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Renal phosphaturia during metabolic acidosis revisited: molecular mechanisms for decreased renal phosphate reabsorption

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Abstract

During metabolic acidosis (MA), urinary phosphate excretion increases and contributes to acid removal. Two Na+-dependent phosphate transporters, NaPi-IIa (Slc34a1) and NaPi-IIc (Slc34a3), are located in the brush border membrane (BBM) of the proximal tubule and mediate renal phosphate reabsorption. Transcriptome analysis of kidneys from acid-loaded mice revealed a large decrease in NaPi-IIc messenger RNA (mRNA) and a smaller reduction in NaPi-IIa mRNA abundance. To investigate the contribution of transporter regulation to phosphaturia during MA, we examined renal phosphate transporters in normal and Slc34a1-gene ablated (NaPi-IIa KO) mice acid-loaded for 2 and 7 days. In normal mice, urinary phosphate excretion was transiently increased after 2 days of acid loading, whereas no change was found in Slc34a1−/− mice. BBM Na/Pi cotransport activity was progressively and significantly decreased in acid-loaded KO mice, whereas in WT animals, a small increase after 2 days of treatment was seen. Acidosis increased BBM NaPi-IIa abundance in WT mice and NaPi-IIc abundance in WT and KO animals. mRNA abundance of NaPi-IIa and NaPi-IIc decreased during MA. Immunohistochemistry did not indicate any change in the localization of NaPi-IIa and NaPi-IIc along the nephron. Interestingly, mRNA abundance of both Slc20 phosphate transporters, Pit1 and Pit2, was elevated after 7 days of MA in normal and KO mice. These data demonstrate that phosphaturia during acidosis is not caused by reduced protein expression of the major Na/Pi cotransporters NaPi-IIa and NaPi-IIc and suggest a direct inhibitory effect of low pH mainly on NaPi-IIa. Our data also suggest that Pit1 and Pit2 transporters may play a compensatory role.

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Acknowledgments

M. Nowik is the recipient of a PhD student fellowship from the Zurich Center for Integrative Human Physiology. This study was supported by grants from the Swiss National Science Research Foundation to J. Biber and H. Murer (31-65397.01) and C. A. Wagner (31-109677/1) and the 6th EU Frame work project EuReGene to J. Biber, H. Murer, and C. A. Wagner. The use of the ZIHP Core Facility for Rodent Physiology is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Physiology and Zurich Center for Human Integrative Physiology (ZHIP), University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Marta Nowik, Gerti Stange, Paola Capuano, Jürg Biber, Heini Murer & Carsten A. Wagner

  2. Institute of Anatomy, University of Zurich, Zurich, Switzerland

    Nicolas Picard

  3. Departments of Pediatrics and Human Genetics, McGill University and Montreal Children’s Hospital Research Institute, Montreal, QC, Canada

    Harriet S. Tenenhouse

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  1. Marta Nowik
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Correspondence to Carsten A. Wagner.

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Supplementary Fig. 1

Characterization of the anti-NaPi-IIc antibody by immunoblotting. Brush border membrane vesicles were prepared from kidneys of Slc34a1+/− and Slc34a1−/− mice to test the specificity and cross-reactivity of new antibodies against NaPi-IIc. The antibody against mouse NaPi-IIc was raised in rabbit, affinity-purified, and recognized a single major band of approximately 75kDa (stripe B). This band was prevented by preincubation with the immunizing peptide (stripe C). The preimmune serum from the rabbit used to obtain the anti-NaPi-IIc antibody did not show any reactivity (stripe D). Stripe A shows NaPi-IIa staining in the same samples (PDF 50 KB).

Supplementary Fig. 2

Characterization of anti-NaPi-IIc antibody by immunohistochemistry. Serial kidney sections of normal mice were stained with antibodies against NaPi-IIa (left panel), NaPi-IIc (middle panel), and NaPi-IIc after preincubation with the immunizing peptide (right panel). No staining was detected with the anti-NaPi-IIc antibody after preincubation with the immunizing peptide. Please note: The background level was enhanced in the right panel to detect possible unspecific staining (PDF 342 KB).

Supplementary Fig. 3

Increased abundance of NaPi-IIc protein in the brush border membrane of kidneys from NaPi-IIa-deficient mice. Blots were probed for NaPi-IIc and β-actin, and data are summarized by normalizing NaPi-IIc abundance against β-actin (mean ± SEM). These data are in agreement with previous work demonstrating that renal NaPi-IIc protein expression is significantly increased in NaPi-Iia-deficient mice when compared with normal littermates [34] (PDF 29 KB).

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Nowik, M., Picard, N., Stange, G. et al. Renal phosphaturia during metabolic acidosis revisited: molecular mechanisms for decreased renal phosphate reabsorption. Pflugers Arch - Eur J Physiol 457, 539–549 (2008). https://doi.org/10.1007/s00424-008-0530-5

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