Impaired solute accumulation in inner medulla of Clcnk1-/- mice kidney

Norikazu Akizuki, Shinichi Uchida, Sei Sasaki, and Fumiaki Marumo

Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1-/- mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1-/- mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/- and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1-/- mice. These results indicate that the polyuria observed in Clcnk1-/- mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1-/- mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1-/- mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1-/- mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1-/- mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1-/- mice. We concluded that NDI in Clcnk1-/- mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems.

chloride channel; knockout mouse; countercurrent system; osmolytes; aquaporin-2 water channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CLC-K1 AND CLC-K2 are kidney-specific CLC chloride channels (1, 27). Although these channels are highly homologous (90% amino acid identity in humans and 80% in rat), their intrarenal localizations were completely different. We recently demonstrated by in situ hybridization that CLC-K1 is present only in the inner medulla and that CLC-K2 is expressed in the distal tubules, connecting ducts, cortical collecting ducts, and medullary thick ascending limb of Henle's loop (mTAL). Because sodium-dependent chloride transporters [NKCC2 (22) and TSC (9)] are present in the apical surfaces of these nephron segments, it had been speculated that a chloride channel had to be present on the basolateral plasma membrane for a vectorial transepithelial chloride transport to occur in these segments (21, 32). Accordingly, CLC-K2 could be a candidate chloride channel involved in chloride reabsorption in the distal nephron. This hypothesis was recently verified by genetic evidence that mutations of the CLC-Kb gene [a human homolog of rat CLC-K2 (26) cause Bartter's syndrome (23)]. On the basis of these observations and the structural similarity of CLC-K1 to CLC-K2, the former could also be involved in transepithelial chloride transport in the inner medulla. We have already clarified by immunohistochemistry that CLC-K1 is present in both apical and basolateral plasma membranes of the thin ascending limb of Henle's loop (tAL) (28). Because this nephron segment has been reported to have an extraordinarily high chloride permeability among nephron segments (12), we explored whether CLC-K1 was responsible for the high chloride permeability in the tAL by comparing the characteristics of CLC-K1 expressed in Xenopus oocytes with those of a native chloride transport in the tAL (16). Anion selectivity, pH sensitivity, and sensitivities to various inhibitors were quite similar in both transport systems, suggesting that CLC-K1 may be a major chloride channel responsible for the transepithelial chloride transport in the tAL (28). In a passive model proposed by Kokko and Rector (16), the tAL constituted a component of countercurrent systems for urinary concentration, and NaCl transport in the tAL could be an important route for supplying NaCl to the interstitium in the inner medulla. Thus we speculated that CLC-K1 was involved in the countercurrent systems in the inner medulla and important for urinary concentration.

To verify the above hypothesis, we generated CLC-K1-knockout mice (Clcnk1-/-) by targeted disruption of the gene (17). As a result, the Clcnk1-/- mice showed overt nephrogenic diabetes insipidus (NDI), clearly demonstrating that CLC-K1 has an essential role for urinary concentration (17). In our previous report (17), however, the exact mechanisms of NDI were not fully studied (17). On the basis of results from a 1-deamino-8-D-arginine vasopressin (dDAVP) infusion test, we speculated that NDI in Clcnk1-/- mice resulted from the impairment of generating hypertonicity in the inner medulla (17). Accordingly, we directly measured tissue osmolarity and solute contents in the inner medulla of Clcnk1-/- mice in this study. We also performed detailed studies on renal function of Clcnk1-/- mice because our previous data on urine were based on urine collected over 24 h, a limited period, which might not have allowed accuracy because of evaporation and contamination with stool, especially when urine volumes were small. Accordingly, we performed an exact clearance study of Clcnk1-/- mice under normal and dehydrated conditions.

We found that the polyuria in the Clcnk1-/- mice is water diuresis and that the Clcnk1-/- mice cannot accumulate osmotic solutes in the inner medulla even in dehydration.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Animals. Wild-type (Clcnk1+/+), heterozygous (Clcnk1+/-), and homozygous mutant (Clcnk1-/-) littermates were generated from F6 heterozygous mice (Clcnk1+/-) (18). Only male mice (5-6 wk old) were used in this study.

Clearance study. A single experiment consisted of 6 mice (2 wild, 2 heterozygous, and 2 homozygous mutant) divided into a normal group (free access to water) and a dehydrated group (24-h water deprivation). The mice were kept awake during the clearance study because anesthetics significantly affected blood pressure and kidney function in initial attempts to perform the experiment. Urination was induced at the beginning of the clearance period by abdominal tapping. Over a 4-h clearance period, each mouse was kept into a clean plastic cage and its urine was collected at every urination. After the clearance period, the mice were anesthetized deeply by nebulization of diethyl ether, blood was collected from their hearts, and their kidneys were isolated for Northern hybridization. Urine in the bladder was also recovered for clearance analysis.

The serum sodium, chloride, and urea concentration were measured by i-STAT (i-STAT, East Windsor, NJ). Urinary concentration of sodium and chloride was measured by a 9180 electrolyte analyzer (AVL Scientific, Roswell, GA). Serum and urinary creatinine and urea concentration were measured by the Creatinine Test Wako and Urea Test Wako, respectively, (Wako, Tokyo, Japan). Osmolarity of serum and urine was measured by a One-Ten Osmometer (Fiske, Norwood, MA).

Tissue analysis. The animals were anesthetized with diethyl ether, and their kidneys were removed. To obtain enough tissue for the analyses for sodium, chloride, and urea contents and osmolarity, eight papillae from four mice were pooled, weighed, and homogenized in 200 µl of water. The supernatants were analyzed after centrifugation at 15,000 rpm for 10 min. Sodium and chloride concentrations were measured by a 9180 electrolyte analyzer, and osmolarity was measured by using a One-Ten Osmometer. The urea content was measured by a Urea Test Wako kit.

Measurements of urinary vasopressin and plasma aldosterone. Urinary vasopressin was measured with a radioimmunoassay kit (AVP-kit Mitsubishi; Mitsubishi Kagaku, Tokyo, Japan) and corrected by creatinine concentration. Plasma aldosterone concentrations were measured with a radioimmunoassay kit (SPAC-S aldosterone kit; Daiichi Radio-isotope, Tokyo, Japan).

Probes. For Northern hybridization, the mouse aldose reductase (mAR) probe was prepared by PCR using kidney cDNA library in lambda gt10 (Clontech, Palo Alto, CA) as a template. The primer sequences were as follows: sense strand, 5'-GTGAACCAGATCGAGTGCCAC-3' (nucleotides 560-580, in mAR cDNA), and antisense strand, 5'-AGTCGTTACGCGTGACCACAG-3' (nucleotides 831-811). The PCR product was subcloned into pGEM-T easy vector (Promega, Madison, WI) and then sequenced. The cDNAs for probe of mouse betaine/GABA transporter (mBGT) and rat sodium/myo-inositol cotransporter (rSMIT) were kindly provided by M. Takenaka (Osaka Univ.). The probe of mouse aquaporin-2 (mAQP2) was obtained as described previously (7).

Northern blots. Total RNA (20 µg/lane) separated by electrophoresis in an agarose gel containing formamide was transferred to nylon membranes (MagnaGraph, MSI, Westborough, MA). The filters were hybridized for 24 h at 42°C in 50% formamide, 6× sodium chloride-sodium phosphate-EDTA, and 5× Denhardt's solution containing 1% SDS and 100 µg/ml salmon sperm DNA, with cDNAs of mAR, mBGT, Rsmit, and mAQP2 labeled with [alpha -32P]dCTP. The filters were washed once in 0.1% SDS and 1× standard sodium citrate (SSC) at room temperature for 20 min and then twice in 0.1% SDS and 0.2× SSC at 50°C for 20 min. They were exposed to film (RX-U, Fujifilm, Tokyo, Japan) at -80°C.

Immunohistochemistry. Clcnk1+/+ and Clcnk1-/- mice were perfused and fixed in a PLP solution (6% paraformaldehyde, 25 mM L-lysine monohydrochloride, and 0.1 M sodium periodate in PBS). Frozen sections were incubated with 1:100 dilution of anti-rat AQP2 antibody obtained as described previously (8) and visualized with cyanine 3-conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) under a LMS-5 laser scanning microscope (Carl Zeiss, Thornwood, NY).

Statistics. ANOVA was used to analyze the differences in data among groups. P values <0.05 were considered to be significant.


    RESULTS
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Clearance study and hormonal data. Figure 1 shows creatinine clearance (Ccr) of Clcnk1+/+, Clcnk1+/- and Clcnk1-/- mice in normal and dehydrated conditions, and the ratio of osmolar clearance to Ccr (FEosm) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal and dehydrated conditions. Ccr of Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice was 5.8 ± 2.1, 5.8 ± 1.2, and 7.7 ± 1.6 (SE) ml · min-1 · g body wt-1 (n = 3) under normal conditions, with no significant difference among groups. These Ccr values are compatible with those previously reported (15), suggesting that a 4-h clearance period was long enough to evaluate overall renal function. Clcnk1-/- mice could preserve normal glomerular filtration rate (GFR) under an euhydrated condition. Ccr after 24-h water deprivation significantly decreased in Clcnk1-/- (1.0 ± 0.2) compared with Clcnk1+/+ (5.4 ± 0.8) and Clcnk1+/- mice (5.8 ± 0.6), indicating that GFR in Clcnk1-/- mice was impaired only by 24-h dehydration. As shown in Fig. 1B, FEosm values were not significantly different among the three groups in normal conditions, clearly demonstrating that polyuria in Clcnk1-/- mice was not osmotic diuresis but water diuresis. Figure 2 shows fractional excretion of sodium (FENa), chloride (FECl), and urea (FEurea) of mice in six groups. These values were not significantly different among the three groups under a normal euhydrated condition, clearly demonstrating that a loss of chloride transport in the tAL did not result in chloride diuresis. FENa significantly decreased after 24-h dehydration in Clcnk1-/- mice (Fig. 2A), a finding that also showed, along with the decreased GFR (Fig. 1A), that 24-h dehydration was sufficient to cause a severe reduction in circulating blood volume in Clcnk1-/- mice.


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Fig. 1.   Creatinine clearance and fractional excretion of osmolar substances. A: creatinine clearance (Ccr; ml · min-1 · g body wt-1) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal and dehydrated conditions. BW, body wt. *P < 0.05 compared with Clcnk1+/+ and Clcnk1+/- mice under dehydrated conditions. B: fractional excretion of osmolarity (FEosm; osmolar clearance/Ccr) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal and dehydrated conditions. Values are means ± SE (n = 3).



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Fig. 2.   Fractional excretion of sodium, chloride, and urea. Fractional excretion of sodium (FENa; A), chloride (FECl; B), and urea (FEurea; C) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal and dehydrated conditions. Values are means ± SE (n = 3). *P < 0.05 compared with Clcnk1+/+ and Clcnk1+/- mice under dehydrated conditions.

Figure 3A shows the plasma aldosterone concentration (PAC) of Clcnk1+/+ and Clcnk1-/- mice in normal and dehydrated conditions. The PAC in Clcnk1-/- mice [2,832.5 ± 686.1 (SE) pg/ml, n = 8] was much higher than that in dehydrated Clcnk1+/+ mice, again supporting the severe volume depletion in Clcnk1-/- mice after dehydration. Figure 3B shows urinary vasopressin excretion (U-AVP) of Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal conditions. The U-AVP was significantly higher in Clcnk1-/- mice [0.54 ± 0.068 (SE) ng/mg Cr, n = 3] than in Clcnk1+/+ and Clcnk1+/- mice even under normal conditions. This suggests that a slight increase in plasma osmolarity occurs in Clcnk1-/- mice even when they can drink water freely. Water deprivation raised U-AVP ~40-fold (19.3 ± 6.0 ng/mg Cr, n = 3) above the normal condition (data not shown) in Clcnk1-/- mice, which was consistent with the high serum osmolarity and/or severe volume depletion in these animals.


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Fig. 3.   Plasma aldosterone concentration and urinary concentration of vasopressin. A: plasma aldosterone concentration (PAC) in the Clcnk1+/+ and Clcnk1-/- mice under normal and dehydrated conditions. Values are expressed as means ± SE (n = 8). *P < 0.05 compared with Clcnk1-/- mice under normal conditions or Clcnk1+/+ mice under dehydrated conditions. B: urinary concentration of vasopressin (U-AVP) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal conditions. Values are means ± SE (n = 3). *P < 0.05 compared with Clcnk1+/+ or Clcnk1+/- mice.

Tissue solute contents. Figure 4 shows the contents of osmolarity, sodium, chloride, and urea, in the tissue of inner medulla of Clcnk1+/+ and Clcnk1-/- mice. Total osmolarity in the inner medulla in Clcnk1-/- mice was about one-half of that of Clcnk1+/+ mice, and it did not increase after the dehydration period (Fig. 4A). These results clearly showed that Clcnk1-/- mice could not generate hypertonicity in the inner medulla even under the dehydrated conditions. Because the tAL was potentially a main site for the supply of NaCl to the interstitium, the decrease in NaCl content in the Clcnk1-/- mice had been anticipated. In fact, the inner medullary contents of sodium and chloride in Clcnk1-/- mice in both normal and dehydrated conditions were significantly smaller than those in Clcnk1+/+ mice (Fig. 4, B and C). Sodium osmolarity in the inner medulla in Clcnk1+/+ and Clcnk1-/- mice under normal conditions was 0.50 ± 0.09 and 0.25 ± 0.05 (SE) mmol/g wet wt, n = 3, respectively. Those in a dehydrated condition were 0.46 ± 0.04 and 0.28 ± 0.02 mmol/g wet wt, respectively. Chloride osmolarity in the inner medulla in Clcnk1+/+ and Clcnk1-/- under normal conditions were 0.23 ± 0.03 and 0.11 ± 0.05 mmol/g wet wt, n = 3, respectively. Those in a dehydrated condition were 0.25 ± 0.02 and 0.07 ± 0.01 mmol/g wet wt, respectively. These values of sodium and chloride contents in wild-type mice under normal conditions are consistent with those in a previous report (3). Furthermore, urea accumulation in the inner medulla in Clcnk1-/- mice was also impaired under normal conditions (Fig. 4D). In contrast to the marked increase in urea content under dehydrated conditions in Clcnk1+/+ mice, urea content in Clcnk1-/- mice was not increased by dehydration (Fig. 4D).


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Fig. 4.   The total osmolarity and electrolyte contents in the inner medulla. The contents of total osmolarity (IM-Osm; A), sodium (IM-Na; B), chloride (IM-Cl; C), and urea (IM-urea; D) in the inner medulla in the Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice under normal and dehydrated conditions. Values are means ± SE (N = 3). WT, weight.*P < 0.05 compared with the values in the Clcnk1+/+ mice. **P < 0.05 compared with the values in the other 3 groups.

Expression of osmolyte-related genes. Figure 5 shows Northern blots of kidney total mRNA (20 µg/lane) from normal and dehydrated Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice probed with AR, BGT, and SMIT. Although all three genes were known to be regulated by tonicity (6), their sites of expression in kidney were different. In previous in situ hybridization studies (19), AR expression was found to be localized in the inner medulla, whereas BGT and SMIT were mainly expressed in the outer medulla. As shown in Fig. 5, AR expression in Clcnk1-/- mice was significantly lower [66.0 ± 9.1 (SE) % of Clcnk1+/+ in normal conditions, n = 3, P < 0.05] than that in Clcnk1+/+ mice in normal conditions, and it was not increased by dehydration (75.7 ± 3.1% of Clcnk1+/+ in normal conditions, n = 3, not significantly different from Clcnk1-/- mice in normal conditions), whereas that in Clcnk1+/+ mice apparently was 140.4 ± 10.8% of Clcnk1+/+ in normal conditions, n = 3, P < 0.05. This result also supports the relative hyposmolarity in the inner medulla of Clcnk1-/- mice. BGT and SMIT mRNA in Clcnk1-/- mice was significantly increased [1.3 ± 0.02 (SE)-fold increase in BGT and 1.2 ± 0.03-fold increase in SMIT, n = 3, P < 0.05] by dehydration, suggesting that the abnormality in the tissue osmolarity in Clcnk1-/- mice only occurred in the inner medulla.


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Fig. 5.   Representative Northern blot of aldose reductase (AR), betaine/GABA transporter (BGT), and sodium-myo-inositol cotransporter (SMIT) in Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice. Twenty micrograms of total mRNA were loaded in each lane. beta -Actin probe was used as a standard. The intensity of bands was measured using a BAS2000 image analyzer (Fuji). As a control for statistics, we used the intensity of bands in the wild-type (Clcnk1+/+) mice under normal conditions.

AQP2 expression and cellular localization. To confirm that NDI in Clcnk1-/- mice is not due to the dysregulation of the AQP2 water channel, we checked the expression of AQP2 in Clcnk1+/+ and Clcnk1-/- mice by Northern analysis and the cellular localization by immunohistochemistry. As shown in Fig. 6, AQP2 expression was slightly increased in Clcnk1-/- mice [1.6 ± 0.1 (SE)-fold increase, n = 3, P < 0.05] than in Clcnk1+/+ mice. Fluorescence immunohistochemical study also revealed that AQP2 was localized in apical membranes of inner medullary collecting ducts in Clcnk1-/- mice as well as in Clcnk1+/+ mice (Fig. 7, A and B). These results suggest that NDI in Clcnk1-/- mice may not be due to the apparent abnormal regulation of AQP2.


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Fig. 6.   Representative Northern blot of aquaporin2 (AQP2) in the Clcnk1+/+ and Clcnk1-/- mice. Twenty micrograms of total RNA were loaded in each lane. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as a control.



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Fig. 7.   Immunolocalization of AQP2 of the Clcnk1+/+ (A) and Clcnk1-/- (B) mice in the inner medulla. AQP2 localized in the apical membranes of the inner medullary collecting ducts of the Clcnk1-/- mice as well as Clcnk1+/+ mice. Magnification, ×400.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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We previously demonstrated that the Clcnk1-/- mice showed an exogenous vasopressin-insensitive urinary concentrating defect (18). We speculated that this NDI could result from the dysfunction of the countercurrent system because the tAL is thought to be an important component of this system (16). Generation and maintenance of a hypertonic environment in the inner medulla is thought to be a major mechanism of urinary concentration, together with a switching mechanism of water permeability in the collecting ducts. The significance of the latter has been verified by the identification of mutations of vasopressin V2 receptor and AQP2 genes in patients with NDI. However, there has been no direct evidence that a countercurrent system(s) in the inner medulla is indeed functioning to generate hypertonicity in vivo. To prove that the deletion of the Clcnk1 resulted in the dysfunction of a countercurrent system and impairment of generating hypertonicity in the inner medulla, in this study we directly measured solute contents in the inner medulla in the Clcnk1-/- mice. In addition, we performed a clearance study to confirm that the polyuria observed in the Clcnk1-/- mice is water diuresis.

In our previous report (17), the urine osmolarity in the Clcnk1-/- mice was not proportionally lower than the value expected from the increased urinary volume. If the data was truly accurate, the increased urinary volume in the Clcnk1-/- mice could be partly due to osmotic diuresis, and this could be expected because the chloride reabsorption in the tAL was thought be to blocked by the Clcnk1 deletion. However, our previous data were based on 24-h collected urine, so the urine volume might have been affected by evaporation, especially in the case of the Clcnk1+/+ and Clcnk1+/- mice due to their scant urine output (~1-2 ml/day). Although we initially tried the clearance study according to the method described by Okubo et al. (20), we could not maintain the blood pressure during operation and clearance period when anesthesia was used. Thus we performed the clearance study while the mice were awake. First, we confirmed that creatinine clearance of the Clcnk1-/- mice was not significantly different from those of the Clcnk1+/+ and Clcnk1+/- mice (Fig. 1A), indicating that the deletion of Clcnk1 gene did not affect the glomerular filtration rate under normal conditions. Next, we measured the osmolar clearances (Fig. 1B) of the Clcnk1+/+, Clcnk1+/-, and Clcnk1-/- mice and found that they were not significantly different. This clearly indicated that osmotic diuresis was not observed in the Clcnk1-/- mice. FECl, as well as FENa and FEurea, did not significantly differ under normal conditions among the three groups. This confirmed the data on osmolar clearance, and it also indicated that, unlike the mutations of CLC-Kb in humans (23), the transepithelial chloride transport in the tAL did not affect chloride clearance. The decrease in chloride reabsorption in the tAL may be compensated for in the thick ascending limb, where there is a powerful, active chloride reabsorption system (10). We previously reported that the Clcnk1-/- mice appeared remarkably lethargic even after 24-h water deprivation. Our clearance study also clearly demonstrated the consequence of water deprivation in the kidney of the Clcnk1-/- mice. FENa decreased significantly in the Clcnk1-/- mice after dehydration (Fig. 2). This showed that the Clcnk1-/- mice were in a state of volume depletion, a finding consistent with the severe hemoconcentration and reduction in body weight reported previously (17).

Because our clearance study confirmed that the polyuria in the Clcnk1-/- mice is water diuresis, we know that the mechanism of NDI could be either a loss of water permeability in the collecting ducts or an impairment of the generation of hypertonicity in the inner medulla. There is no reason to suspect that the molecules involved in regulating water permeability in the collecting ducts were impaired in the knockout of CLC-K1 in the tAL, and accordingly there was no need in the gene-knockout experiments to check whether all molecules other than the targeted molecule are intact in the body, nor is there in fact a way to perform such an investigation. However, we did check the expression and cellular localization of AQP2, a major molecule regulating water permeability in the collecting ducts, in the Clcnk1-/- mice (Figs. 6 and 7). The expression of AQP2 in the Clcnk1-/- mice confirmed by Northern analysis was even higher than that in the Clcnk1+/+ mice. We and others reported that AQP2 expression was mainly regulated by vasopressin through the V2 receptor and a cAMP-responsive element in the AQP2 gene promoter (11, 18, 31). Because we observed that U-AVP was higher in the Clcnk1-/- mice (Fig. 3B), it is conceivable that AQP2 expression was increased by the increased secretion of vasopressin. As for cellular localization, AQP2 was clearly stained on the apical surface of collecting ducts, and the staining appeared to be even stronger than that in the Clcnk1+/+ mice. These results at least show that NDI in the Clcnk1-/- mice is not due to the secondary effect of the CLC-K1 knockout on AQP2.

Measurement of total osmolarity in the inner medulla clearly showed that levels of osmotic substances in the inner medulla of the Clcnk1-/- mice were lower than those in the Clcnk1+/+ mice (Fig. 4A), suggesting the inability of generation of hypertonicity in the inner medulla in the Clcnk1-/- mice. These results were more impressive when the osmolarity at the tip of papilla (Ctip) was calculated from the measured osmolarity in papilla homogenates (Cpap) on the basis of the equation by Kettyle and Valtin (14). To make this calculation we need to determine the distance from the papillary tip to the corticomedullary junction (hm), that distance from the papillary tip to the papilla base (hp), and the osmolarity at the corticomedullary junction (Cm)
C<SUB>tip</SUB>=C<SUB>m</SUB>+ <FR><NU>1</NU><DE>1−3&cjs0823;  4 · hp<SUP>−1</SUP><IT> · </IT>hm<SUP>−1</SUP></DE></FR> (<IT>C<SUB>pap</SUB>−C<SUB>m</SUB></IT>) (14)
We cited these values from the aforementioned paper (14) and simply assumed that the ratio of hp/hm was 0.6 (no dimensional change was noted in the kidney of Clcnk1-/- mice) and that the Cm was 320 mosmol/kgH2O. On the basis of the values in Fig. 4A, Ctip of the Clcnk1+/+ and Clcnk1-/- mice under normal conditions were calculated to be 1,976 and 698 (SE) mosmol/kgH2O, respectively. Those under dehydrated conditions were 2,894 and 878 mosmol/kgH2O, respectively (Fig. 8). Measured urine osmolarity in each condition was as follows: 814 ± 59.7 in Clcnk1+/+ and 650 ± 60.5 mosmol/kgH2O in Clcnk1-/- under normal conditions; 3,158 ± 190 in Clcnk1+/+ and 850 ± 6.8 mosmol/kgH2O in Clcnk1-/- under dehydrated conditions (n = 3). Under normal conditions, the calculated tissue Ctip was ~1,300 higher in Clcnk1+/+ mice than in Clcnk1-/- mice even when the urine osmolarity differed only slightly (814 vs. 650 mosmol/kgH2O). This suggests that the relatively low tissue osmolarity in the Clcnk1-/- mice was not the result of hypotonic urine, but rather the principal cause of the relative low urine osmolarity. This was also evident when the osmotic disequilibrium between urine osmolarity and Ctip was taken into consideration. The osmotic disequilibrium was only observed in the Clcnk1+/+ mice under normal conditions, and Ctip surprisingly matched the measured urine osmolarity in the dehydrated Clcnk1+/+ mice and normal and dehydrated Clcnk1-/- mice. Accordingly, we can conclude, first, that there is no defect in regulating water permeability in collecting ducts in the Clcnk1-/- mice and, second, that NDI in the Clcnk1-/- mice resulted from the impairment of the generation of hypertonicity in the inner medulla. The near-complete absence of osmotic disequilibrium (650 vs. 698 mosmol/kgH2O) in the Clcnk1 -/- mice under normal conditions was consistent with the data on increased U-AVP (Fig. 3B) and the immunohistochemistry of AQP2 (Fig. 7) in the Clcnk1-/- mice.


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Fig. 8.   Osmolarity at the tip of papilla (Ctip) and urine osmolarity of the Clcnk1+/+ and Clcnk1-/- mice. Ctip was calculated from the measured osmolarity in papilla homogenates on the basis of the equation in a previous report (14) and measured urine osmolarity in the Clcnk1+/+ and Clcnk1-/- mice under normal and dehydrated conditions. The water permeability of collecting ducts and gradient of tonicity in the inner medulla are shown by arrows and shading, respectively.

The next problem to solve was to identify the accumulation of which solute was impaired in the Clcnk1-/- mice. Decreased sodium and chloride accumulations were expected to occur because the tAL is a nephron segment that has high NaCl permeability (13). As shown in Fig. 4, B and C, sodium and chloride accumulations were impaired in the Clcnk1-/- mice even under dehydrated conditions. In addition, urea accumulation was also impaired in the Clcnk1-/- mice (Fig. 4D) under both normal and dehydrated conditions. Because a marked increase in urea accumulation was observed in the Clcnk1+/+ mice under dehydrated conditions, urea must be an important solute that constitutes hypertonicity in the inner medulla in mice. The absence of any increase of urea accumulation by dehydration in the Clcnk1-/- mice was surprising, and the exact mechanisms of this phenomenon remain to be determined. Impairment of urea accumulation as well as NaCl in the Clcnk1-/- mice implies that the mechanisms for generation of hypertonicity in the inner medulla is not a sum of transport systems independently functioning, but rather a system consisting of various transport systems interacting with each other, i.e., countercurrent systems. From this viewpoint, this study for the first time verified that a countercurrent system(s) is really functioning in vivo.

Finally, we confirmed the relative hypoosmolarity in the inner medulla of the Clcnk1-/- mice by examining the expression of osmolyte-related genes. The genes involved in the accumulation of osmolytes are known to be regulated by tonicity in vitro and in vivo (24, 29). Accordingly, the expressions of these genes are good markers of tissue osmolarity. The major organic osmolyte in the inner medulla was sorbitol generated by AR from glucose (2, 5). Previous reports identified that the AR gene is present only in the inner medulla in the kidney (4), whereas myo-inositol and betaine are major osmolytes located mainly in the outer medulla (19, 30). Figure 5 clearly showed that AR expression was lower in the Clcnk1-/- mice than in Clcnk1+/+ mice and that the upregulation by dehydration observed in the Clcnk1+/+ mice was minimal in the Clcnk1-/- mice. This coincides with the measured tissue osmolarity (Fig. 4A) and further supports the relative hypoosmolarity in the inner medulla of the Clcnk1-/- mice. In contrast to AR, expression of SMIT and BGT was increased under dehydrated conditions in the Clcnk1-/- mice. Considering that the main site of expression of SMIT and BGT is in the outer medulla (19), these data suggest that the reduction of tissue osmolarity in the Clcnk1-/- mice only occurs in the inner medulla. The increase in SMIT and BGT in the Clcnk1-/- under normal and dehydrated conditions may be caused by the dehydration and/or increased vasopressin secretion.

In conclusion, we demonstrated that the gene knockout of the CLC-K1 chloride channel impaired the solute accumulation in the inner medulla without affecting water permeability in the collecting duct and that the proposed countercurrent system(s) is actually functioning in vivo.


    ACKNOWLEDGEMENTS

This work was supported by the Salt Science Research Foundation and Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Uchida, Second Dept. of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: suchida.med2{at}med.tmd.ac.jp).

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.

Received 7 February 2000; accepted in final form 29 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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