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Am J Physiol Renal Physiol 293: F1231-F1237, 2007. First published July 25, 2007; doi:10.1152/ajprenal.00038.2007
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Effects of furosemide on renal calcium handling

Chien-Te Lee,1,2 Hung-Chun Chen,2 Li-Wen Lai,3 Kim-Chong Yong,3 and Yeong-Hau H. Lien3

1Division of Nephrology, Department of Medicine, Chang-Gung Memorial Hospital, Kaohsiung Medical Center, Chang-Gung University College of Medicine, Kaohsiung, Taiwan; 2Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan; and 3Department of Medicine, University of Arizona, Tucson, Arizona

Submitted 20 January 2007 ; accepted in final form 20 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Furosemide is a loop diuretic agent that has been used to treat hypercalcemia because it increases renal calcium excretion. The effect of furosemide on calcium transport molecules in distal tubules has yet to be investigated. We conducted studies to examine the effects of furosemide on renal calcium excretion and expression of calcium transport molecules in mice. Mice were administered with a single dose of furosemide (15 mg/kg) and examined 4 h later or were given twice-daily furosemide injections for 3 days. To evaluate the effects of volume depletion, drinking water was supplemented with salt. Our results showed that, in acute experiments, furosemide enhanced urinary calcium excretion, which was associated with a significant increase in mRNA levels of TRPV5, TRPV6, and calbindin-D28k but not calbindin-D9k as measured by real-time PCR (TRPV5 and TRPV6 are transient receptor potential vanilloid 5 and 6). Chronic furosemide administration induced three- to fourfold increases in urinary calcium excretion and elevated mRNA levels of TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k without or with salt supplement. Similar upregulation of calcium transport molecules was observed in mice with gentamicin-induced hypercalciuria. Coadministration of chlorothiazide decreased furosemide-induced calciuria, either acutely or chronically, although still accompanied by upregulation of these transport molecules. Immunofluorescent staining studies revealed comparably increased protein abundance in TRPV5 and calbindin-D28k. We conclude that furosemide treatment enhances urinary calcium excretion. Increased abundance of calcium transport molecules in the distal convoluted tubule represents a solute load-dependent effect in response to increased calcium delivery and serves as a compensatory adaptation in the downstream segment.

thiazides; TRPV5; TRPV6; calbindin-D28k; calbindin-D9k

FUROSEMIDE IS A POTENT DIURETIC agent that decreases sodium chloride reabsorption by competing for the chloride site of sodium potassium chloride cotransporter (NKCC2) in the thick ascending limb of loop of Henle (TALH) (29). By diminishing sodium reabsorption, the concentrating process that occurs in the medulla is thus impaired and fluid loss ensues. Long-term furosemide treatment is usually indicated primarily for its natriuretic action for the treatment of a variety of clinical conditions such as hypertension, edematous status, and chronic renal insufficiency (6).

Administration of furosemide not only elicits diuresis but is also associated with adverse effects, such as volume contraction, alterations in electrolyte and acid-base homeostasis, glucose intolerance, and lipid abnormality (11). It has been well established that furosemide and other loop diuretics promote urinary calcium and magnesium excretion (9). About 20% ultrafiltered calcium and 60–80% magnesium are reabsorbed in the TALH. Loop diuretics reduce the lumen-positive transepithelial voltage and consequently diminish paracellular transport of calcium and magnesium (12). Patients with Bartter syndrome caused by NKCC2 mutation manifest similar electrolyte disturbances (30). Because the calciuric effect is rapid and effective, furosemide has been used as a first-line therapy for patients with hypercalcemia in the absence of volume contraction (2).

The distal convoluted tubule (DCT) is an important nephron segment for renal handling of salt, calcium, and magnesium (3). Recently, the novel apical calcium channel specifically expressed in DCT was identified (14). Soon after that, another calcium channel was cloned and found to be expressed in the DCT and also in more distal tubules (23, 26). Subsequent studies have revealed that these channels exhibit characteristics of the superfamily of transient receptor potential (TRP) and are renamed as TRPV5 (TRP vallinoid) and TRPV6 (15). With their colocalization with intracellular calcium binding proteins, such as calbindin-D28k, and the basolateral transporters, such as sodium/calcium exchanger and calcium pump, these transport molecules constitute the fundamental machinery for calcium transport in the DCT (15). In the present study, we investigated the acute and chronic effects of furosemide, and effects of the combination of furosemide and thiazides, on renal calcium handling and expression of calcium channels and calcium binding proteins in the kidney.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male C57/BL6 mice, 6–8 wk old weighing ~20 g, were used. All animals were allowed free access to both food and drinking water. The contents of food included 0.3% sodium, 1.2% calcium, and 0.16% magnesium. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona and the Chang-Gong Memorial Hospital, and all animal procedures were performed according to the IACUC policy.

Study Design

Acute effects of furosemide. The mice (n = 4) were administered with a single dose of furosemide (15 mg/kg), or furosemide with chlorothiazide (25 mg/kg), by intraperitoneal injection and killed 4 h later. Urine samples were collected in metabolic cages. The whole kidney was used for total RNA isolation.

Chronic effects of furosemide. The mice were divided into five groups(n = 6–8 in each group): 1) control group, 2) furosemide group (15 mg/kg) every 12 h for 3 days, 3) furosemide with salt supplement group (same dosage as group 2 and drinking water with 0.8% NaCl and 0.1% KCl was offered), 4) salt supplement group (mice were given drinking water with 0.8% NaCl and 0.1% KCl but no furosemide treatment), and 5) coadministration of furosemide and chlorothiazide group (with same dosage as in acute experiment every 12 h for 3 days). On the fourth day, urine samples were collected in metabolic cages, and blood samples were collected for hematocrit and biochemical measurements. The kidneys were harvested for isolation of total RNA or protein and for immunofluorescence study.

Gentamicin treatment. To verify the effects of furosemide-induced hypercalciuria on DCT calcium transport molecules, we added a gentamicin-treated group (n = 8 mice) as a hypercalciuric control because gentamicin can induce hypercalciuria without renal tubular damages (25). The mice were administered with a single subcutaneous dose of gentamicin (40 mg/kg) daily for 4 days. The collection of urine, blood, and kidney samples was performed as described in the furosemide experiments.

Biochemical Assays and Hematocrit

Creatinine and calcium levels were measured in urine and blood samples according to manufacturer's protocols as described previously (19). The urinary excretion of calcium was expressed as calcium-to-creatinine (Ca/Cr) ratio. Hematocrit was measured before and after treatment, with diuretics as an indicator of volume status.

Total RNA Isolation and cDNA Synthesis

The whole kidney was homogenized, and total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription for first-strand cDNA synthesis was performed with a reverse transcription system (SuperScript system, Invitrogen).

Real-Time PCR

The mRNA levels of apical calcium channels TRPV5 and TRPV6, calbindin-D28k, and calbindin-D9k were measured by quantitative real-time RT-PCR as described previously (20). The first-strand cDNA was subject to real-time PCR using the Cepheid Smart Cycle II system (Cepheid, Sunnyvale, CA) and a fluorescent dye (SYBR green; Molecular Probes, Eugene, OR) as a tracer. beta-Actin gene was used as an internal control. For a given target gene, the relative gene expression was determined as 2(beta-actin Ct – target gene Ct), where Ct is the first cycle at which the reporter signal significantly exceeds the preset signal. Each PCR reaction was performed in triplicate and averaged. Melting curve analysis was carried out at the end of a 40-cycle amplification to validate the size of the PCR products. The primer sequences used for the studied genes are listed in Table 1.


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  		Table 1. List of mice primers used for real-time PCR

 
Immunofluorescence Study

Frozen kidney cortex tissue was used for assessing calbindin-D28k and TRPV5 protein expression in four groups of animals. Sections of 5 µm in thickness were fixed with 1% periodate-lysine-paraformaldehyde for 2.0 h and incubated with primary antibody (1:5,000 monoclonal anti-mouse calbindin-D28k antibody, Sigma; 1:50 rabbit anti-rat ECaC1, Alpha Diagnostic International, San Antonio, TX) for 1.5 h and then with FITC-conjugated secondary antibodies for calbindin-D28k (Jackson ImmunoResearch Laboratories, West Grove, PA) and FITC-conjugated secondary antibody for TRPV5 (ZYMED Laboratories, San Francisco, CA) for 1.0 h. The immunofluorescence pictures were then taken with a Zeiss fluorescence microscope connected to a digital photo camera (Evolution VF, MediaCybernetics, Silver Spring, MD). Semiquantitative determination of the protein expression was performed with the Image-Pro Plus 5.0 image analysis software. Amount of protein was expressed as mean of integrated optimal density (21). The alternation is expressed as percentage of results for control animal.

Immunoblotting Study

The frozen renal tissue was homogenized at 4°C in protein lysis buffer solution containing protease inhibitor cocktail (Roche, Penzberg, Germany). Immunoblotting for quantification of calbindin-D28k was performed as described previously (21). The primary antibody was goat anti-mouse calbindin-D28k monoclonal antibody (1:2,000, Sigma). After washing and blocking were completed, the membrane was incubated with goat anti-rabbit antibody conjugated with horseradish peroxidase. The abundance of studied protein was then quantified by densitometric analyses. The changes in protein abundance are presented as percentages of control animal values. beta-Actin was used as the internal control in this study.

Statistical Analysis

All values are expressed as means ± SE. Statistical analyses of the data were performed with SPSS software. Unpaired Student's t-tests were used to compare differences between two groups. To determine the significant difference among controls, furosemide-treated, furosemide plus salt supply, salt only, and furosemide plus chlorothiazide groups, one-way ANOVA and Tukey's test were used. P < 0.05 was considered statistically significant for all tests.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute Effects of Furosemide

A significant increase in urinary calcium excretion was observed 4 h after a single dose treatment of furosemide (urine Ca/Cr: 1.98 ± 0.18 vs. 0.21 ± 0.09; P < 0.05). Coadministration of chlorothiazide partially reversed furosemide-induced hypercalciuria (urine Ca/Cr: 1.16 ± 0.05; P < 0.05 vs. control and furosemide only). This increased calcium excretion in the furosemide group was accompanied with elevated mRNA levels of TRPV5 and TRPV6 (174 ± 10% and 183 ± 9% of control; both P < 0.05) and calbindin-D28k (222 ± 13%; P < 0.05) but no significant change in calbindin-D9k (144 ± 10%) mRNA abundance. Significant upregulation was observed in mice treated with furosemide plus chlorothiazide (TRPV5: 181 ± 5%, TRPV6: 169 ± 8%, calbindin-D28k: 179 ± 10%, and calbindin-D9k: 158 ± 5%; all P < 0.05).

Chronic Effects of Furosemide

After administration of furosemide for 3 days, a fourfold increase in urinary calcium excretion was observed (Table 2). A similar increase in urinary excretion of calcium was also observed in the group treated with furosemide plus salt supplement and in mice with salt supplement only; however, the increase was less in the latter group. The calciuretic effect of furosemide is decreased in mice by the addition of chlorothiazide (urine Ca/Cr: 0.81 ± 0.09 for furosemide only and 0.47 ± 0.15 for furosemide plus chlorothiazide; P < 0.05). No significant changes in serum creatinine or calcium levels were found in any of the treatment groups. There was a significant increase in hematocrit and decrease in body weight in the furosemide-treated and furosemide plus chlorothiazide-treated groups compared with the control animals. The hematocrit values and weight loss (4% in furosemide group and 3.4% in furosemide plus chlorothiazide group) were not significantly different between these two groups. The hematocrit values of mice in the furosemide plus salt or salt only groups were not different from control mice. They also showed no significant weight changes after treatment (Table 2).


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  		Table 2. Effects of diuretics on body weight, hematocrit, and serum and urine chemistry

 
The increases in calcium excretion in furosemide only, salt only, and furosemide plus salt groups are associated with a significant increase in mRNA abundance of TRPV5 (170 ± 10%, 212 ± 11%, 236 ± 9% of the control, respectively), TRPV6 (210 ± 9%, 182 ± 10, 194 ± 10% of the control, respectively), calbindin-D28k (310 ± 14%, 290 ± 12%, 264 ± 10% of the control, respectively), and calbindin-D9k (220 ± 13%, 254 ± 9%, 230 ± 9% of the control, respectively). In the furosemide plus chlorothiazide group, except for calbindin-D9k, all of the other transport molecules showed a >50% increase in gene expression: 171 ± 8% for TRPV5, 170 ± 11% for TRPV6, and 167 ± 7% of control for calbindin-D28k (all P < 0.05 vs. control), with a marginal increase found in calbindin-D9k (140 ± 6% of control; Fig. 1).


Figure 1
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  		Fig. 1. Gene expression of TRPV5 (A), TRPV6 (B), calbindin-D28k (C), calbindin-D9k (D) among the 5 groups of mice: control (Ctrl), furosemide treatment (F), furosemide treatment with salt supplementation (F+S), salt supplementation only (S), and coadministration with furosemide and chlorothiazide (F+C). TRPV5 and TRPV6, transient receptor potential vanilloid 5 and 6; CBD-28k and CBD-9k: calbindin-D28k and calbindin-D9k. *P < 0.05 compared with the controls.

 
To assess whether protein expression was also increased, we performed immunofluorescence studies with antibodies against TRPV5 and calbindin-D28k. Figure 2 shows that there are more TRPV5- and calbindin-D28k-positive tubules in kidneys from mice treated with furosemide, furosemide with salt supplementation, salt alone, and furosemide with chlorothiazide. Semiquantitative studies showed significant increases in both TRPV5 protein (furosemide only: 210 ± 9%, furosemide plus salt: 201 ± 12%, salt only: 223 ± 7%, furosemide plus chlorothiazide: 190 ± 7% vs. controls; all P < 0.05; Fig. 2, top) and calbindin-D28k protein (furosemide only: 212 ± 10%, furosemide with salt: 227 ± 9%, salt only: 197 ± 12%, furosemide plus chlorothiazide: 185 ± 10% vs. controls; all P < 0.05; Fig. 2, bottom).


Figure 2
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  		Fig. 2. Immunofluorescent staining of representative kidney sections using anti-TRPV5 (top) and anti-CBD-28k (bottom) antibodies in the 5 treatment groups (Ctrl, F, F+S, S, and F+C). There are more positive-staining tubules in all treated groups than shown in the control kidney. Bottom right of top and bottom show results of semiquantitative studies. *P < 0.05 compared with the controls.

 
The increased protein expression of calbindin-D28k was further confirmed by immunoblotting studies (Fig. 3). Furosemide treatment was associated with increased abundance of calbindin-D28k (170 ± 6% of control; P < 0.05). A similar increase was observed in furosemide with salt, salt alone, and coadministration with furosemide and chlorothiazide groups (169 ± 5%, 189 ± 8% and 173 ± 6%, respectively; all P < 0.05; Fig. 3).


Figure 3
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  		Fig. 3. Immunoblotting results of CBD-28k protein in the 5 treatment groups (Ctrl, F, F+S, S, and F+C). The relative CBD-28k protein abundance is presented as CBD-28k-to-beta-actin ratio. The ratio of control animals is set as 100%. *P < 0.05 compared with the controls.

 
Effects of Gentamicin

Gentamicin injection produced marked calciuria (urine Ca/Cr: 2.0 ± 0.11 vs. 0.19 ± 0.03; P < 0.05) without changing serum calcium (9.0 ± 0.1 mg/dl) or creatinine (0.41 ± 0.9 mg/dl) levels significantly. Similar to furosemide, gentamicin treatment increased mRNA expression of TRPV5 (175 ± 6%), TRPV6 (160 ± 8%), calbindin-D28k (180 ± 5%), and calbindin-D9k (168 ± 6%; all P < 0.05 vs. control). Immunofluorescence studies (Fig. 4) showed that gentamicin treatment was associated with increased abundance of TRPV5 (170 ± 9%) and calbindin-D28k (196 ± 8%; both P < 0.05 vs. control). The increased protein expression of calbindin-D28k was further confirmed by immunoblotting studies (Fig. 5). Gentamicin treatment was associated with increased abundance of calbindin-D28k (183 ± 9% of control; P < 0.05).


Figure 4
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  		Fig. 4. Immunofluorescence studies demonstrate that gentamicin (GM) treatment increases TRPV5 and CBD-28k (CBD) in the kidney. Ctrl, control mouse.

 

Figure 5
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  		Fig. 5. Immunoblotting results of CBD-28k protein. The relative CBD-28k protein abundance is presented as CBD-28k-to-beta-actin ratio. The ratio of control animals is set as 100%. *P < 0.05 compared with the controls.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding in the present study is that furosemide-induced hypercalciuria is accompanied by an increased abundance of calcium transporters in the DCT for both acute and chronic treatments. The immediate response illustrates the rapidness of the reaction in DCT transport. In the chronic experiments, furosemide upregulates the expression of TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k, and these effects are not affected by the volume status of the mice.

As furosemide inhibits NKCC2, the lumen-positive charge is reduced because of diminished active sodium reabsorption and potassium recycling. Consequently, the paracellular transport of calcium in the TALH is reduced and more calcium is delivered into downstream segments beyond the TALH. In vivo micropuncture study has shown that intraluminal furosemide perfusion in the TALH resulted in a load-dependent increase in absolute calcium reabsorption in superficial distal tubules (27). In the presence of increased solute load, a two- to threefold increases in calcium binding proteins, calbindin-D9k and calbindin-D28k, TRPV5, and TRPV6 were observed in our study. It has been proposed that vitamin D might mediate the furosemide-induced increase in calbindin-D28k (28). However, other studies did not find any alternation in calciotropic hormones including vitamin D (4, 13). We believe that the upregulation of these calcium transporters is more likely due to the increased solute delivery to the DCT. Similar upregulation of DCT calcium transport molecules induced by hypercalciuria was observed in gentamicin-treated mice in the present studies, as well as in streptozotocin-induced diabetic rats (21). The upregulation of downstream transporters has also been observed in sodium and water transporters after furosemide administration (18). In response to the increased sodium load, structural and function adaptation have been observed in the DCT and collecting duct with increased transport capacity (1, 17). Previous studies have revealed conflicting results that calbindin-D28k was unchanged (13) or upregulated (28) by loop diuretics. It is likely that the variations in the degree of calciuria and different methods may explain these varied results.

To evaluate the effect of volume depletion and sodium deficiency, mice treated with furosemide were given salt supplementation in the drinking water. Our results demonstrate that salt supplement corrects volume deficit but does not affect furosemide-induced hypercalciuria. These findings are consistent with previous studies that showed that salt supplement in furosemide-treated young rats did not affect calciuria and nephrocalcinosis (10). The upregulation of calcium transport molecules are also similar between these two groups of mice, as shown in the present study. In fact, salt supplement alone induces an increase of urinary calcium content, although the change is smaller than that in furosemide-treated mice. With increased salt intake, the calcium reabsorption in the proximal tubule is diminished (22). It appears that increased distal delivery of calcium induced by reduced reabsorption in either proximal tubule or TALH upregulates calcium transport molecules in the distal tubules. These findings support the compensatory mechanism to reduce calcium loss by increasing DCT calcium reabsorption machinery.

Clinically, prolonged use of furosemide is associated with persistent hypercalciuria and nephrocalcinosis especially in infants (7). These patients do not develop hypocalcemia because the serum calcium level is highly regulated by the parathyroid hormone, vitamin D, and calcitonin. However, diminished bone mineral content was observed in longer treatment, indicating a negative calcium balance (8). To maintain calcium homeostasis, compensatory mechanisms must be activated to prevent calcium loss. It has been shown that intestinal calcium absorption is enhanced during chronic furosemide treatment without alternation in serum vitamin D level (4). The upregulation of calcium channels and calcium binding proteins in the DCT is likely to be another compensatory mechanism to reduce calcium loss.

Thiazides have been used to diminish furosemide-induced nephrocalcinosis in infants. Hufnagle et al. (16) reported that furosemide (2 mg·kg–1·day–1) increased urine calcium 10- to 20-fold compared with that shown in untreated infants. The addition of chlorothiazide (20 mg·kg–1·day–1) resulted in a 4- to 15-fold decrease in calcium excretion (16). In the present study, we demonstrate similar calcium conservation effects in mice: chlorothiazide reduces furosemide-induced renal calcium wasting by 40%. Clearly, these two drugs are used synergistically to enhance the diuretic effect, but they affect renal calcium handling in opposite directions.

The mechanisms of hypocalciuric action of thiazides have been controversial. Bindels and colleagues (24) indicated that thiazides increase calcium reabsorption only in the proximal tubule because of volume depletion. They reported the same hypocalciuric response to thiazide in the TRPV5 knockout mice, which do not reabsorb calcium in the DCT. We have shown previously that, when the proximal tubule effect is blocked by salt supplementation, thiazides still reduce urine calcium excretion in wild-type mice (20). Because furosemide causes hypercalciuria in the presence of volume depletion, it provides an opportunity for testing whether the thiazide effect is limited to the proximal tubule. The coadministration of chlorothiazide and furosemide did not result in a higher hematocrit or more weight loss than furosemide-treated mice (Table 2). These data indicate that the degree of volume depletion is similar between the two groups. Under these conditions, chlorothiazide still reduces furosemide-induced calcium wasting, suggesting that thiazides may increase calcium reabsorption in the DCT. The enhancement of DCT calcium reabsorption by inhibition of sodium chloride cotransporter NCC is further supported by a recent study reported by Cheng et al. (5) in patients with Gitelman syndrome due to loss-of-function mutations in the NCC. They demonstrated that hypocalciuria in these patients is minimally corrected with volume repletion by saline infusion. These findings indicate that hypocalciuria in Gitelman syndrome, a condition similar to NCC inhibition by thiazides, is in part due to increased calcium reabsorption in the DCT. We should point out that our data only provide indirect evidence of thiazide effects on DCT calcium reabsorption. Further studies are needed to prove these effects with direct measurement of DCT calcium reabsorption, particularly in conditions with a high distal calcium load as described in our studies.

In conclusion, our studies demonstrate that furosemide treatment induces an upregulation of calcium transport molecules in the DCT. These changes may be stimulated by a load-dependent effect and represent a compensatory adaptation in the DCT. Our finding that addition of chlorothiazide reduces furosemide-induced hypercalciuria without affecting volume status suggests that thiazides may increase calcium reabsorption in the DCT. Further studies are needed to confirm the direct effects of thiazides on calcium transport in the DCT.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the National Science Council of the Republic of China (NSC93-2314-B-182A-107) and a grant from Dialysis Clinic, a nonprofit organization, to Y.-H. H. Lien.


   ACKNOWLEDGMENTS
 
Part of this work has been presented at the 2005 World Congress of Nephrology (Singapore).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-H. H. Lien, Nephrology Section, Dept. of Medicine, Univ. of Arizona Health Sciences Center, Tucson, AZ 85724 (e-mail: lien{at}u.arizona.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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