Impaired urine concentration and absence of tissue ACE: involvement of medullary transport proteins

Janet D. Klein1,*, D. Le Quach2,*, Justin M. Cole2, Kevin Disher2, Anne K. Mongiu2, Xiaodan Wang1, Kenneth E. Bernstein2, and Jeff M. Sands1,3

1 Renal Division, Department of Medicine, 2 Department of Pathology, and 3 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · kg-1 · 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); alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Blood chemistries in wild-type and ACE.2 mice


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Urine chemistries after 24 h of water deprivation

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.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   UT-A1 protein in the inner medulla. A: representative Western blot of protein from the inner medullas of wild-type (WT) and ACE.2 knockout (KO) mice probed with an antibody to UT-A1. Each lane shows protein from both kidneys of an individual mouse. Arrows, UT-A1 glycoproteins at 117 and 97 kDa. B: summary of densitometric analysis expressed as %WT control value. Each glycoprotein form was evaluated separately. The 117-kDa UT-A1 glycoprotein was significantly reduced to 28 ± 5% of WT control values. However, there was no significant change in the abundance of the 97-kDa glycoprotein. Values are means ± SE; n = 14 WT and 12 KO mice. *P < 0.05.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   ClC-K1 protein in the inner medulla. A: representative Western blot of protein from the inner medullas of WT and KO mice probed with an antibody to ClC-K1. Each lane shows protein from both kidneys of an individual mouse. Arrow, 80-kDa ClC-K1 protein band. B: summary of densitometric analysis expressed as %WT control value. The abundance of ClC-K1 in the KO mice was significantly reduced to 6 ± 6% of WT control values. Values are means ± SE; n = 5 mice/group. * P < 0.03.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Aquaporin-1 (AQP1) protein in the inner and outer medulla. A: representative Western blot of AQP1 protein from inner (top) and outer (bottom) medullas of WT and KO mice probed with an antibody to AQP1. Each lane shows protein from both kidneys of an individual mouse. Arrows, glycoprotein smears from the 35- to 50-kDa and the nonglycosylated 28-kDa bands that are characteristic of AQP1. B: summary of densitometric analysis in inner (left; IM) and outer medulla (right; OM) expressed as %WT control value. The 2 AQP1 protein bands were combined for analysis. The abundance of AQP1 in the KO mice was significantly reduced to 29 ± 6 and 39 ± 5% of WT control values in IM and OM, respectively. Values are means ± SE; n = 6 WT mice and 5 KO mice. P < 0.05.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   AQP2 protein in IM and OM. A: representative Western blot AQP2 protein from IM (top) and OM (bottom) of WT and KO mice probed with an antibody to AQP2. Each lane shows protein from both kidneys of an individual mouse. Arrows, glycoprotein smears from the 35- to 50-kDa and the nonglycosylated 29-kDa bands that are characteristic of AQP2. B: summary of densitometric analysis in IM (left) and OM (right) expressed as %WT control value. The 2 AQP2 protein bands were combined for analysis. There was no significant difference in the abundance of AQP2 protein in WT compared with KO mice in either the IM or OM or in the cortex. Values are means ± SE; n = 13 WT and 12 KO mice.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   UT-B protein in the IM and OM. A: representative Western blot of UT-B protein from IM (top) and OM (bottom) of WT and KO mice probed with an antibody to UT-B. Each lane shows protein from both kidneys of an individual mouse. Arrows, characteristic smears of UT-B glycoproteins from 40-50 kDa. B: summary of densitometric analysis in IM (left) and OM (right) expressed as %WT control value. There was no significant difference in the abundance of UT-B protein in WT compared with KO mice in either the IM or OM. Values are means ± SE; n = 10 mice/group.

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 alpha -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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Na+-K+-2Cl- cotransporter (NKCC2/BSC1) protein in OM. A: representative Western blot of OM protein from WT and KO mice probed with an antibody to NKCC2/BSC1. Each lane shows protein from both kidneys of an individual mouse. Arrow, 150-kDa NKCC2/BSC1 protein band. B: summary of densitometric analysis expressed as %WT control value. The abundance of NKCC2/BSC1 in the KO mice was significantly reduced to 56 ± 11% of WT control values. Values are means ± SE; n = 12 mice/group. * P < 0.01.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Na+-K+-ATPase (alpha -1 subunit) protein in OM. A: representative Western blot of OM protein from WT and KO mice probed with an antibody to the alpha -1 subunit of Na+-K+-ATPase. Each lane shows protein from both kidneys of an individual mouse. Arrow, 96-kDa Na+-K+-ATPase alpha -1 subunit protein band. B: summary of densitometric analysis expressed as %WT control value. There was no significant change in the abundance of Na+-K+-ATPase alpha -1 subunit protein in KO compared with WT mice. Values are means ± SE; n = 7 mice/group.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   UT-A2 protein in OM. A: representative Western blot of OM protein from WT and KO mice probed with an antibody to UT-A2. Each lane shows protein from both kidneys of an individual mouse. Arrow, 55-kDa UT-A2 protein band. B: summary of densitometric analysis expressed as %WT control value. There was no significant change in the abundance of UT-A2 protein in KO compared with WT mice. Values are means ± SE; n = 7 mice/group.

In marked contrast, the protein abundance of ROMK (Fig. 9) was significantly increased in ACE.2 mice to 711 ± 187% of the level in wild-type mice. The identity of the 70-kDa band that is consistently detected by this antibody is unknown (3).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 9.   Renal outer medullary potassium channel (ROMK) protein in OM. A: representative Western blot of OM protein from WT and KO mice probed with an antibody to ROMK. Each lane shows protein from both kidneys of an individual mouse. Arrow, 45-kDa ROMK protein band. The identity of the 70-kDa band is unknown but is consistently detected with this antibody (3). B: summary of densitometric analysis expressed as %WT control value. The abundance of ROMK in the KO mice was significantly increased to 711 ± 178% of WT control values. Values are means ± SE; n = 6 mice/group. * P < 0.02.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

ROMK is expressed in the apical membrane of the thick ascending limb and is responsible for secreting K+ into the thick ascending limb lumen (15, 17), where it is needed to establish the appropriate electrochemical gradients for NaCl reabsorption by NKCC2/BSC1 (reviewed in Refs. 8 and 27). In contrast to the decrease in ROMK protein in hypercalcemic rats (36), we found a large increase in ROMK in the outer medulla of ACE.2 mice, regardless of background strain. Consistent with previous studies of ACE knockout mice (6), the ACE.2 mice in this study have mild hyperkalemia, elevated urinary potassium excretion, and low, but measurable, plasma aldosterone levels. This suggests that some hyperkalemia is necessary to stimulate angiotensin II-independent aldosterone secretion. However, how ACE knockout mice are able to maintain hyperkalemia despite an increase in urinary potassium excretion is unknown. One possibility is dietary potassium, because placing angiotensinogen knockout mice on a potassium-restricted diet is lethal (5, 25). Because an increase in ROMK, coupled with a decrease in NKCC2/BSC1, could contribute to an increase in urinary potassium excretion, we speculate that the increase in ROMK protein in the outer medulla could be important for preventing lethal levels of hyperkalemia while permitting serum potassium to be high enough to stimulate some aldosterone secretion. Future studies will be necessary to elucidate the potassium homeostatic mechanisms present in ACE knockout mice.

In summary, ACE.2 mice have decreased abundance of UT-A1, ClC-K1, NKCC2/BSC1, and AQP1 proteins, which could contribute to the urine-concentrating defect by reducing urea delivery to the inner medullary interstitium, NaCl reabsorption from the thin and thick ascending limbs, and water transport across the thin descending limb and vasa recta, respectively. 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 transport proteins.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Chou, CL, Knepper MA, Van Hoek AN, Brown D, Yang BX, Ma TH, and Verkman AS. Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice. J Clin Invest 103: 491-496, 1999[Abstract/Free Full Text].

2.   Cole, J, Ertoy D, Lin H, Sutliff RL, Ezan E, Guyene TT, Capecchi M, Corvol P, and Bernstein KE. Lack of angiotensin II-facilitated erythropoiesis causes anemia in angiotensin-converting-enzyme-deficient mice. J Clin Invest 106: 1391-1398, 2000[Abstract/Free Full Text].

3.   Ecelbarger, CA, Kim GH, Knepper MA, Liu J, Tate M, Welling PA, and Wade JB. Regulation of potassium channel Kir 1.1 (ROMK) abundance in the thick ascending limb of Henle's loop. J Am Soc Nephrol 12: 10-18, 2001[Abstract/Free Full Text].

4.   Ecelbarger, CA, Sands JM, Doran JJ, Cacini W, and Kishore BK. Expression of salt and urea transporters in rat kidney during cisplatin-induced polyuria. Kidney Int 60: 2274-2282, 2001[ISI][Medline].

5.   Ertoy, D, and Bernstein KE. Targeted interruption of the renin-angiotensin system. In: Drugs, Enzymes and Receptors of the Renin-Angiotensin System: Celebrating a Century of Discovery, edited by Husain A, and Graham RM.. Amsterdam: Harwood Academic, 2000, p. 205-224.

6.   Esther, CR, Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, and Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74: 953-965, 1996[ISI][Medline].

7.   Esther, CR, Jr, Marrero MB, Howard TE, Machaud A, Corvol P, Capecchi MR, and Bernstein KE. The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice. J Clin Invest 99: 2375-2385, 1997[Abstract/Free Full Text].

8.   Giebisch, G. Renal potassium channels: function, regulation, and structure. Kidney Int 60: 436-445, 2001[ISI][Medline].

9.   Hebert, SC. Roles of Na-K-2Cl and Na-Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism. Am J Physiol Renal Physiol 275: F325-F327, 1998[Abstract/Free Full Text].

10.   Kato, A, Klein JD, Zhang C, and Sands JM. Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs. Am J Physiol Renal Physiol 279: F835-F840, 2000[Abstract/Free Full Text].

11.   Kim, G-H, Ecelbarger CA, Mitchell C, Packer RK, Wade JB, and Knepper MA. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop. Am J Physiol Renal Physiol 276: F96-F103, 1999[Abstract/Free Full Text].

12.   Kishore, BK, Krane CM, Di Iulio D, Menon AG, and Cacini W. Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria. Kidney Int 58: 701-711, 2000[ISI][Medline].

13.   Knepper, MA, Kim GH, Fernández-Llama P, and Ecelbarger CA. Regulation of thick ascending limb transport by vasopressin. J Am Soc Nephrol 10: 628-634, 1999[Free Full Text].

14.   Kokko, JP, and Rector FC. Countercurrent multiplication system without active transport in inner medulla. Kidney Int 2: 214-223, 1972[ISI][Medline].

15.   Lee, W-S, and Hebert SC. ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1124-F1131, 1995[Abstract/Free Full Text].

16.   Liu, W, Moritomo T, Kondo Y, Iinuma K, Uchida S, Sasaki S, Marumo F, and Imai M. Analysis of NaCl transport in the ascending thin limb of Henle's loop in CLC-K1 null mice. Am J Physiol Renal Physiol 282: F451-F457, 2002[Abstract/Free Full Text].

17.   Lu, M, and Wang WH. Two types of K+ channels are present in the apical membrane of the thick ascending limb of the mouse kidney. Kidney Blood Press Res 23: 75-82, 2000[ISI][Medline].

18.   Ma, TH, Yang BX, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem 273: 4296-4299, 1998[Abstract/Free Full Text].

19.   Matsumura, Y, Uchida S, Kondo Y, Miyazaki H, Ko SBH, Hayama A, Morimoto T, Liu W, Arisawa M, Sasaki S, and Marumo F. Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel. Nature Genet 21: 95-98, 1999[ISI][Medline].

20.   Mount, DB, Baekgaard A, Hall AE, Plata C, Xu J, Beier DR, Gamba G, and Hebert SC. Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization. Am J Physiol Renal Physiol 276: F347-F358, 1999[Abstract/Free Full Text].

21.   Naruse, M, Klein JD, Ashkar ZM, Jacobs JD, and Sands JM. Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517-523, 1997[Abstract].

22.   Nielsen, S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663-11667, 1993[Abstract].

23.   Nielsen, S, Kwon TH, Christensen BM, Promeneur D, Frokiaer J, and Marples D. Physiology and pathophysiology of renal aquaporins. J Am Soc Nephrol 10: 647-663, 1999[Abstract/Free Full Text].

24.   Nielsen, S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495-5500, 1996[Abstract/Free Full Text].

25.   Okubo, S, Niimura F, Nishimura H, Takemoto F, Fogo A, Matsusaka T, and Ichikawa I. Angiotensin-independent mechanism for aldosterone synthesis during chronic extracellular fluid volume depletion. J Clin Invest 99: 855-860, 1997[Abstract/Free Full Text].

26.   Pallone, TL, Kishore BK, Nielsen S, Agre P, and Knepper MA. Evidence that aquaporin-1 mediates NaCl-induced water flux across descending vasa recta. Am J Physiol Renal Physiol 272: F587-F596, 1997[Abstract/Free Full Text].

27.   Palmer, LG. Potassium secretion and the regulation of distal nephron K channels. Am J Physiol Renal Physiol 277: F821-F825, 1999[Abstract/Free Full Text].

28.   Sands, JM. Regulation of renal urea transporters. J Am Soc Nephrol 10: 635-646, 1999[Abstract/Free Full Text].

29.   Sands, JM, and Knepper MA. Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium. J Clin Invest 79: 138-147, 1987[ISI][Medline].

30.   Sands, JM, and Kokko JP. Current concepts of the countercurrent multiplication system. Kidney Int 50: S-93-S-99, 1996.

31.   Sands, JM, Timmer RT, and Gunn RB. Urea transporters in kidney and erythrocytes. Am J Physiol Renal Physiol 273: F321-F339, 1997[Abstract/Free Full Text].

32.   Stephenson, JL. Concentration of urine in a central core model of the renal counterflow system. Kidney Int 2: 85-94, 1972[ISI][Medline].

33.   Terris, J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414-F422, 1996[Abstract/Free Full Text].

34.   Timmer, RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, and Sands JM. Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am J Physiol Cell Physiol 281: C1318-C1325, 2001[Abstract/Free Full Text].

35.   Wang, W. Regulation of the ROMK channel: interaction of the ROMK with associate proteins. Am J Physiol Renal Physiol 277: F826-F831, 1999[Abstract/Free Full Text].

36.   Wang, WD, Kwon T-H, Li CL, Frokiaer J, Knepper MA, and Nielsen S. Reduced expression of Na-K-2Cl cotransporter in medullary TAL in vitamin D-induced hypercalcemia in rats. Am J Physiol Renal Physiol 282: F34-F44, 2002[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(3):F517-F524
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society