<i>P2x</i> subunit expression in response to the loss of <i>P2x6</i> function in the kidney.

<p>a-f) The mRNA expression levels of <i>P2x1</i> (a), <i>P2x2</i> (b), <i>P2x3</i> (c), <i>P2x4</i> (d), <i>P2x5</i> (e), <i>P2x7</i> (f), in kidney of <i>P2x6</i>^<i>+/+</i> (Black bars), <i>P2x6</i>^<i>+/-</i> (Striped bars), <i>P2x6</i>^<i>-/-</i> (white bars) mice were measured by quantitative RT-qPCR and normalized for <i>Gapdh</i> expression. Data (n = 10) represent mean ± SEM and are expressed as the fold difference when compared to the expression in <i>P2x6</i>^<i>+/+</i> mice.</p

Metabolic Parameters of P2x6 Mice.

<p>Metabolic Parameters of P2x6 Mice.</p

P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

<div><p>ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg²⁺ and Na⁺ reabsorption, but its role in ion transport remains unknown. In this study, <i>P2x6</i> knockout <i>(P2x6</i>^<i>-/-</i>) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The <i>P2x6</i>^<i>-/-</i> animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na⁺, K⁺, Mg²⁺ and Ca²⁺ were not detected between <i>P2x6</i>^+/+, <i>P2x6</i>^+/- and <i>P2x6</i>^-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other <i>P2x</i> subunits and electrolyte transporters, including <i>P2x1-5</i>, <i>P2x7</i>, <i>Trpm6</i>, <i>Ncc</i>, <i>Egf</i>, <i>Cldn16</i>, <i>Scnn1</i>, <i>Slc12a3</i>, <i>Slc41a1</i>, <i>Slc41a3</i>, <i>Cnnm2</i>, <i>Kcnj10 and Fxyd2</i>. Additionally, protein levels of P2X2 and P2X4 were assessed in <i>P2x6</i>^<i>+/+</i> and <i>P2x6</i>^<i>-/-</i> mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from <i>P2x6</i>^<i>-/-</i> mice. Except for a significant (P<0.05) upregulation of <i>P2x2</i> in the heart of <i>P2x6</i>^<i>-/-</i> mice compared to the <i>P2x6</i>^<i>+/+</i> mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions.</p></div

Basolaterally expressed compensatory mechanisms for the loss of P2x6 function in the kidney.

<p>a-d) The mRNA expression levels of <i>Fxyd2</i> (a), <i>Kcjn10</i> (b), <i>Slc41a1</i> (c), <i>Slc41a3</i> (d) in kidney of <i>P2x6</i>^<i>+/+</i> (Black bars), <i>P2x6</i>^<i>+/-</i> (Striped bars), <i>P2x6</i>^<i>-/-</i> (white bars) mice were measured by quantitative RT-qPCR and normalized for <i>Gapdh</i> expression. Data (n = 10) represent mean ± SEM and are expressed as the fold difference when compared to the expression in <i>P2x6</i>^<i>+/+</i> mice.</p

Protein abundance of P2x2 and P2x4 in response to the loss of P2x6.

<p>a) Western blots of membrane and cytosol fractions of <i>P2x6</i>^<i>-/-</i> (KO) and <i>P2x6</i>^<i>+/+</i> (WT) mice. The upper blot shows the immune-staining for P2x2 in mouse kidney material. To the right a western blot of HEK293 cells transiently transfected with HA-tagged P2x4 (P4) and mock (M) constructs is displayed. Middle, two western blots below are immune-stained for P2x4, left depicts <i>P2x6</i>^<i>-/-</i> (KO) and <i>P2x6</i>^<i>+/+</i> (WT) material stained for P2x4, right represents a P2x4 blot on HEK293 material transiently transfected with human P2X4 and a mock construct. Bottom, displays a ß-actin immune-staining used as a loading control. Ladders (ez-run prestained marker (ThermoScientific, Breda, The Netherlands) represent protein size in kilo Dalton (kD). b) Protein expression levels for the P2x2 membrane lysate, P2x4 membrane lysate, P2x4 cytosol lysate, ß-actin membrane and ß-actin cytosol lysates in <i>P2x6</i>^<i>-/-</i> and <i>P2x6</i>^<i>+/+</i> mice. Data (n = 3) represents mean ± SEM and are expressed as the % of total band intensity.</p

Gene expression of renal electrolyte transporters was not altered in <i>P2x6</i>^<i>-/-</i> mice.

<p>a-f) The mRNA expression levels of <i>Trpm6</i> (a), <i>Egf</i> (b), <i>Cldn16</i> (c), <i>Cnnm2</i> (d), <i>Scnn1a</i> (e), <i>Slc12a3</i> (f) in kidney of <i>P2x6</i>^<i>+/+</i> (Black bars), <i>P2x6</i>^<i>+/-</i> (Striped bars), <i>P2x6</i>^<i>-/-</i> (white bars) mice were measured by quantitative RT-PCR and normalized for <i>Gapdh</i> expression. Data (n = 10) represent mean ± SEM and are expressed as the fold difference when compared to the expression in <i>P2x6</i>^<i>+/+</i> mice.</p

Normal renal electrolyte handling in <i>P2x6</i>^<i>-/-</i> mice.

<p>a) Serum Mg²⁺ concentrations of wild-type, heterozygous and knockout P2x6 mice. b) 24 hrs urinary Mg²⁺ excretion of wild-type, heterozygous and knockout P2x6 mice. c) Serum Ca²⁺ concentrations. d) 24 hrs urinary Ca²⁺ excretion. e) Serum Na⁺ concentrations. f) 24 hrs urinary Na⁺ excretion. g) Serum K⁺ concentrations. h) 24 hrs urinary K⁺ excretion. Values (n = 10) are presented as means ± SEM.</p

Characteristics of the <i>P2x6</i>^<i>-/-</i> mouse.

<p>a) Targeted insertion of the knockout (KO) cassette. Top: <i>P2x6</i> locus on chromosome 16. Bottom: targeted allele in which the KO cassette is inserted within exon 2. Grey boxes indicate exons, arrows depict genotype primers. a-c) SA: Splice acceptor site, IRES: internal ribosome entry site, LacZ: ß-galactosidase, NEO: neomycin cassette, pA: polyA. b) Identification of the mouse genotype by PCR analysis of ear-derived DNA. The PCR product size ± 478 bp shows the presence of the wild-type allele (+/+), using primers A and C; the PCR product sized ± 800 bp shows the KO allele (-/-) using primers B and C. Both alleles are detected in heterozygous animals (+/-). c) cDNA isolated from murine heart samples were used to amplify exons 1–12 of <i>P2x6</i> with PCR. The top agarose gels show the PCR products for exons 1–12 in two <i>P2x6</i>^<i>+/+</i> animals. The lower gels represent the PCR products for exons 1–12 in two <i>P2x6</i>^<i>-/-</i> animals.</p

Compensatory mechanisms for the loss of P2x6 function in the heart.

<p>a-h) The mRNA expression levels of <i>P2x1</i> (a), <i>P2x2</i> (b), <i>P2x3</i> (c), <i>P2x4</i> (d), <i>P2x5</i> (e), <i>P2x7</i> (f), <i>Trpm7</i> (g), <i>Cnnm2</i> (h), in heart of <i>P2x6</i>^<i>+/+</i> (Black bars), <i>P2x6</i>^<i>+/-</i> (Striped bars), <i>P2x6</i>^<i>-/-</i> (white bars) mice were measured by quantitative RT-qPCR and normalized for <i>Gapdh</i> expression. Data represent mean (n = 10) ± SEM and are expressed as the fold difference when compared to the expression in <i>P2x6</i>^<i>+/+</i> mice. * P< 0.05 indicates a significant difference from <i>P2x6</i>^<i>+/+</i> mice.</p