Metabolic acidosis regulates rat renal Na-Si cotransport activity

Krishna Puttaparthi1, Daniel Markovich2, Nabil Halaihel1, Paul Wilson1, Hubert K. Zajicek1, Huamin Wang1, Jürg Biber3, Heini Murer3, Thomas Rogers1, and Moshe Levi1

1 Department of Internal Medicine, University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center, Dallas, Texas 75216; 3 Institute of Physiology, University of Zürich, 8057 Zürich, Switzerland; and 2 Department of Physiology and Pharmacology, University of Queensland, Brisbane 4072, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, we cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (Si) transport protein (Na-Si cotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-Si cotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of Si, which was associated with a 2.4-fold decrease in BBM Na-Si cotransport activity. The decrease in Na-Si cotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Si cotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-Si cotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Si reabsorption by causing decreases in BBM Na-Si cotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-Si cotransport activity during MA.

sodium-inorganic sulfate cotransport; brush-border membrane; serum sulfate; urinary excretion; Xenopus laevis oocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

METABOLIC ACIDOSIS (MA) causes a number of adaptive changes in the renal tubules that induce an increase in the net urinary excretion of acid. Chronic MA leads to a large increase in proximal tubular reabsorption of bicarbonate (23) and increases in the activities of the apical Na/H exchanger (1, 14, 21, 43, 50), the vacuolar H-ATPase (12), and the basolateral Na-3HCO3 cotransporter (1, 43).

Inorganic sulfate (Si) is an important anion for growth and development; its homeostasis is maintained through renal clearance mechanisms. Si is freely filtered at the glomerulus and is actively reabsorbed in the proximal convoluted tubule of the kidney. Stop-flow experiments suggest that the kidney proximal tubule is the major site of Si reabsorption. The active Si reabsorption process is capacity limited and saturable (40, 51). Human plasma Si concentration is maintained within a narrow range of 1.0-1.5 mM (39). Renal clearance of Si increases with increasing serum Si concentrations, reaching approximately the glomerular filtration rate (GFR). Because Si is not extensively bound to serum proteins (8), this suggests that Si is not secreted to any significant extent. At a physiological serum Si concentration of 0.7-1.0 mM in the rat (22, 39, 44), renal Si clearance is ~30% of the GFR. At decreased serum Si concentrations due to conjugation reactions with exogenous compounds, a decrease in renal clearance, i.e., an increase in the renal tubular reabsorption of Si, is observed (22).

Regulation of the renal excretion of Si is mediated mainly at the level of the proximal tubule via a Na gradient reabsorption of Si (Na-Si cotransport) (19, 40, 51). Recently we have cloned the rat renal proximal tubular Na-Si cotransport system (NaSi-1) (33, 34). Expression of NaSi-1 in Xenopus oocytes revealed that the kinetic parameters of the expressed Na-Si cotransport are similar to those found in transport experiments performed with proximal tubular brush-border membrane (BBM) (11, 33). RT-PCR experiments using microdissected nephron segments from rat kidney indicated proximal expression of NaSi-1 (15). Furthermore, immunohistochemical studies using a highly specific NaSi-1 antibody indicated that the NaSi-1 protein is exclusively expressed in the apical membranes of the proximal tubules (28).

The purposes of this study were to determine whether MA alters renal Si transport and whether the regulation takes place at the level of the proximal tubular BBM Na-Si cotransport system (NaSi-1). In this study we show that MA causes an increase in the urinary excretion of Si, which is associated with a 2.4-fold decrease in BBM Na-Si cotransport activity, a 2.8-fold decrease in BBM NaSi-1 protein abundance, and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. In addition, in Xenopus oocytes injected with mRNA from rats with MA, there is also a similar decrease in the resultant Na-Si cotransport activity. Our results therefore indicate that decreases in the expression of NaSi-1 mRNA and protein, at least in part, play an important role in the inhibition of Na-Si cotransport activity during MA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals and dietary conditions. All experiments were performed with male Sprague-Dawley rats weighing 175-225 g. During the experiments the animals were housed in individual metabolic cages to control food and fluid intake and to collect urine for the determination of Si and creatinine levels. Experiments were started after the animals were allowed to adapt to the metabolic cages for 3-5 days.

For acute acidosis (6-24 h) experiments, the rats were trained to eat their food and drink their water between 7:00 and 11:00 a.m. The rats were trained for three consecutive days, and on the fourth day (the day of the experiment), the rats were given either control chow and deionized water or control chow supplemented with 16 mg NH4Cl/g diet and 0.28 M NH4Cl in deionized water. Control and acid-fed rats were then killed 6, 12, or 24 h later.

For chronic acidosis (10 days) experiments, control and experimental rats were pair fed either control rat chow (controls) or control rat chow supplemented with 16 mg NH4Cl/g diet. Control animals received deionized water; the acid-fed animals received 0.28 M NH4Cl in deionized water.

At the end of the experiments the rats were anesthetized with intraperitoneal pentobarbital, and an aortic puncture was performed to obtain blood for measurement of arterial blood gas and Si and creatinine levels. Both kidneys were then rapidly removed; one-half of each kidney was used for BBM isolation, and the other half was used for RNA isolation. We used at least six rats (n = 6 BBM or RNA samples) from the control and acid-treated groups.

Plasma and urine electrolyte measurements. Urine and blood samples were analyzed for Si concentration with an ion chromatograph (Dionex, Sunnyvale, CA) and for creatinine concentration (autoanalyzer) (32). The total urinary excretion of Si and the fractional excretion of Si were calculated by standard clearance formulas.

BBM isolation and Na-Si uptake measurements. Renal BBMs were isolated according to the Mg precipitation method described previously (9, 47). Determinations of BBM Na-Si cotransport in freshly isolated BBM vesicles were performed by measurement of radiotracer uptake of Na235SO4 (DuPont NEN Research Products, Boston, MA) and an inwardly directed Na gradient (120 mM NaCl), followed by rapid filtration (32, 35). Uptake was stopped after 10 s; this uptake represented the initial linear rate. BBM Na-Si cotransport activity is presented as picomoles of 35SO4 per 10 seconds per milligram of BBM protein. The protein concentration of BBM was measured by the Lowry method (30).

Western blotting. Western blotting experiments using BBM with the NaSi-1 polyclonal antibody were performed as described previously (28, 32, 36). BBM protein (20 µg/lane) was separated after SDS-gel electrophoresis (24) and transferred onto nitrocellulose membranes (49). Anti-NaSi-1 antiserum was used at a dilution of 1:5,000 (28), and primary antibody binding was visualized with anti-rabbit IgG conjugated to horseradish peroxidase by enhanced chemiluminescence (Pierce, Bradford, IL). Fusion protein (2 µg/ml) was added, together with the anti-NaSi-1 antiserum, for the peptide protection experiments. The resulting bands were imaged and quantitated by a phosphorimager equipped with a chemiluminescence detector and densitometry software (Bio-Rad, Richmond, CA).

RNA isolation and Northern blotting. Rat renal cortical RNA was isolated by the guanidinium thiocyanate technique (13), as described previously (3, 32, 35). RNA gel electrophoresis and Northern blotting were performed exactly as described previously (16, 32, 35) with a full-length NaSi-1 cDNA probe (32-36), as well as 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, and beta -actin cDNA probes, as previously described (2, 3). The resulting bands were imaged and quantitated by a phosphorimager equipped with densitometry software (Bio-Rad).

mRNA isolation, injection into Xenopus laevis oocytes, and uptake measurements. Rat renal cortical poly(A)+ mRNA was purified through an oligo(dT) column, and this mRNA (0.2 µg/µl) was injected into X. laevis oocytes as described previously (32-36). Oocyte transport measurements were performed with K235SO4 (30 µCi/ml) with 0.1 mM K2SO4, as described previously (32-36).

Immunohistochemistry. For these studies four additional control rats and rats with MA were anesthetized with 100 mg/kg body wt of thiopental and the kidneys were fixed by perfusion as described previously (26, 28, 29). Kidney samples were immediately fixed in 5% paraformaldehyde, dehydrated through a graded series of ethanol solutions, and embedded in paraffin. Sections (4 µm thick) were cut and mounted onto gelatin-coated glass slides. Immunohistochemical detection of NaSi-1 protein was performed with a 1:800 dilution of NaSi-1 antiserum and avidin-biotin peroxidase complexes (Vector Laboratories, Burlingame, CA). The sections were incubated with the rabbit anti-rat NaSi-1 antibody overnight, washed with PBS for 15 min, and incubated with biotinylated goat anti-rabbit immunoglobulin serum for 1 h. After a 15-min wash with PBS, the slides were immersed in PBS containing 3% hydrogen peroxide for 3 min. After a 15-min wash with PBS, the kidney sections were incubated with avidin-biotin peroxidase complexes for 1 h. After extensive washing for 30 min with PBS, the sections were incubated with the substrate solution (0.05% diaminobenzidine, 0.03% hydrogen peroxide, and 0.05% nickel chloride in PBS buffer) for 10 min. Sections were then rinsed for 15 min with PBS, dehydrated in a graded series of alcohol solutions, and mounted. The sections were also counterstained with 0.1% fast green FCF dye solution for cytoplasmic staining.

Data analysis. All data are presented as means ± SE; n = 6 rats in each experimental group. The statistical significance of results for control rats and rats with MA was determined by an unpaired Student t-test, with significance accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of NH4Cl diet on arterial blood pH and [HCO3]. For rats receiving NH4Cl in their diet and drinking water, significant decreases in arterial blood pH and HCO3 concentration ([HCO3]) were evident as early as in 6 h. These changes persisted at 12 h and became more marked after 24 h and 10 days of treatment (Table 1).

                              
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Table 1.   Effect of acid diets on arterial blood pH and [HCO3]

Effect of MA on BBM Na-Si cotransport and urinary Si excretion. In rats with MA there was no effect on BBM Na-Si cotransport activity after 6 or 12 h. However, there was a significant decrease in Na-Si cotransport activity after 24 h (Fig. 1). The decrease in BBM Na-Si cotransport was associated with a significant increase in the fractional excretion of Si (FESi; Fig. 2A). The decrease in BBM Na-Si cotransport activity and the increase in urinary Si excretion also persisted in rats with MA for 10 days (Fig. 2B).


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Fig. 1.   Effects of 6, 12, and 24 h of metabolic acidosis (MA) on brush-border membrane (BBM) vesicle Na-inorganic sulfate (Si) cotransport activity. Initial rate of Na gradient-dependent Si (Na/Si) uptake in BBM vesicles isolated from control rats (CON) or rats with MA (ACID) was determined as detailed in MATERIALS AND METHODS. Data are means ± SE for 6 rats per condition. NS, not significant.




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Fig. 2.   Effects of 24-h (A) and 10-day (B) MA on fractional excretion of inorganic sulfate (FESi) and BBM vesicle Na-Si cotransport activity. FESi {[urine Si/serum Si]/[urine creatinine (Cr)/serum Cr] × 100} was calculated during a 6-h period of urine collection from control rats (CON) or rats with MA (ACID). Data are means ± SE for 6 rats per condition.

Role of Na-Si protein and mRNA in the regulation of Na-Si cotransport activity by MA. The effect of MA to increase the urinary excretion of Si and to decrease BBM Na-Si cotransport activity was associated with a significant decrease in BBM NaSi-1 protein abundance. This effect was seen after 24 h (Fig. 3A), as well as after 10 days (Fig. 4A), of MA. The Western blot data were further confirmed by immunohistochemistry data, which revealed a significant decrease in proximal tubular apical membrane expression of NaSi-1 protein in rats with MA for 24 h or 10 days (Fig. 5). Similarly, MA was also associated with significant decreases in renal cortical NaSi-1 mRNA abundance after 24 h (Fig. 3B) and 10 days (Fig. 4B) of MA. In addition, when mRNA from rats with MA was injected into X. laevis oocytes, there was a marked decrease in the induced Na-Si cotransport activity compared with Na-Si cotransport activity induced by mRNA from control rats (Fig. 6). As we have previously reported (2), MA also caused a decrease in Na-Pi cotransport activity (Fig. 6); however, there was no change in L-arginine transport activity (Fig. 6).



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Fig. 3.   Effects of 24 h of MA on BBM NaSi-1 protein abundance (A) and renal cortical NaSi-1 mRNA abundance (B). Western blot and Northern blot analyses of samples from control rats (CON) and rats with MA (ACID) were performed, and corresponding bands were quantified with a phosphorimager equipped with densitometry software, as detailed in MATERIALS AND METHODS. NaSi-1 mRNA signal was normalized for 18S signal. Data are means ± SE for 6 BBM or RNA samples from 6 rats per condition.




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Fig. 4.   Effects of 10 days of MA on BBM NaSi-1 protein abundance (A) and renal cortical NaSi-1 mRNA abundance (B). Western blot and Northern blot analyses of samples from control rats (Con) and rats with MA (Acid) were performed and quantified as detailed in MATERIALS AND METHODS. Data are means ± SE for 6 BBM or RNA samples from 6 rats per experimental condition.




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Fig. 5.   Effects of MA on NaSi-1 protein expression in perfusion-fixed kidneys. In perfusion-fixed kidney slices, immunohistochemistry using NaSi-1 antibody shows that there is a marked decrease in proximal tubular apical BBM expression of NaSi-1 protein in rats with MA (B) compared with control rats (A).



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Fig. 6.   mRNA-induced Si, Pi, and L-arginine transport in Xenopus oocytes injected with mRNA isolated from control rats (CON) or rats with chronic MA (CMA). Oocytes were injected with 50 nl water or 50 nl water containing kidney cortex mRNA (0.2 µg/µl) from control rats or rats with CMA. Uptake experiments were performed on day 3 postinjection at room temperature for 35 min in presence of Na (100 mM). A: Si transport: K235SO4 uptake (30 µCi/ml) with 0.1 mM K2SO4. B: Pi transport: K2H32PO4 uptake (20 µCi/ml) with 0.1 mM K2HPO4. C: L-arginine transport: L-[3H]arginine uptake (20 µCi/ml) with 0.1 mM L-arginine. Data are means ± SE for 7-10 oocytes per group, with similar data obtained in 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The kidney plays a major role in the regulation of Si homeostasis. Si is reabsorbed in the proximal convoluted tubule by a Na gradient-dependent process (Na-Si cotransport). MA leads to a number of adaptive changes in renal tubules that contribute to increased urinary net acid excretion. Bicarbonate reabsorption is increased in the proximal tubule after chronic acid loading (23). This adaptation is associated with parallel increases in uptake activities of the apical Na/H exchanger (1, 14, 21, 43, 50), the vacuolar H-ATPase (12), and the basolateral Na-3HCO3 cotransporter (1, 43).

The purpose of the present study was to determine if MA modulates Na-Si cotransport activity and if the effect is mediated through regulation of renal expression of the recently cloned NaSi-1 protein and/or mRNA (33, 34). Compared with control rats, rats subjected to acidosis had an increased excretion of Si, as a consequence of a decrease in the renal tubular Si reabsorption and a parallel decrease in BBM vesicular Na-Si cotransport activity. The decrease in Na-Si cotransport activity was paralleled by a significant decrease in BBM NaSi-1 protein abundance. In addition there was also a significant decrease in NaSi-1 mRNA abundance. We showed that renal cortical mRNA from rats with acidosis, when injected into Xenopus oocytes, led to a marked decrease in Na-Si cotransport activity compared with mRNA isolated from control rats, with no changes in L-arginine transport. Our study therefore indicates that MA causes a decrease in renal cortical NaSi-1 mRNA abundance, which results in diminished expression of the NaSi-1 protein at the level of the proximal tubular apical membrane. At the present time, it is not known whether the decrease in NaSi-1 mRNA abundance in rats with MA is mediated by differential mRNA stability, by transcriptional or posttranscriptional mechanisms, or by a combination of these.

In rats with MA it is possible that, in addition to the decrease in the proximal tubular apical BBM NaSi-1 protein, there is also a decrease in the activity of the Si/oxalate/bicarbonate exchanger (sat-1) localized in the basolateral membrane of the proximal tubule (10, 20).

One interesting observation of our study is that in rats with MA we see the inhibition of Na-Si cotransport activity after 24 h, but not after 12 h, of MA. Although blood pH values are identical at 12 and 24 h, at 24 h there is a further decrease in blood bicarbonate levels. Although this may imply that the downregulation of Na-Si cotransport activity is in response to a change in serum bicarbonate rather than pH per se, it may also indicate that it takes >12 h for the pH- or bicarbonate-induced regulatory mechanisms to cause the down-regulation of NaSi-1 protein and mRNA abundances.

The results of our study are seemingly different from those of an earlier study of the rat that showed that acute MA causes an increase in the renal tubular reabsorption of Si (18). This study was performed with rats, under anesthesia, initially perfused with NaHCO3 (alkalosis, pH 7.53, HCO3 concn 38.2 meq/l) and then with NH4Cl (acidosis, pH 7.29, HCO3 concn 19.4 meq/l), or vice versa (initially perfused with NH4Cl and then NaHCO3). Interestingly, that study also showed that acidosis caused an increase in the renal tubular reabsorption of Pi, which is in contrast to two well-known effects of acidosis: phosphaturia and inhibition of Na-Pi cotransport activity (Ref. 2 and references therein). At this time it is very likely that differences in the experimental design account for the differences in the results. Furthermore, in our study of conscious (rather than anesthetized) rats fed (rather than infused) an NH4Cl diet, the effect of acidosis on Na-Si cotransport activity was not seen until after 24 h.

It is interesting to contrast the mechanisms involved in the adaptive response of the Na-Si cotransporter to MA to what was previously described as the adaptive responses of the type II Na-Pi cotransporter to acidosis in the rat (2). The effect of acidosis on Na-Pi cotransport was evident as early as after 6 h of acidosis. The decrease in BBM Na-Pi cotransport activity was associated with a similar decrease in BBM NaPi-2 protein abundance, which however occurred independently of changes in NaPi-2 mRNA abundance. However, in response to a chronic (>12 h) acidosis, the decrease in Na-Pi cotransport activity was associated with parallel decreases in NaPi-2 protein and mRNA levels (2).

MA has recently been shown to affect another renal Na-coupled transporter, the Na-citrate cotransporter (NaDC-1), which was upregulated both at the mRNA and protein levels by acidosis in rats (4). Recently, the Na/H exchanger (NHE3) was also shown to be up-regulated at the protein and mRNA levels in response to chronic MA (25). Thus it can be observed that MA regulates the expression and function of a multitude of renal transporters.

In addition to MA, various other conditions have been shown to modulate renal Na-Si cotransport at the level of the proximal tubular BBM. Dietary Si intake (7, 32, 41, 42), thyroid hormone (in the mouse but not in the rat) (6, 48), glucocorticoids (45, 46), aging (5), acetaminophen (27), nonsteroidal anti-inflammatory agents (37), and salicylic acid (38) have been shown to modulate urinary Si excretion and/or BBM Na-Si cotransport activity. A recent study has shown that vitamin D also modulates renal Na-Si cotransport (17). Vitamin D-deficient rats showed lower plasma Si levels and an increased fractional excretion of Si, which correlated with decreases in BBM Na-Si cotransport activity and NaSi-1 protein and mRNA abundances (17). We have also recently shown that NaSi-1 mRNA and protein levels are downregulated in rats fed high-Si (32) or potassium-deficient (36) diets. All these studies suggest that the NaSi-1 cotransporter is a highly regulated physiological transport protein in the renal proximal tubules.

In conclusion, our study demonstrates, for the first time, that MA is an important modulator of renal Na-Si cotransport activity. We have shown that acidosis decreases rat renal proximal tubular Si reabsorption and Na-Si cotransport activity, at least in part by decreasing the abundance of the renal cortical NaSi-1 mRNA and expression of NaSi-1 protein in the apical membrane of the rat proximal tubule. This is of particular physiological and pathophysiological relevance because it demonstrates that Na-Si cotransport activity and mRNA and protein abundances can be regulated under conditions necessitating increased renal acidification (such as MA and/or dietary potassium deficiency).


    ACKNOWLEDGEMENTS

We thank Dr. Sakhaee and Dr. Pak at the University of Texas Southwestern Medical Center, Dallas, TX, for the measurement of serum and urinary Si, Teresa Autrey for secretarial assistance, and the Medical Media Department at the Department of Veterans Affairs Medical Center for the illustrations.


    FOOTNOTES

This work was supported by the Department of Veterans Affairs Merit Review and the National Kidney Foundation (M. Levi), a Department of Veterans Affairs Minority Initiative Grant (to K. Puttaparthi and M. Levi), National Institutes of Health National Research Service Award Grant 1-F32-DK-09689-01 to H. Zajicek, National Health and Medical Research Council of Australia Grant 961188 to D. Markovich, and Swiss National Funds Grant 32-30-785-91 to H. Murer.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Levi, 4500 South Lancaster Rd., MC 151, Dallas, TX 75216 (E-mail: mmjjl{at}aol.com).

Received 9 December 1998; accepted in final form 22 March 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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