Kidney transcriptome reveals altered steroid homeostasis in NaS1 sulfate transporter null mice

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Abstract

Sulfate is essential for human growth and development, and circulating sulfate levels are maintained by the NaS1 sulfate transporter which is expressed in the kidney. Previously, we generated a NaS1-null (Nas1−/−) mouse which exhibits hyposulfatemia. In this study, we investigated the kidney transcriptome of Nas1−/− mice. We found increased (n = 25) and decreased (n = 60) mRNA levels of genes with functional roles that include sulfate transport and steroid metabolism. Corticosteroid-binding globulin was the most up-regulated gene (110% increase) in Nas1−/− mouse kidney, whereas the sulfate anion transporter-1 (Sat1) was among the most down-regulated genes (≥50% decrease). These findings led us to investigate the circulating and urinary steroid levels of Nas1−/− and Nas1+/+ mice, which revealed reduced blood levels of corticosterone (≈50% decrease), dehydroepiandrosterone (DHEA, ≈30% decrease) and DHEA-sulfate (≈40% decrease), and increased urinary corticosterone (≈16-fold increase) and DHEA (≈40% increase) levels in Nas1−/− mice. Our data suggest that NaS1 is essential for maintaining a normal metabolic state in the kidney and that loss of NaS1 function leads to reduced circulating steroid levels and increased urinary steroid excretion.

Introduction

Inorganic sulfate (SO42−) is the fourth most abundant anion in mammalian plasma and is essential for numerous metabolic and cellular processes (reviewed in [1]). SO42− conjugation or sulfonation is an important step in the metabolism of drugs and xenobiotics, and in the biotransformation of endogenous compounds, including cholesterol and steroid hormones [2]. In particular, sulfonation markedly changes the physicochemical properties of steroids, and in most cases, decreases the biological activity of the steroid by preventing their binding to steroid receptors (reviewed in [3], [4]). In addition, steroid sulfonates form a circulating and intracellular reservoir, which can be activated via steroid sulfatases (reviewed in [5]). Importantly, sufficient levels of SO42− and its universal sulfonate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS), need to be maintained for sulfonation reactions to function efficiently (reviewed in [1]).

The kidneys play an essential role in maintaining plasma SO42− levels. SO42− is filtered in the glomerulus and actively reabsorbed in the proximal tubule (reviewed in [1]). The first step of renal SO42− reabsorption is mediated by the Na+ sulfate cotransporter-1 (NaS1) on the apical brush border membrane [6] and the second step via the sulfate anion transporter-1 (Sat1) on the basolateral membrane [7], [8]. We have cloned the mouse NaS1 gene, designated Slc13a1 [9], and generated NaS1-null (Nas1−/−) mice, which have hyposulfatemia, increased urinary sulfate excretion, reduced growth and hepatic glycogen levels, increased serum LDL-cholesterol levels, fatty liver and a decreased hepatic glutathione and sulfonation capacity [10], [11], [12]. One approach to understanding these abnormal features in the Nas1−/− mouse, is to determine the kidney transcriptional profile of Nas1−/− mice and compare those to the wild-type kidney where NaS1 is primarily expressed. Transcriptional profiling has become a valuable tool for investigating gene expression in mammalian physiology. In particular, studies of the kidney transcriptome have led to the identification of genes that are involved in kidney development [13] and chronic renal dysfunction [14]. Among the most abundant transcripts in the kidney, are those belonging to the solute linked carrier (SLC) gene families, which have functional roles in the transport of various substrates, including ions, amino acids and glucose, across the renal tubular epithelium.

Studies of the Nas1−/− mouse, can give us an insight into the consequences of a disrupted SLC13A1 gene, yet to be linked to human disease. Of particular relevance to these studies, is that we recently identified two polymorphisms (R12X and N174S) in human SLC13A1, which lead to 100% and 60% loss of SO42− transport function, respectively [12]. The aims of this study were to determine the kidney transcriptional profile of mice lacking Slc13a1 and compare those to wild-type mice. Our findings show changes in the mRNA expression of genes involved in steroid metabolism in the kidneys of Nas1−/− mice, as well as reduced circulating steroid levels and increased urinary steroid concentrations.

Section snippets

Mice

We previously generated Nas1 knock-out (Nas1−/−) mice in which the NaS1 gene was disrupted by targeted mutagenesis [10]. Male Nas1−/− and wild-type Nas1+/+ (controls) mice were group housed at a constant temperature (23 ± 1 °C) with a 12 h/12 h light/dark cycle (lights on at 06.00 h and off at 18.00 h). Male mice were weaned at 3-wk of age and were then fed a standard rodent chow (no. AIN93G: Glen Forrest Stockfeeders, Glen Forest, Western Australia) and water ad libitum for 1 wk, and were used for

Kidney transcriptional profile of Nas1−/− mice

Since the kidneys play an important role in sulfate reabsorption (reviewed in [1]), as well as being the major tissue of NaS1 expression [9], [22], we investigated the kidney gene expression profile of Nas1−/− and Nas1+/+ mice using cDNA microarrays, which revealed transcriptional differences in the Nas1−/− mice. We have listed all genes assessed as having a B statistic score greater than zero (Table 2, Table 3). Of these, 19 transcripts were up-regulated and 39 down-regulated in Nas1−/− mice

Discussion

In this study, we determined the kidney mRNA profile of Nas1−/− mice, which revealed transcriptional changes for 85 genes, including genes with functional roles in steroid metabolism. These findings led us to quantitate circulating and urinary steroid concentrations, which revealed altered corticosterone and DHEA homeostasis in the Nas1−/− mice. In addition, we found decreased Sat1 sulfate transporter mRNA levels in the Nas1−/− kidney, and demonstrated that sulfate administration was unable to

Acknowledgements

We thank Drs. M. Waters, T. Walker and H. Cooper (University of Queensland, Brisbane, Australia) for valuable discussions. Cbg and Cyp11a1 antibodies were kind gifts from Drs. G.L. Hammond (Child and Family Research Institute, Vancouver, Canada) and B. Chung (Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan), respectively. This work was supported by the Australian National Health and Medical Research Council, Australian Research Council, and a Project grant awarded by the

References (50)

  • M.J. Meaney et al.

    Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response

    J. Steroid Biochem. Mol. Biol.

    (1991)
  • G.J. Chader et al.

    Steroid-protein interactions. XXVI. Studies on the polymeric nature of the corticosteroid-binding globulin of the rabbit

    J. Biol. Chem.

    (1972)
  • P.A. Dawson et al.

    Behavioural abnormalities of the hyposulfataemic Nas1 knock-out mouse

    Behav. Brain Res.

    (2004)
  • F. Labrie et al.

    DHEA and its transformation into androgens and estrogens in peripheral target tissues: intracrinology

    Front. Neuroendocrinol.

    (2001)
  • A. Lee et al.

    NaSi-1 and Sat-1: structure, function and transcriptional regulation of two genes encoding renal proximal tubular sulfate transporters

    Int. J. Biochem. Cell Biol.

    (2005)
  • V.F. Price et al.

    Effects of sulfur-amino acid-deficient diets on acetaminophen metabolism and hepatotoxicity in rats

    Toxicol. Appl. Pharmacol.

    (1989)
  • D. Markovich

    Physiological roles and regulation of mammalian sulfate transporters

    Physiol. Rev.

    (2001)
  • C.N. Falany

    Enzymology of human cytosolic sulfotransferases

    FASEB J.

    (1997)
  • C.A. Strott

    Steroid sulfotransferases

    Endocr. Rev.

    (1996)
  • C.A. Strott

    Sulfonation and molecular action

    Endocr. Rev.

    (2002)
  • D. Markovich et al.

    Expression cloning of rat renal Na+/SO42− cotransport

    Proc. Natl. Acad. Sci. U.S.A.

    (1993)
  • L.P. Karniski et al.

    Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney

    Am. J. Physiol.

    (1998)
  • P.A. Dawson et al.

    Hyposulfatemia, growth retardation, reduced fertility and seizures in mice lacking a functional NaSi-1 gene

    Proc. Natl. Acad. Sci. U.S.A.

    (2003)
  • P.A. Dawson et al.

    Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice

    Physiol. Genomics

    (2006)
  • S. Lee et al.

    Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity

    Hepatology

    (2006)
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