Nephrology Dialysis Transplantation

NDT Advance Access originally published online on June 24, 2006
Nephrology Dialysis Transplantation 2006 21(10):2762-2767; doi:10.1093/ndt/gfl335

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Defect in parathyroid-hormone-induced luminal calcium absorption in connecting tubules of Klotho mice

Shuichi Tsuruoka^1,, Kenta Nishiki¹, Takashi Ioka¹, Hitoshi Ando¹, Yuichiro Saito², Masahiko Kurabayashi², Ryozo Nagai³ and Akio Fujimura¹

¹Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University, Tochigi ²Second Department of Internal Medicine, Gumma University School of Medicine, Gumma and ³Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Correspondence and offprint requests to: Shuichi Tsuruoka, MD, Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University, 3311 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Email: tsuru{at}jichi.ac.jp

	Abstract

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Background. Homozygous Klotho mutant mice (KL^–/– mice) exhibit multiple phenotypes resembling human ageing. Increases in the ratio of urinary calcium to urinary creatinine (uCa/uCr) and in serum Ca concentration and decreases in urinary Cr excretion and serum parathyroid hormone (PTH) concentration were reported; however, precise information about renal Ca handling was not reported in these animals.

Methods. We evaluated the PTH-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) in cells of isolated perfused connecting tubules (CNTs) of KL^–/– mice. We also determined fractional excretion of Ca from the urine and serum samples of the same animals (n = 7), and compared them with KL^+/+ mice and hemi-nephrectomized KL^–+/+ mice (n = 10 in each) as controls.

Results. FECa was significantly higher in KL^–/– mice than in controls (0.67 ± 0.13 vs 0.20 ± 0.04%). The PTH (10 nM)-induced increase in [Ca²⁺]i was diminished in KL^–/– mice (58 ± 5 vs 231 ± 15 nM). Addition of 10 nM of 8-(4-chlorophenylthio)-cyclic adenosine 3',5'-monophosphate had a similar effect. The PTH-induced increase had completely disappeared by the removal of Ca from lumen and bath in both groups of animals. Removal of sodium (Na) from the solution increased [Ca²⁺]i to a similar extent in both groups.

Conclusion. We conclude that renal Ca excretion estimated by determining FECa was defective in the KL^–/– mice. Impairment of Ca absorption from the lumen by stimulation of PTH in CNTs is one of the mechanisms of this defect. Activity of the basolateral Na/Ca exchanger was preserved in this strain. Therefore, the pathway downstream after generation of second messengers following stimulation of PTH (such as the sorting of transporters of Ca absorption) might be impaired by disruption of the Klotho gene.

Keywords: calcium absorption; connecting tubule; Klotho; PTH

	Introduction

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Klotho mice (KL^–/–), which have a homogeneous disruption of the Klotho gene, exhibit multiple phenotypes (such as short life span, osteoporosis, arteriosclerosis, bone malformation, soft tissue calcification and renal insufficiency) resembling human ageing [1]. The Klotho gene encodes a putative membrane protein that shares an amino acid similarity with ß-galactosidase, and its messenger RNA (mRNA) is predominantly expressed in distal tubules in the kidney and the choroid plexus in the brain [1,2]. The KL^–/– mice also have abnormal calcium (Ca) homeostasis. Compared with the wild type, Ca²⁺, phosphorus (P), calcitonin and 1,25-dihydroxyvitamin D concentrations are higher and intact parathyroid hormone (PTH) concentration is lower in KL^–/– mice [3,4]. The ratio of urinary Ca to urinary creatinine (uCa/uCr) is reported to be higher than in the wild type [3]. However, it is uncertain whether renal excretion of Ca is truly impaired, because serum Ca²⁺ concentration is higher in KL^–/– mice and hence the filtered load of Ca²⁺ must also be higher. Reduced serum PTH concentration may decrease urinary Ca excretion. In the KL^–/– mice, mRNA expression of 1-hydroxylase, 24-hydroxylase, stanniocalcin-1 and stanniocalcin-2 in the kidney is enhanced [3,4], also increasing Ca excretion. Evaluation of fractional excretion of Ca (FECa) permits accurate determination of Ca excretion, although use of this approach in this situation has not been reported until now.

Maintenance of Ca²⁺ balance in the body is one of the important roles of the kidney. About 60% of filtered Ca²⁺ is reabsorbed from the urine in the proximal tubule, while 30% is reabsorbed in the Henle loop and distal nephron segments [5,6]. The connecting tubule (CNT) is one of the most important segments for Ca handling in the distal nephron [7,8]. This segment absorbs Ca from the luminal fluid into the blood [6], and the absorption is regulated by PTH under physiological conditions [9,10]. In vitro microperfusion studies involving measurement of intracellular Ca²⁺ concentration ([Ca²⁺]i) confirmed that elevation of [Ca²⁺]i by PTH in CNT cells is mainly caused by enhancement of Ca absorption from the tubular lumen [9–11]. The Na/Ca exchanger at the basolateral membrane is the transporter responsible for the exit of Ca from CNT cells [12]. Regarding Ca entry at the luminal membrane, the epithelial Ca channel has been reported as a possible transporter [9]; however, this theory is not fully approved.

The purpose of this study was to determine, by evaluating FECa, whether urinary Ca excretion by the kidney was primarily impaired in Klotho mice. We also measured [Ca²⁺]i in cells from isolated perfused CNT segments and evaluated the mechanism of reduced Ca absorption in this segment in Klotho mice.

	Methods

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Animals
KL^–/– mice were generated from mating pairs of KL^+/– mice. Their genotypes were determined by Southern blot analysis and polymerase chain reaction at age 1 week [1,13,14]. KL^–/– mice and KL^+/+ mice without nephrectomy were used for the following experiments at age 7–14 days. Hemi-nephrectomy in KL^+/+ mice as controls was performed at age 7 days and clearance study was done at age 20–21days. All mice were anesthetized with ether. Urine was obtained by bladder puncture. Blood was taken directly from the heart, and both kidneys were removed after the animals were killed. All procedures were conducted in accordance with the Jichi Medical School guide for laboratory animals.

Measurement of Ca and Cr concentrations in serum and urine
Ca and Cr concentrations in serum and urine were measured by the o-cresolphthalein complexone method [15] and the ammonium molybdate method [16] with an autoanalyser. Cr concentration was measured by the modified Jaffé reaction with an autoanalyser.

In vitro microperfusion and Ca²⁺ measurement
The CNTs from the animals were isolated and perfused according to the method of Burg et al. [17] as previously slightly modified [18–20] kidney slices were placed with modified Collins solution containing 140 mM NaCl, 44 mM K₂HPO₄, 14 mM KH₂PO₄, 9 mM NaHCO₃ and 160 mM sucrose (pH 7.4). The CNT was isolated at 4°C with fine forceps according to the criteria reported previously by Imai [8]. Usually, the length of isolated CNT was 0.3–0.4 mm.

Isolated tubules were transferred to a perfusion chamber mounted on an inverted microscope and were perfused in vitro at 37°C. Tubules were hooked up to the holding pipette, and a single-barrel perfusion pipette was inserted into the tubular lumen. Triple-barrel polyethylene tubing was inserted into the perfusion pipette to allow rapid exchange of perfusion fluid. The perfusion rate was controlled at 10–20 nl/min by adjusting the height of the fluid reservoir, which was connected to the back end of the perfusion pipette. We used a relatively constant and high perfusion rate throughout the study to maximize the effect of PTH [18]. A flow-through system was used to allow rapid exchange of solution. The solution was maintained at 37°C. The flow rate was 3–5 ml/min, which allowed the solution to be exchanged within 2 s. The perfusing solution consisted of 135 mM NaCl, 5 mM KCl, 1.0 mM Na₂HPO₄, 1.0 mM NaH₂PO₄, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 5 mM L-alanine and 10 mM N-(2-hydroxyethyl)piperazine-N'-2 ethanesulfonic acid (HEPES), which was adjusted to pH 7.4 by adding tris(hydroxymethyl)aminomethane (Tris). To inhibit entry of Ca²⁺ from the solution, Ca²⁺ was eliminated from the solution and 0.1 mM ethyleneglycoltetraacetic acid (EGTA) was added when we evaluated the effect of PTH and 8-(4-chlorophenylthio)-cyclic adenosine 3',5'-monophosphate (CPT-cAMP). Na⁺-free solution was prepared by replacing NaCl with choline chloride and sodium phosphate with potassium phosphate.

[Ca²⁺]i of microperfused CNT segment was measured with Fura-2 [21,22] with a slight modification [18]. An aliquot of the stock solution (1 mM in dimethyl sulfoxide) of the acetoxymethyl ester of Fura-2 (Fura-2/AM) was added to the Ca²⁺-free solution (30 µM at final concentration) and incubated for 20 min at room temperature. The Fura-2-loaded CNT was rinsed by perfusing with a Ca²⁺-free solution at 37°C for 10 min. Thereafter, [Ca²⁺]i was measured using a microscopic fluorescence photometry system (OSP-3; Olympus, Tokyo, Japan). The large diameter (75 µm) of the beam of light focused on the tubule exceeded the diameter of the CNT in order to minimize the effect on tubular movement when luminal fluid was changed [18]. The fluorescent dye was excited at 340 and 380 nm with light supplied from a xenon lamp. The fluorescence emission was measured at 510 nm. Emission signals excited at 340 nm (F340) and 380 nm (F380) were sampled every 10 ms; then 100 signals were averaged to obtain one data point every 0.5 s. Before calculating the fluorescence ratio (R = F340/F380), we subtracted the background autofluorescence at each wavelength from each emission signal. A neutral density filter (ND6W18; Olympus) was used to equalize the emission light.

The calibration curve for free Ca²⁺ as a function of R was constructed by adding various amounts of CaCl₂ to a standard solution containing 1 mM ethylene glycol tetraacetic acid and 30 µM Fura-2. We employed the following equation for [Ca²⁺]i:

where R_min and R_max are fluorescence ratios at zero Ca²⁺ and saturated Ca²⁺ concentrations, respectively, and F_min and F_max are the fluorescence intensities at 380 nm at zero Ca²⁺ and Ca²⁺ saturation in the solution, respectively. In our system, we used a value of 224 mmol/l for the effective dissociation constant (K_d) of Fura-2, 17.8 for F_min/F_max. R_min and R_max were measured as 0.47 and 16.23, respectively, in the presence of 1 mM EGTA and 1 mM CaCl₂. Because it has been reported that the K_d of Fura-2 for Ca²⁺ varies in the presence of protein [21,22], we should note that the calibrated values for [Ca²⁺]i provide only relative changes in Ca²⁺ concentration.

To examine the effect of human PTH (1-34) (Peptide Institute, Osaka, Japan) and CPT-cAMP, we added 10 nM PTH or 0.1 mM CPT-cAMP to the Ca²⁺-free solution. Ruthenium red (Sigma), an inhibitor of transient receptor potential (TRP) valinoid4, TRPV5 and TRPM6 [23–25], at 10 µM was also used. In some experiments, CaCl₂ was omitted from the luminal solution and 0.1 mM EGTA was added.

Statistics
All data are presented as mean ± standard error. Statistical analysis was performed by ANOVA with StatView 5 for Windows (SAS Institute Inc., USA). P < 0.05 was regarded as significant.

	Results

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Serum and urine Ca concentrations and serum Cr concentrations are shown in Table 1. KL^–/– mice showed significantly higher concentrations of serum Ca and Cr and a higher uCa/uCr ratio compared with control animals. Hemi-nephrectomized mice showed almost similar serum Cr concentration as KL^–/– mice. However, the calculated FECa was significantly higher in KL^–/– mice than the other two groups. Body weight was significantly lower in KL^–/– mice than in the age-matched controls.

View this table: [in this window] [in a new window]   Table 1. Serum and urine values in KL–/– mice and wild-type controls

 
In vitro microperfusion study of CNTs
First, we evaluated the effect of PTH on solution in CNTs from control (KL^+/+) mice. Ca²⁺-free solution was used in the basolateral side to inhibit the entry of Ca²⁺ from the basolateral side. A representative tracing is shown in Figure 1. As reported previously in rabbits, PTH increased [Ca²⁺]i within a few minutes and stabilized within 10 min. The same manoeuvre in CNTs from KL^–/– mice did not affect [Ca²⁺]i (Figure 1). Mean [Ca²⁺]i before and 10 min after addition of PTH is summarized in Figure 1. The PTH (10 nM)-induced increase in [Ca²⁺]i was diminished in KL^–/– mice (58 ± 5 vs 231 ± 15 nM).

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. (A) Representative tracings of [Ca2+]i. [Ca2+]i became stable within 10 min, and mean [Ca2+]i before and 10 min after addition of PTH is summarized. (B) Effect of 10 nM PTH on intracellular Ca2+ concentration ([Ca2+]i) in isolated perfused connecting tubules (CNTs). Emission signals were sampled every 10 ms, and then 100 signals were averaged to obtain one data point every 0.5 s. Ca2+ was removed from bath and 1 mM ethyleneglycoltetraacetic acid (EGTA) was added to the solution.

 
Because it has been reported that cAMP is the second messenger after stimulation of PTH, we evaluated the effect of CPT-cAMP, an analogue of cAMP. Ca²⁺-free solution was used in the basolateral side to inhibit the entry of Ca²⁺ from the basolateral side. As shown in Figure 2, CPT-cAMP did not increase [Ca²⁺]i in KL^–/– mice but significantly increased it in control mice.

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Effect of 10 nM 8-(4-chlorophenylthio)-cyclic adenosine 3',5'-monophosphate (CPT-cAMP) on [Ca2+]i in isolated perfused CNTs. [Ca2+]i became stable within 10 min, and mean [Ca2+]i before and 10 min after addition of PTH is summarized. Ca was removed from bath and 1 mM EGTA was added to the solution.

 
Next, we examined the effect of Ca²⁺ removal from luminal solution as well as basolateral solution. Removal of Ca²⁺ from both bath and lumen diminished the PTH-induced increase in [Ca²⁺]i in CNTs from control mice but did not affect the PTH-induced increase in KL^–/– mice (Figure 3). Furthermore, we evaluated ruthenium red, an inhibitor of TRPV4, TRPV5 and TRPM6 [23–25] on PTH-induced Ca²⁺ increase. We found that the pre-treatment (3 min before the addition of PTH) of ruthenium red inhibited PTH-induced [Ca²⁺]i increase in wild type but not in KL^–/– mice (Figures 1 and 4).

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. Effect of luminal Ca removal on PTH-induced change in [Ca2+]i in isolated perfused CNTs. Ca was also removed in bath solution and 1 mM EGTA was added to the solution.

 

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. Effect of pre-treatment of luminal ruthenium red (10 µM) on PTH-induced change in [Ca2+]i in isolated perfused CNTs. Ruthenium red was added about 3 min before the addition of PTH. Ca was also removed in bath solution. Comparing with the results in Figure 1, we found that the ruthenium red significantly inhibited PTH-induced [Ca2+]i increase in wild type but not in KL–/– mice.

 
Finally, we evaluated the contribution of basolateral Na/Ca exchanger in CNTs of KL^–/– mice. Removal of Na from basolateral fluid in the presence of luminal Ca increased [Ca²⁺]i in KL^–/– and control mice to a similar degree (Figure 5), indicating that the Na/Ca exchanger activity in the basolateral membrane was preserved in KL^–/– mice.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. Effect of basolateral sodium (Na) removal on [Ca2+]i in isolated perfused CNTs. Ca was added to the solution.

 

	Discussion

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In this study, we found that FECa was increased in KL^–/– mice compared with control mice and hemi-nephrectomized normal mice. Serum Cr concentrations of KL^–/– mice and hemi-nephrectomized KL^+/+ mice increased a similar extent; however, FECa was significantly higher in KL^–/– mice. This is the first direct evidence of impairment of renal Ca handling in this strain. We also confirmed a previous report [3,4] that concentrations of serum Ca and Cr in KL^–/– mice are higher than in age-matched controls. Because FECa is high, the increase in serum Ca concentration in this strain cannot be due to renal Ca handling alone. Therefore, the increase in Ca absorption from bone through osteoporosis and/or the increase in Ca absorption from intestine might be greater than the amount of renal Ca excretion. Reduction of concentrations of humoral mediators, such as PTH, calcitonin and 1,25-dihydroxyvitamin D, also affects net Ca handling into urine.

We also found that PTH-induced Ca²⁺ entry from the luminal membrane in CNT cells was impaired in KL^–/– mice. This finding was made in in vitro experiments and thus was not affected by circulating hormones. Several reports indicate that urinary excretion of electrolytes and expression of mRNA and proteins (such as Klotho protein and 1-hydroxylase) in the kidney are altered in this strain [3,4]. However, this is the first study to obtain direct evidence about the impairment of renal tubular function in these mice. Because the CNT is believed to be an important segment for regulating urinary Ca excretion [7,8], we think that the increase in FECa in these mice is primarily due to an abnormality in Ca excretion by PTH in this segment.

It has also been reported that the effect of PTH on Ca handling in the kidney and intestine is impaired with ageing in animals and humans [26,27]. The precise mechanisms of the age-related abnormality have not been reported, especially in the kidney. Our finding is compatible with the hypothesis that KL^–/– mice are a model of ageing.

At this time, it is uncertain how the reduction in Klotho protein expression in this strain leads to insensitivity to PTH. Because CPT-cAMP, an analogue of cAMP, showed a similar effect, the pathway downstream after generation of second messengers (such as the transporters of Ca absorption) might be impaired by disruption of the Klotho gene. The mechanism of renal failure in these mice is not certain at the present time. However, it was reported that the Klotho mice are weak against trigger of renal damage such as ischaemia and activation of renin–angiotensin systems [28,29], which may contribute to progression of renal failure.

The transporter responsible for Ca absorption in luminal membrane of CNT is not known. The epithelial Ca channel is a likely candidate [9]; however, some transporters belonging to the TRP family have been reported as candidates [30]. We found that the ruthenium red, an inhibitor of TRPV4, TRPV5 and TRPM6 [23–25], inhibited PTH-induced Ca²⁺ increase in both mice. These results may indicate that TRPV5, TRPV4 or TRPM6 will be responsible for Ca transporter in luminal membrane of CNTs in normal rats and that disruption of Klotho gene leads the reduced function of the transporter in the KL^–/– mice. Studies are needed to evaluate the expression and function of these transporters in this strain. It has also been reported that the thick ascending limb of Henle's loop is important for the regulation of Ca excretion, especially in rodents [5]. We did not examine this segment in our study, and studies are needed. Cyclic guanosine 3',5'-monophosphate-dependent Ca absorption was reported in CNTs [31]. Further study is needed to evaluate the contribution of this signal transduction pathway in this strain, especially because reductions in nitric oxide synthesis in other organs were reported in these mice [14].

In conclusion, we found for the first time that renal Ca excretion was impaired in KL^–/– mice. We also found that the PTH-induced [Ca²⁺]i increase was impaired in isolated perfused CNT segments, the most important segments in the regulation of renal Ca handling. This is one of the important mechanisms of the reduced FECa in these mice. Activity of the basolateral Na/Ca exchanger was preserved in this strain. The pathway downstream after generation of second messengers following stimulation of PTH (such as sorting of the transporter of Ca absorption) might be impaired by disruption of the Klotho gene. The observation provides a unique insight into the relationship between the abnormality of Ca handling in this strain and ageing.

Conflict of interest statement. None declared.

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Received for publication: 3.10.05
Accepted in revised form: 15. 5.06

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