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Background Hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase Transient Receptor Potential Melastatin type 6. Patients have very low serum Mg2+ levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without diagnosis. Methods In this study, two families with a HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7 identified variants on Mg2+ transport was examined. Results For the first time, variants in TRPM7 were identified in two families as potential cause for hereditary hypomagnesemia with secondary hypocalcemia. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcemia. In the first family, a splice-site variant caused the incorporation of intron 1 sequences in the TRPM7 mRNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit reduced cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. Conclusion We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesemia and secondary hypocalcemia. Screening of unresolved patients with hypomagnesemia and secondary hypocalcemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.

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Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1 −/− mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1 −/− mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1 −/− iMCD3 cells to EVs derived from Pkd1 −/− mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1 −/− cells to mDCT15 Pkd1 −/− EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.