight) from day 1 for 2 wk rescued approximately 50% of the −/− mice. The life span of the −/− mice that survived to weaning was close to normal, although their body weights were approximately 20% lower than WT at 4 mo 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. The newborn −/− pups from this mating did not have any obvious pathologic changes.
BP, Plasma, and the Renin Angiotensin Aldosterone System in Ren1c−/−
The BP of −/− mice is approximately 30 mmHg lower than WT counterparts at 4 mo of age (Table 1). They showed metabolic acidosis and high plasma creatinine and blood urea nitrogen, indicating a substantial reduction in GFR. Renin and angiotensins I and II in plasma are undetectable. Daily urinary aldosterone excretion is <10% of WT. Aldosterone synthase mRNA expression in 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).
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 daily than WT (Table 1). Daily urinary Na+, K+, Cl−, and creatinine excretion is indistinguishable from WT (data not shown). Daily urinary albumin excretion in the −/− tends to be higher 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 determining whether the −/− mice can concentrate urine in response to dehydration, they were deprived of water for 12 h. Although this deprivation induces a loss of >20% of body weight, the −/− mice do not increase the concentration of their urine. This could be due to a structural kidney defect and/or to unresponsiveness to vasopressin. To test the latter possibility, we administered an antidiuretic hormone analogue, desmopressin (1 ng/g intraperitoneally). Figure 2 shows that this treatment did 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 increased five times (583 ± 44 to 2630 ± 174; n = 12; P < 0.0001). We next tested the possibility that angiotensin II is required for the kidney to respond to antidiuretic signals by administering angiotensin II subcutaneously using an osmotic minipump. Angiotensin II (4 ng/kg per min) restored the BP of the −/− mice to levels not different from WT (without angiotensin II, 86.4 ± 2.8 mmHg; with angiotensin II, 107.4 ± 2.8 mmHg; n = 6; P < 0.0005) and tended to decrease the daily urine volume and increase urine osmolality (without angiotensin II, 5.6 ± 0.6 ml/24 h per 20 g body weight, 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.
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 varying degrees (Figure 3, A through C). The severely affected kidneys have a thin medulla with an atrophic/hypoplastic papilla and a dilated renal pelvis. The surface of these −/− kidneys looks granular. The kidneys of the −/− mice are smaller than normal (142.5 ± 11.6 versus 194.8 ± 6.9 mg in WT; n = 6 each; P = 0.006), although the kidney/body weight 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 afferent arterioles are thickened as a result of an increased number of cells (Figure 3E). These vascular abnormalities are confined to the kidney. Other tissues, including the hearts, adrenal glands, and lungs, seem histologically normal. In particular, there is no evidence of arterial or arteriolar thickening at these sites.
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 in the +/− heterozygotes affects BP and renal function. Table 2 presents the surprising result that BP and PRC of the +/− mice are not different from WT. Body weight and kidney weight/body weight also are not different. Likewise, daily urine volume, Na+, K+, Cl−, creatinine excretion, and urine osmolality of the +/− are not different from WT (data not shown).
Ren1c+/− phenotypea
Renin Expression of Heterozygous Ren1c+/−
Past experience with heterozygous null mice showed that they have levels of mRNA that are 50% of WT. We therefore expected the 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 expression is significantly different (P < 0.0001) from the expected 50%. This suggests the possibility that the number of renin-producing cells in the kidneys of Ren1c+/− mice is twice WT, but we found that the numbers of renin-producing cells are not different between WT and heterozygous animals on normal or on low-salt diets. However, because the number of renin-producing cells is small, we thought that the differences between the two genotypes might be difficult to detect. We therefore stimulated renin production by treating the mice with enalapril. This treatment leads to the generation of hypertrophic renin-producing cells along the preglomerular arterioles (Figure 5). The number of renin-producing cells again was not different between WT and heterozygous animals that were treated with enalapril (WT 1202 ± 90; +/− 1078 ± 79; P = 0.24).
Renin mRNA expression in kidneys of Ren1c+/− and WT mice. Renin mRNA was quantified using real-time reverse transcriptase–PCR with β-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.
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+/−.
Discussion
We have generated mice that lack renin by disrupting the Ren1c gene using ES cells derived from C57BL/6 mice, a strain that has only a single renin gene. The Ren1c−/− mice are born in expected numbers but have reduced survival (approximately 20%), low BP, impaired ability to concentrate urine, and abnormal kidney structure, a phenotype virtually identical to that of homozygous 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 as a result of their decreased BP and inability to conserve salt and water normally. The absence of a difference in the plasma Na+ concentration between Ren1c−/− and WT and the rescue of some pups by saline injection suggest that the Ren1c−/− pups die of fluid volume depletion consequent to NaCl losses as also seen in mineralocorticoid receptor −/− pups (22). This contrasts with the dehydration as a result of water loss seen in kidney-specific NaK2Cl cotransporter (NKCC2) −/− pups (6). Although renin is normally expressed in the reproductive organs of both male and female mice, Ren1c−/− male and female mice with no renin expression are fertile, indicating that renin is dispensable for fertility and that fertility is not dependent on angiotensin production.
The low BP of the Ren1c−/− mice and the absence of renin and plasma angiotensins indicate that renin is the only 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 II is 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) is sufficient for normal aldosterone production.
The kidneys of the Ren1c−/− mice develop varying degrees of hydronephrosis. Previous investigations have shown that loss of angiotensin II receptors leads to the absence of ureteral 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 ureter and may account for the subsequent development of the medullary damage and hydronephrosis. The development of hydronephrosis could also be predicted by their higher urine output compared with WT combined with the dramatic increase in urine production that normally occurs shortly after birth. These factors may exceed the maximum capability of the ureter to remove urine, causing an increase in backward pressure in the renal pelvis and leading to hydronephrosis, inflammation, fibrosis, and atrophy of the papilla and enlarged calyx (26).
The Ren1c−/− mice drink several times more water than WT and excrete a corresponding amount of urine. Because water deprivation does not increase the osmolality of their urine, they excrete more urine not because they drink more but because they are unable to concentrate urine. Polyuria and polydipsia can be due either to a decrease in vasopressin from the brain or to failure of the kidney to respond to vasopressin. Because the Ren1c−/− mice do not respond to desmopressin, their polyuria is nephrogenic. This is largely due to the medullary damage that disables generation of axial osmotic gradient necessary to concentrate urine. In addition, angiotensin II may be required for vasopressin to concentrate urine, because Agtr1a−/− mice also show an impaired ability to concentrate urine even though they have no hydronephrosis (27). Agtr1a−/− mice have decreased expression of the thiazide-sensitive NaCl co-transporter and the α 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.
Another phenotype observed in the Ren1c−/− mice is medial thickening of the small arteries in the kidney but not of the vessels outside the kidney. This phenotype has been described by others in renin-deficient mice (5) as well as in mice deficient of Agt (15,17,28), Ace (18,19), Agtr1a (29), and Agtr1a/1b (20,21). Mononuclear cell infiltration surrounding the thickened arteries is evident, suggesting that some common pathogenic mechanism causes vascular thickening and perivascular inflammation. Because both are seen in the renin-deficient mice, it is clear that neither renin nor the angiotensins are necessary for this phenotype. Mice in which all cells that express or have previously expressed renin have been ablated do not show this phenotype (30). It therefore is likely that the hypertrophy that we see in the renal vessels of the Ren1c−/− mice involves cells that during development would normally synthesize renin. This suggests that renin-producing cells per se may contribute to the vessel thickening possibly by excreting some factor(s) other than renin that is coordinately regulated by the expression level of renin.
Because our Ren1c+/− mice are derived from C57BL/6 ES cells and are maintained on the pure C57BL/6 genetic background, we have been able to make and interpret quantitative measurements in the Ren1c+/− mice that are not possible with heterozygous Ren1c+/− mice that have a mixed genetic background. The heterozygous gene disruption of other genes in the renin angiotensin system, such as Agt and Agtr1a, shows lower BP than WT (15,31). In contrast, the most striking finding in Ren1c disruption is that there is no difference between the Ren1c+/− and WT in BP, kidney function, or PRC. This is surprising because, in general, the expression of a gene product is proportional to the number of copies of the corresponding gene (32,33). Thus, many previous observations have shown that the expression of a protein is usually close to 50% of normal in individuals who are heterozygous for a loss of function mutation (32). However, the amount of renin mRNA in the Ren1c+/− kidney is almost as high as WT and does not differ significantly from WT, but it is significantly different from 50% WT. Therefore, some feedback regulation must have restored the kidney renin mRNA level back to the WT level. Because in earlier studies we had observed that the number of renin-producing cells is severalfold greater in Agt+/− mouse kidney compared with WT (7), we looked to see whether the number of renin-producing cells in the kidney of Ren1c+/− kidney is two times that in WT. It is surprising that it is indistinguishable from WT, and this equality is still maintained even when renin synthesis is stimulated by low salt or enalapril. We conclude, therefore, that the production of renin mRNA and renin protein by individual renin-producing cells in the kidney is the same in Ren1c+/− and WT mice. Possible explanations for this regulation at the cellular level in the kidney include an increase in the transcription rate of renin mRNA from a single renin gene in the Ren1c+/− and/or a decrease in the rate of degradation of renin mRNA in the Ren1c+/− animals. Further studies are needed to determine how these or other changes are executed in the renin-producing cells of the Ren1c+/− mice. However, regardless of the details, the conclusion is clear: Kidney expression of renin mRNA is very tightly regulated. We are not aware of other examples in which heterozygous mice have WT levels of protein except in the case of NKCC2+/− mice (10). This is partly because heterozygotes without phenotype often are not studied carefully. Heterozygotes can have WT levels of protein at least three ways: By upregulation of mRNA production or stability in each cell, an increase in the number of cells making the protein (7), or posttranscriptional regulation (10). There is a need for more studies of heterozygotes to determine how often these types of compensation occur.
In summary, we have generated a mouse disrupted for Ren1c using C57BL/6 ES cells. Ren1c−/− mice have hypotension and impaired ability to concentrate urine. Ren1c+/− mice have the same BP, kidney renin mRNA level, and the number of renin-producing cells in the kidney as WT. These Ren1c-deficient mice will be useful for studies of regulation of BP and renin expression.
Acknowledgments
This work is supported by grants from the National Institutes of Health (HL49277) to O.S., and from the Burroughs Wellcome Fund (Innovation Awards in Functional Genomics 1001254) and the 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 (J Am Soc Nephrol 14, 29A, 2003) and presented at the Annual Meeting of the American Society of Nephrology, November 15, 2003, San Diego, California.
We thank John Hagaman, Sylvia Hiller, and Lonquan Xu for excellent assistance.
- © 2005 American Society of Nephrology
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