1 Renal Division, Department of Medicine, 2 Department of Pathology, and 3 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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ACE.2 mice lack all tissue
angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE
activity. They exhibit the urine-concentrating defect and hyperkalemia
present in mice that lack all ACE, but in contrast to the complete
knockout, ACE.2 mice have normal medullary histology and creatinine
clearance. To explore the urine-concentrating defect in ACE.2 mice,
renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1)
were significantly reduced to 28 ± 5, 6 ± 6, and 39 ± 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B
were unchanged. In the outer medulla,
Na+-K+-2Cl cotransporter
(NKCC2/BSC1) and AQP1 were significantly reduced to 56 ± 11 and
29 ± 6%, respectively, whereas
Na+-K+-ATPase, UT-A2, UT-B, and AQP2 were
unchanged, and renal outer medullary potassium channel was
significantly increased to 711 ± 187% of the level in wild-type
mice. The abnormal expression of these transporters was similar in
ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not
rescued by ANG II infusion. We conclude that the urine-concentrating
defect in ACE.2 mice is associated with, and may result from,
downregulation of some or all of these key urea, salt, and water
transport proteins.
urine-concentrating mechanism; angiotensin; urea; sodium chloride; potassium; aquaporin; angiotensin-converting enzyme
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INTRODUCTION |
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THE PRODUCTION OF CONCENTRATED or dilute urine requires transport mechanisms for the independent control of water and sodium excretion (reviewed in Ref. 30). In recent years, the cloning of cDNAs for several key transport proteins in the renal medulla and the production of polyclonal antibodies to these transporters have improved our understanding of the molecular basis for the urine-concentrating mechanism (reviewed in Refs. 13, 23, and 28).
In the outer medulla, the thick ascending limb of the loops of Henle
actively reabsorb NaCl, thereby diluting the luminal fluid and
providing NaCl to increase the osmolality of the medullary interstitium. NaCl is reabsorbed by the action of the
Na+-K+-2Cl cotransporter
(NKCC2/BSC1) in the apical membrane of the thick ascending limb and the
sodium pump (Na+-K+- ATPase) in the
basolateral membrane; the reabsorbed potassium is secreted back into
the thick ascending limb lumen by the renal outer medullary potassium
channel (ROMK) Kir 1.1 (reviewed in Refs. 8,
9, and 35).
In the inner medulla, osmolality continues to increase from the inner-outer medullary border to the papillary tip. Although the precise mechanism by which this occurs remains controversial, the most widely accepted mechanism remains the passive mechanism hypothesis proposed in 1972 by Kokko and Rector (14) and by Stephenson (32). The passive mechanism hypothesis proposes that NaCl can be reabsorbed passively from the thin ascending limb of the loops of Henle because the inner medullary interstitium has a lower NaCl concentration, but a higher urea concentration, than fluid within its lumen. This permits NaCl to be reabsorbed from the thin ascending limb down its concentration gradient. Chloride is reabsorbed by the chloride channel, ClC-K1, in the thin ascending limb; the molecular mechanism for sodium reabsorption is unknown (reviewed in Ref. 30).
The passive mechanism also requires a very high urea concentration within the inner medullary interstitial fluid to balance the higher NaCl concentration in the lumen of the thin ascending limb. The source of this urea is reabsorption from the terminal inner medullary collecting duct (IMCD; reviewed in Refs. 28 and 31). The countercurrent configuration of nephron segments and vessels allows the generation of a medullary osmolality gradient along the corticomedullary axis. In the presence of a hypertonic medulla and vasopressin [AVP; also called antidiuretic hormone (ADH)], the entire collecting duct becomes highly permeable to water due to the insertion of aquaporin-2 (AQP2) water channels into the apical membrane (reviewed in Ref. 23), water is reabsorbed, and a concentrated urine is produced.
Several studies have documented that knockout mice with a genetic lack of angiotensinogen, angiotensin-converting enzyme (ACE), or both angiotensin type 1a (AT1a) and 1b (AT1b) receptors have a urine-concentrating defect due, in part, to a marked underdevelopment of the renal medulla (reviewed in Ref. 5). Previously, we described a mouse, termed ACE.2, in which homologous recombination in embryonic stem cells resulted in an animal expressing a truncated form of ACE (7). This protein, while catalytic, lacks the COOH-terminal domain necessary for ACE incorporation into cell membranes. Thus ACE.2 mice have no tissue ACE and very low angiotensin II levels but possess ~33% of normal circulating (plasma) levels of ACE (7). While ACE.2 mice have a very low blood pressure, equivalent to that of mice lacking all ACE, light microscopy shows that they typically have normal development of the renal medulla (7). ACE null mice have some degree of renal insufficiency, as indicated by an elevation of serum creatinine, but ACE.2 mice have serum creatinine and creatinine clearance values that are indistinguishable from wild-type littermate control mice (7). Despite normal renal histology and renal function, ACE.2 mice have a profound urine-concentrating defect, equivalent to that present in mice lacking all ACE expression (7). To gain insight into the molecular basis for the urine-concentrating defect in the ACE.2 mice, we measured the abundance of several of the major solute and water transport proteins in the renal inner and outer medulla.
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METHODS |
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ACE.2 knockout mice. ACE.2 knockout mice were generated using targeted homologous recombination as previously described (7). The mice used in this study were from the fifth and sixth generations of backcrossing onto a C57BL/6 background; wild-type littermates were used as controls (7). In addition, mice were backcrossed onto a Swiss background (Taconic, Germantown, NY). The genotype of the mice was confirmed by standard PCR analysis. Blood was drawn by cardiac puncture at the time of death and assayed for sodium, potassium, chloride, and bicarbonate by the Emory University Pathology Department Clinical Laboratory.
Urine-concentrating ability. ACE.2 and wild-type mice were water deprived for 24 h but given food ad libitum. Urine was collected during this 24-h period and assayed for creatinine, sodium, potassium, chloride, and pH by the Emory University Pathology Department Clinical Laboratory. At the end of the 24-h period, a spot urine was collected and its osmolality was measured using a vapor pressure osmometer (model 5500, Wescor, Logan, UT). The mice were allowed to rehydrate for 3 wk before death.
Angiotensin II infusion.
Angiotensin II (0.5 mg · kg1 · day
1 dissolved in
sterile 0.9% saline) or vehicle (saline) was infused via osmotic
minipump into ACE.2 and wild-type mice for 2 wk (2). Blood
pressure was measured by tail cuff as previously described
(2). Two days before death, a spot urine sample was
collected to measure osmolality, and then the mice were water
restricted for 8 h, after which a second spot urine sample was
collected for osmolality measurement. The mice were allowed to
rehydrate for 48 h before death. Blood was collected at death, and
aldosterone was measured using a radioimmunoassay (Diagnostic Products,
Los Angeles, CA).
Preparation of kidney samples. Kidneys from ACE.2 and wild-type mice were dissected into cortex, outer medulla, and inner medulla as described (21), except that the inner medulla was not subdivided into base and tip portions due to the small size of the inner medulla in mice. Tissues from both kidneys of a single mouse were pooled and placed into ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, 0.1 mg/ml PMSF, pH 7.6, 0.025-0.1 g tissue/ml isolation buffer), homogenized, sheared with a 26-gauge needle, and diluted 1:1 with 1% SDS for Western blot analysis of total cell lysate. Total protein in each sample was measured by the Bradford method (DC Protein Assay Kit, Bio-Rad, Richmond, CA).
Western blot analysis. Proteins (10 µg/lane, except 30 µg/lane for ClC-K1) were size separated by SDS-PAGE using 7.5, 10, or 12% polyacrylamide gels. Proteins were blotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris · HCl, 0.5 M NaCl, pH 7.5) at room temperature for 30 min, then incubated with primary antibody overnight at 4°C. Blots were washed three times in TBS with 0.5% Tween 20 (TBS/Tween) and then incubated with horseradish peroxidase-linked goat anti-rabbit IgG at a dilution of 1:4,000-1:5,000 (Amersham, Arlington Heights, IL) for 2 h at room temperature. Blots were washed twice with TBS/Tween, and then the bound secondary antibody was visualized using chemiluminescence (ECL kit, Amersham). Blots were quantified using an Imaging Densitometer GS670 and Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). In all cases, parallel gels were stained with Coomassie blue and showed uniformity of loading (data not shown). Results are expressed as arbitrary units per microgram protein loaded.
Antibodies.
Western blots were probed with antibodies (diluted in TBS/Tween) to the
following proteins: NKCC2/BSC1 (generous gift of Dr. B. K. Kishore, Univ. of Utah) (4, 11); -1 subunit of
Na-K-ATPase (Upstate Biotechnologies, Lake Placid, NY)
(22); ROMK (generous gift of Dr. M. A. Knepper,
National Institutes of Health) (3); UT-A1 and UT-A2
(21); UT-B (34); AQP2 (generous gift of Dr. B. K. Kishore) (12, 22); ClC-K1 (Alomone Labs,
Jerusalem, Israel); and AQP1 (generous gift of Dr. M. A. Knepper)
(33).
Statistics. Data are presented as means ± SE, and n is the number of mice. An unpaired Student's t-test was used to test for statistical significance, with P < 0.05 indicating statistical significance.
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RESULTS |
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Blood and urine chemistries.
Serum potassium was significantly higher in ACE.2 than wild-type mice
(Table 1). There was no difference in
serum sodium, chloride, or bicarbonate (Table 1). To verify that the
ACE.2 mice in this study had a urine-concentrating defect
(7), we measured urine osmolality after water deprivation.
Maximal urine osmolality was significantly lower in the ACE.2 mice
(1,020 ± 10 mosmol/kgH2O) than in the wild-type mice
(3,300 ± 10 mosmol/kgH2O, Table
2). Urine volume and urine potassium
excretion were significantly increased, and urine pH was significantly
more acidic in the ACE.2 mice, compared with wild-type mice (Table 2).
Urine creatinine, sodium, and chloride excretion were similar in
wild-type and ACE.2 mice (Table 2).
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Inner medulla.
In ACE.2 mice, the protein abundances of the 117-kDa urea transporter
UT-A1 (Fig. 1), ClC-K1, the ~80-kDa
chloride channel (Fig. 2), and AQP1 (Fig.
3) were significantly reduced to 28 ± 5, 6 ± 6, and 39 ± 5%, respectively, of the levels in
wild-type mice. The protein, abundances of the 97-kDa UT-A1 protein
(Fig. 1), the water channel AQP2 (Fig.
4), and the urea transporter UT-B (Fig.
5) were not different between kidneys
from ACE.2 and wild-type mice.
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Outer medulla.
In ACE.2 mice, the protein abundances of NKCC2/BSC1, the
Na+-K+-2Cl cotransporter (Fig.
6), and the AQP1 water channel (Fig. 3)
were significantly reduced to 56 ± 11 and 29 ± 6% of the
level in wild-type mice, respectively. However, the protein abundances
of the
-1 subunit of Na+-K+-ATPase (Fig.
7), UT-A2 (Fig.
8), UT-B (Fig. 5), and AQP2 (Fig. 4)
were not different between kidneys from ACE.2 and wild-type mice.
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Cortex. The abundance of AQP1 and AQP2 proteins was not significantly different between kidneys from ACE.2 and wild-type mice (data not shown).
Swiss background. To ensure that the preceding changes were due to the ACE.2 knockout rather than the C57BL/6 background, we repeated these studies using ACE.2 mice bred onto a Swiss background (n = 7 wild-type and 7 knockout mice). All of the results in the Swiss mice were the same as in C57BL/6 mice (data not shown).
Angiotensin II infusion. To determine whether the preceding changes could be rescued by angiotensin II infusion, minipumps with angiotensin II or vehicle were implanted into ACE.2 mice bred onto the C57BL/6 and Swiss backgrounds. Angiotensin II increased blood pressure and plasma aldosterone (vehicle: 1.9 ± 0.3 nM; angiotensin II: 5.2 ± 0.7 nM, n = 4, P < 0.01) in the ACE.2 mice, indicating that the pumps worked correctly but had no effect on urine osmolality or the abundance of any transport protein (data not shown).
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DISCUSSION |
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The major finding of this study is that the abundances of several major solute transport proteins responsible for generating a hypertonic medullary interstitium and of those of the AQP1 water channel are significantly decreased in genetically engineered mice that lack tissue ACE. These effects are independent of background (C57BL/6 vs. Swiss), strongly suggesting that the changes are a specific result of ACE.2 knockout. In addition, the changes in urine osmolality and transporter protein abundances are not corrected by angiotensin II infusion, suggesting that angiotensin II administration to adult ACE.2 mice does not rescue these phenotypic features.
The marked (>80%) decreases in UT-A1 and ClC-K1 in ACE.2 mice could contribute to the urine-concentrating defect by reducing the amount of urea reabsorbed from the terminal inner medullary collecting duct and NaCl reabsorption from the thin ascending limb, respectively, thereby reducing the inner medullary contribution of these osmotically active solutes to the generation of a hypertonic medullary interstitium. The modest decrease in NKCC2/BSC1 may also contribute to the concentrating defect by reducing NaCl reabsorption from the thick ascending limb. The decrease in AQP1 may reduce water transport across the thin descending limb and descending vasa recta (1, 26). Although we measured protein abundance in this study, rather than transport by perfused tubule, we and others have found an excellent correlation between the abundance of solute and water transporter proteins by Western blot analysis and transport as measured in isolated perfused kidney tubules (reviewed in Refs. 13, 23, and 28). While it is tempting to conclude that the urine-concentrating defect in these ACE.2 mice results from the decrease in the abundance of these transport proteins, especially in the inner medulla, the present studies can only establish associations between changes in transporter abundance and the urine-concentrating defect rather than establishing a direct cause-and-effect relationship.
Inner medulla. According to the passive mechanism hypothesis (14, 32), the production of concentrated urine in the inner medulla requires the delivery of large quantities of urea to the inner medullary interstitium to establish the chemical gradients necessary for passive NaCl reabsorption from the thin ascending limb. We have shown that urea reabsorption from the terminal IMCD is the source of urea for delivery to the inner medullary interstitium (29). The UT-A1 urea transporter is expressed only in the apical membrane of the IMCD (24). Thus the large decrease in UT-A1 protein could contribute to the urine-concentrating defect by reducing urea delivery to the inner medullary interstitium.
In normal rats, angiotensin II (in the presence of vasopressin) increases the phosphorylation of UT-A1 and urea permeability in perfused terminal IMCDs (10). Thus the lack of angiotensin II in the ACE.2 mice may also result in submaximal function of the small amount of UT-A1 protein that is present in their inner medulla. ClC-K1 is normally expressed in the thin ascending limb and is responsible for chloride reabsorption (16). ClC-K1 is important for producing concentrated urine, because knockout mice that lack ClC-K1 (Clcnk1 mice) have a severe urine-concentrating defect, and treating these mice with vasopressin does not improve urine-concentrating ability (19). The magnitude of the urine-concentrating defect in the ACE.2 mice and the Clcnk1 mice is very similar, suggesting that the >90% decrease in ClC-K1 protein in the ACE.2 mice may be the major cause of their concentrating defect. AQP1 is normally expressed in the thin descending limb and descending vasa recta and is responsible for water transport (1, 26). AQP1 is important for producing concentrated urine, because knockout mice that lack AQP1 have a severe urine-concentrating defect (18). The two-thirds reduction in AQP1 protein in both the outer and inner medulla may contribute to the urine-concentrating defect in the ACE.2 mice. Another surprising finding was the lack of change in AQP2, because AQP2 protein abundance is generally reduced when urine-concentrating ability is reduced (reviewed in Ref. 23). Although the reabsorption of sodium and urea is crucial to the production of a hypertonic medulla, the excretion of concentrated urine requires the reabsorption of water (in excess of solute). The present study does not address whether ACE.2 mice have an abnormality in the regulated trafficking of AQP2, despite normal AQP2 protein abundance, nor whether there is a change in the abundance of AQP3 or AQP4 (located in the basolateral membrane of the collecting duct), and future studies will be needed to test these possibilities.Outer medulla.
NKCC2/BSC1 is expressed only in the apical membrane of the thick
ascending limb (20). In combination with
Na+-K+-ATPase in the basolateral membrane,
NKCC2/BSC1 contributes to the active NaCl reabsorption that is needed
to generate a hypertonic medullary interstitium (reviewed in Refs.
9 and 13). Knepper and colleagues (36)
recently showed that NKCC2/BSC1 protein is decreased in hypercalcemic
rats to 36% of control values in the inner stripe of the outer medulla
and concluded that this reduction in NKCC2/BSC1 contributes to the
concentrating defect in hypercalcemia (36). In this study,
we found that NKCC2/BSC1 protein was reduced to 56% of the level in
wild-type mice in whole outer medulla. The decrease in NKCC2/BSC1 does
not appear to result from nonspecific damage to the thick ascending
limb, because neither we (present study) nor Knepper and colleagues
(36) found a change in the abundance of the -1 subunit
of Na+-K+-ATPase that would occur if there were
cell damage. Although the decrease in NKCC2/BSC1 protein is modest
compared with the changes in UT-A1 and ClC-K1 in the inner medulla, it
is similar to the decrease in NKCC2/BSC1 (and urine osmolality) in
hypercalcemic rats (36), suggesting that the decrease in
NKCC2/BSC1 may also contribute to the urine-concentrating defect in the
ACE.2 mice.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. B. K. Kishore (Univ. of Utah) and M. A. Knepper (National Institutes of Health) for generously providing antibodies and Dr. William E. Mitch (Emory Univ.) for critically reading this manuscript.
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FOOTNOTES |
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* J. D. Klein and D. Le Quach contributed equally to this work.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-51445, R01-DK-55503, and R37-DK-39777 (to K. E. Bernstein) and R01-DK-41707, R01-DK-63657, and P01-DK-50268 (to J. M. Sands).
Portions of this work have been published as abstracts (J Am Soc Nephrol 10: 42A, 1999; J Am Soc Nephrol 12: 33A-34A, 2001; and FASEB J 16: A51, 2002) and presented at the 32nd Annual Meeting of the American Society of Nephrology, November 5-8, 1999, Miami, FL; the 34th Annual Meeting of the American Society of Nephrology, October 14-17, 2001, San Francisco, CA; and Experimental Biology 2002, April 20-24, 2002, New Orleans, LA.
Address for reprints and other correspondence: J. M. Sands, Emory Univ. School of Medicine, Renal Div., WMRB Rm. 338, 1639 Pierce Drive, NE, Atlanta, GA 30322 (E-mail: jsands{at}emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 2, 2002;10.1152/ajprenal.00326.2001
Received 30 March 2001; accepted in final form 30 March 2002.
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