Transcription factor HNF1ß regulates expression of the calcium-sensing receptor in the thick ascending limb of the kidney.

Mutations in hepatocyte nuclear factor 1ß (HNF1ß) cause autosomal dominant tubulointerstitial kidney disease (ADTKD-HNF1ß), and patients tend to develop renal cysts, maturity-onset diabetes of the young (MODY), and suffer from electrolyte disturbances, including hypomagnesemia, hypokalemia, and hypocalciuria. Previous HNF1ß research focused on the renal distal convoluted tubule (DCT) to elucidate the ADTKD-HNF1ß electrolyte phenotype, although 70% of Mg2+ is reabsorbed in the thick ascending limb of Henle's loop (TAL). An important regulator of Mg2+ reabsorption in the TAL is the calcium-sensing receptor (CaSR). This study used several methods to elucidate the role of HNF1ß in electrolyte reabsorption in the TAL. HNF1ß ChIP-seq data revealed a conserved HNF1ß binding site in the second intron of the CaSR gene. Luciferase-promoter assays displayed a 5.8-fold increase in CaSR expression when HNF1ß was present. Expression of the HNF1ß p.Lys156Glu mutant, which prevents DNA binding, abolished CaSR expression. Hnf1ß knockdown in an immortalized mouse kidney TAL cell line (MKTAL) reduced expression of the CaSR and Cldn14 (claudin 14) by 56% and 48%, respectively, while Cldn10b expression was upregulated 5.0-fold. These results were confirmed in a kidney-specific HNF1ß knockout mouse, which exhibited downregulation of the Casr by 81%. Cldn19 and Cldn10b expression levels were also decreased by 37% and 83%, respectively, whereas Cldn3 was upregulated by 4.6-fold. In conclusion, HNF1ß is a transcriptional activator of the CaSR. Consequently, patients with HNF1ß mutations may have reduced CaSR activity in the kidney, which could explain cyst progression and hyperabsorption of Ca2+ and Mg2+ in the TAL resulting in hypocalciuria.

Inhibition of PRL-2·CNNM3 Protein Complex Formation Decreases Breast Cancer Proliferation and Tumor Growth.

The oncogenic phosphatase of regenerating liver 2 (PRL-2) has been shown to regulate intracellular magnesium levels by forming a complex through an extended amino acid loop present in the Bateman module of the CNNM3 magnesium transporter. Here we identified highly conserved residues located on this amino acid loop critical for the binding with PRL-2. A single point mutation (D426A) of one of those critical amino acids was found to completely disrupt PRL-2·human Cyclin M 3 (CNNM3) complex formation. Whole-cell voltage clamping revealed that expression of CNNM3 influenced the surface current, whereas overexpression of the binding mutant had no effect, indicating that the binding of PRL-2 to CNNM3 is important for the activity of the complex. Interestingly, overexpression of the CNNM3 D426A-binding mutant in cancer cells decreased their ability to proliferate under magnesium-deprived situations and under anchorage-independent growth conditions, demonstrating a PRL-2·CNNM3 complex-dependent oncogenic advantage in a more stringent environment. We further confirmed the importance of this complex in vivo using an orthotopic xenograft breast cancer model. Finally, because molecular modeling showed that the Asp-426 side chain in CNNM3 buries into the catalytic cavity of PRL-2, we showed that a PRL inhibitor could abrogate complex formation, resulting in a decrease in proliferation of human breast cancer cells. In summary, we provide evidence that this fundamental regulatory aspect of PRL-2 in cancer cells could potentially lead to broadly applicable and innovative therapeutic avenues.

Loss of transcriptional activation of the potassium channel Kir5.1 by HNF1ß drives autosomal dominant tubulointerstitial kidney disease.

Hepatocyte nuclear factor 1 homeobox B (HNF1ß) is an essential transcription factor for the development and functioning of the kidney. Mutations in HNF1ß cause autosomal dominant tubulointerstitial kidney disease characterized by renal cysts and maturity-onset diabetes of the young (MODY). Moreover, these patients suffer from a severe electrolyte phenotype consisting of hypomagnesemia and hypokalemia. Until now, genes that are regulated by HNF1ß are only partially known and do not fully explain the phenotype of the patients. Therefore, we performed chIP-seq in the immortalized mouse kidney cell line mpkDCT to identify HNF1ß binding sites on a genome-wide scale. In total 7,421 HNF1ß-binding sites were identified, including several genes involved in electrolyte transport and diabetes. A highly specific and conserved HNF1ß site was identified in the promoter of Kcnj16 that encodes the potassium channel Kir5.1. Luciferase-promoter assays showed a 2.2-fold increase in Kcnj16 expression when HNF1ß was present. Expression of the Hnf1ß p.Lys156Glu mutant, previously identified in a patient with autosomal dominant tubulointerstitial kidney disease, did not activate Kcnj16 expression. Knockdown of Hnf1ß in mpkDCT cells significantly reduced the appearance of Kcnj16 (Kir5.1) and Kcnj10 (Kir4.1) by 38% and 37%, respectively. These results were confirmed in a HNF1ß renal knockout mouse which exhibited downregulation of Kcnj16, Kcnj10 and Slc12a3 transcripts in the kidney by 78%, 83% and 76%, respectively, compared to HNF1ß wild-type mice. Thus, HNF1ß is a transcriptional activator of Kcnj16. Hence, patients with HNF1ß mutations may have reduced Kir5.1 activity in the kidney, resulting in hypokalemia and hypomagnesemia.

FAM111A is dispensable for electrolyte homeostasis in mice.

Autosomal dominant mutations in FAM111A are causative for Kenny-Caffey syndrome type 2. Patients with Kenny-Caffey syndrome suffer from severe growth retardation, skeletal dysplasia, hypoparathyroidism, hypocalcaemia, hyperphosphataemia and hypomagnesaemia. While recent studies have reported FAM111A to function in antiviral response and DNA replication, its role in regulating electrolyte homeostasis remains unknown. In this study, we assessed the role of FAM111A in the regulation of serum electrolyte balance using a Fam111a knockout (Fam111a-/-) C57BL/6 N mouse model. Fam111a-/- mice displayed normal weight and serum parathyroid hormone (PTH) concentration and exhibited unaltered magnesium, calcium and phosphate levels in serum and 24-hour urine. Expression of calciotropic (including Cabp28k, Trpv5, Klotho and Cyp24a1), magnesiotropic (including Trpm6, Trpm7, Cnnm2 and Cnnm4) and phosphotropic (Slc20a1, Slc20a2, Slc34a1 and Slc34a3) genes in the kidneys, duodenum and colon were not affected by Fam111a depletion. Only Slc34a2 expression was significantly upregulated in the duodenum, but not in the colon. Analysis of femurs showed unaffected bone morphology and density in Fam111a-/- mice. Kidney and parathyroid histology were also normal in Fam111a-/- mice. In conclusion, our study is the first to characterise the function of FAM111A in vivo and we report that mice lacking FAM111A exhibit normal electrolyte homeostasis on a standard diet.

P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis.

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, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- 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 P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- 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 P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ 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.

Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells.

BACKGROUND: Adrenal chromaffin cells are a widely used model system to study regulated exocytosis and other membrane-associated processes. Alterations in the amount and localization of the proteins involved in these processes can be visualized with fluorescent probes that report the effect of different stimuli or genetic modifications. However, the quantitative analysis of such images remains difficult, especially when focused on specific locations, such as the plasma membrane. NEW METHOD: We developed an image analysis algorithm, named plasma membrane analysis in chromaffin cells (PlasMACC). PlasMACC enables automatic detection of the plasma membrane region and quantitative analysis of multi-fluorescent signals from spherical cells. PlasMACC runs in the image analysis software ImageJ environment, it is user-friendly and freely available. RESULTS: PlasMACC delivers detailed information about intensity, thickness and density of fluorescent signals at the plasma membrane of both living and fixed cells. Individual signals can be compared between cells and different signals within one cell can be correlated. PlasMACC can process conventional laser-scanning confocal images as well as data obtained by super-resolution methods such as structured illumination microscopy. COMPARISON WITH EXISTING METHOD(S): By comparing PlasMACC to methods currently used in the field, we show more consistent quantitative data due to the fully automated algorithm. PlasMACC also provides an expanded set of novel analysis parameters. CONCLUSION: PlasMACC enables a detailed quantification of fluorescent signals at the plasma membrane of spherical cells in an unbiased and reliable fashion.