Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA

Background: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl− cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. Methods: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. Results: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. Conclusion: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies

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

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

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

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

<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