Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells.

Purinergic signaling regulates several renal physiological and pathophysiological processes. Extracellular vesicles (EVs) are nanoparticles released by most cell types, which, in non-renal tissues, modulate purinergic signaling. The aim of this study was to investigate the effect of EVs from renal proximal tubule (HK2) and collecting duct cells (HCD) on intra- and intersegment modulation of extracellular ATP levels, the underlying molecular mechanisms, and the impact on the expression of the alpha subunit of the epithelial sodium channel (αENaC). HK2 cells were exposed to HK2 EVs, while HCD cells were exposed to HK2 and HCD EVs. Extracellular ATP levels and αENaC expression were measured by chemiluminescence and qRT-PCR, respectively. ATPases in EV populations were identified by mass spectrometry. The effect of aldosterone was assessed using EVs from aldosterone-treated cells and urinary EVs (uEVs) from primary aldosteronism (PA) patients. HK2 EVs downregulated ectonucleoside-triphosphate-diphosphohydrolase-1 (ENTPD1) expression, increased extracellular ATP and downregulated αENaC expression in HCD cells. ENTPD1 downregulation could be attributed to increased miR-205-3p and miR-505 levels. Conversely, HCD EVs decreased extracellular ATP levels and upregulated αENaC expression in HCD cells, probably due to enrichment of 14-3-3 isoforms with ATPase activity. Pretreatment of donor cells with aldosterone or exposure to uEVs from PA patients enhanced the effects on extracellular ATP and αENaC expression. We demonstrated inter- and intrasegment modulation of renal purinergic signaling by EVs. Our findings postulate EVs as carriers of information along the renal tubules, whereby processes affecting EV release and/or cargo may impact on purinergically regulated processes.

Functional tests to guide management in an adult with loss of function of type-1 angiotensin II receptor.

BACKGROUND: Genetic loss of function of AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), or AGTR1 (type-1 angiotensin II receptor) leads to renal tubular dysgenesis (RTD). This syndrome is almost invariably lethal. Most surviving patients reach stage 5 chronic kidney disease at a young age. METHODS: Here, we report a 28-year-old male with a homozygous truncating mutation in AGTR1 (p.Arg216*), who survived the perinatal period with a mildly impaired kidney function. In contrast to classic RTD, kidney biopsy showed proximal tubules that were mostly normal. During the subsequent three decades, we observed evidence of both tubular dysfunction (hyperkalemia, metabolic acidosis, salt-wasting and a urinary concentrating defect) and glomerular dysfunction (reduced glomerular filtration rate, currently ~30 mL/min/1.73 m2, accompanied by proteinuria). To investigate the recurrent and severe hyperkalemia, we performed a patient-tailored functional test and showed that high doses of fludrocortisone induced renal potassium excretion by 155%. Furthermore, fludrocortisone lowered renal sodium excretion by 39%, which would have a mitigating effect on salt-wasting. In addition, urinary pH decreased in response to fludrocortisone. Opposite effects on urinary potassium and pH occurred with administration of amiloride, further supporting the notion that a collecting duct is present and able to react to fludrocortisone. CONCLUSIONS: This report provides living proof that even truncating loss-of-function mutations in AGTR1 are compatible with life and relatively good GFR and provides evidence for the prescription of fludrocortisone to treat hyperkalemia and salt-wasting in such patients.

Novel Aspects of Extracellular Vesicles in the Regulation of Renal Physiological and Pathophysiological Processes.

Extracellular vesicles (EV) are nanosized particles released by a large variety of cells. They carry molecules such as proteins, RNA and lipids. While urinary EVs have been longer studied as a source of biomarkers for renal and non-renal disorders, research on EVs as regulatory players of renal physiological and pathological processes has experienced an outbreak recently in the past decade. In general, the microenvironment and (patho)physiological state of the donor cells affect the cargo of the EVs released, which then determines the effect of these EVs once they reach a target cell. For instance, EVs released by renal epithelial cells modulate the expression and function of water and solute transporting proteins in other cells. Also, EVs have been demonstrated to regulate renal organogenesis and blood flow. Furthermore, a dual role of EVs promoting, but also counteracting, disease has also been reported. EVs released by renal tubular cells can reach fibroblasts, monocytes, macrophages, T cells and natural killer cells, thus influencing the pathogenesis and progression of renal disorders like acute kidney injury and fibrosis, nephrolithiasis, renal transplant rejection and renal cancer, among others. On the contrary, EVs may also exert a cytoprotective role upon renal damage and promote recovery of renal function. In the current review, a systematic summary of the key studies from the past 5 years addressing the role of EVs in the modulation of renal physiological and pathophysiological processes is provided, highlighting open questions and discussing the potential of future research.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/- ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1-/- mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1-/- mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

Sensing of tubular flow and renal electrolyte transport.

The kidney is a remarkable organ that accomplishes the challenge of removing waste from the body and simultaneously regulating electrolyte and water balance. Pro-urine flows through the nephron in a highly dynamic manner and adjustment of the reabsorption rates of water and ions to the variable tubular flow is required for electrolyte homeostasis. Renal epithelial cells sense the tubular flow by mechanosensation. Interest in this phenomenon has increased in the past decade since the acknowledgement of primary cilia as antennae that sense renal tubular flow. However, the significance of tubular flow sensing for electrolyte handling is largely unknown. Signal transduction pathways regulating flow-sensitive physiological responses involve calcium, purinergic and nitric oxide signalling, and are considered to have an important role in renal electrolyte handling. Given that mechanosensation of tubular flow is an integral role of the nephron, defective tubular flow sensing is probably involved in renal disease. Studies investigating tubular flow and electrolyte transport differ in their methodology, subsequently hampering translational validity. This Review provides the basis for understanding electrolyte disorders originating from altered tubular flow sensing as a result of pathological conditions.

Effect of Dapagliflozin Treatment on the Expression of Renal Sodium Transporters/Channels on High-Fat Diet Diabetic Mice.

BACKGROUND: Inhibition of the Na+/glucose co-transporter 2 is a new therapeutic strategy for diabetes. It is unclear how proximal loss of Na+ (and glucose) affects the subsequent Na+ transporters in the proximal tubule (PT), thick ascending limb of loop of Henle (TAL), distal convoluted tubule (DCT) and collecting duct (CD). METHODS: Mice on a high fat diet were administered 3 doses streptozotocin 6 days prior to oral dapagliflozin administration or vehicle for 18 days. A control group of lean mice were also included. Body weight and glucose were recorded at regular intervals during treatment. Renal Na+ transporters expression in nephron segments were analyzed by RT-qPCR and Western blot. RESULTS: Dapagliflozin treatment resulted in a significant reduction in body weight and blood glucose compared to vehicle-treated controls. mRNA results showed that Na+-hydrogen antiporter 3 (NHE3), Na+/phosphate cotransporter (NaPi-2a) and epithelial Na+ channel expression was increased, Ncx1, ENaCß and ENaCγ expression declined (p all < 0.05), respectively, in dapagliflozin-treated mice when compared with saline vehicle mice. Na-K-2Cl cotransporters and Na-Cl cotransporter mRNA expression was not affected by dapagliflozin treatment. Na+/K+-ATPase (Atp1b1) expression was also increased significantly by dapagliflozin treatment, but it did not affect Atp1a1 and glucose transporter 2 expression. Western blot analysis showed that NaPi-2a, NHE3 and ATP1b1 expression was upregulated in dapagliflozin-treated diabetic mice when compared with saline vehicle mice (p < 0.05). CONCLUSION: Our findings suggest that dapagliflozin treatment augments compensatory changes in the renal PT in diabetic mice.

Polycystin-1 dysfunction impairs electrolyte and water handling in a renal precystic mouse model for ADPKD.

The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1-/-) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1-/- mice display reduced serum levels of magnesium (Mg2+), calcium (Ca2+), sodium (Na+), and phosphate (Pi) compared with control ( Pkd1+/+) mice and renal Mg2+, Ca2+, and Pi wasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1-/- mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1-/- mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1-/- mice, although a water reabsorption defect was observed in iKsp- Pkd1-/- mice. In conclusion, our data indicate that PC1 is involved in renal Mg2+, Ca2+, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.

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.

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.

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA.

Isoform 3 of the Na+-Ca2+ exchanger (NCX3) participates in the Ca2+ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca2+]i or [Na+]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca2+ uptake after a rise in [Ca2+]i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca2+ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca2+-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.

Determinants of hypomagnesemia in patients with type 2 diabetes mellitus.

BACKGROUND: Hypomagnesemia (plasma magnesium (Mg2+) concentration <0.7 mmol/L) has been described in patients with type 2 diabetes. Polypharmacy is inevitable when treating a complex disease such as type 2 diabetes and could explain disturbances in the plasma Mg2+ concentration. In this study, we aimed to establish the extent of hypomagnesemia in a cohort of type 2 diabetes patients and to identify the determinants of plasma Mg2+ levels. METHODS: Patient data and samples of 395 type 2 diabetes patients were investigated. Plasma Mg2+ concentrations were measured using a spectrophotometric assay. Using Pearson correlation analyses, variables were correlated to plasma Mg2+ levels. After excluding confounding variables, all parameters correlating (P < 0.1) with plasma Mg2+ were included in a stepwise backward regression model. RESULTS: The mean plasma Mg2+ concentration in this cohort was 0.74 ± 0.10 mmol/L. In total, 121 patients (30.6%) suffered from hypomagnesemia. Both plasma triglyceride (r = -0.273, P < 0.001) and actual glucose levels (r = -0.231, P < 0.001) negatively correlated with the plasma Mg2+ concentration. Patients using metformin (n = 251, 62%), proton pump inhibitors (n = 179, 45%) or ß-adrenergic receptor agonists (n = 31, 8%) displayed reduced plasma Mg2+ levels. Insulin use (n = 299, 76%) positively correlated with plasma Mg2+ levels. The model predicted (R2) 20% of all variance in the plasma Mg2+ concentration. CONCLUSIONS: Hypomagnesemia is highly prevalent in type 2 diabetes patients. Plasma triglycerides and glucose levels are major determinants of the plasma Mg2+ concentration, whereas only a minor part (<10%) of hypomagnesemia can be explained by drug intake, excluding polypharmacy as a major cause for hypomagnesemia in type 2 diabetes.

Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

Calpain-3-mediated regulation of the Na⁺-Ca²âº exchanger isoform 3.

Ca(2+) disturbances are observed when Ca(2+)-dependent cysteine proteases malfunction, causing muscle weakness and wasting. For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na(+)-Ca(2+) exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca(2+) content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca(2+) imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca(2+) uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca(2+) ([Ca(2+)]i) or Na(+) ([Na(+)]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca(2+) uptake of NCX3 might participate in the Ca(2+) refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca(2+) storage observed in LGMD2A.

Hypomagnesemia in Type 2 Diabetes: A Vicious Circle?

Over the past decades, hypomagnesemia (serum Mg(2+) <0.7 mmol/L) has been strongly associated with type 2 diabetes mellitus (T2DM). Patients with hypomagnesemia show a more rapid disease progression and have an increased risk for diabetes complications. Clinical studies demonstrate that T2DM patients with hypomagnesemia have reduced pancreatic ß-cell activity and are more insulin resistant. Moreover, dietary Mg(2+) supplementation for patients with T2DM improves glucose metabolism and insulin sensitivity. Intracellular Mg(2+) regulates glucokinase, KATP channels, and L-type Ca(2+) channels in pancreatic ß-cells, preceding insulin secretion. Moreover, insulin receptor autophosphorylation is dependent on intracellular Mg(2+) concentrations, making Mg(2+) a direct factor in the development of insulin resistance. Conversely, insulin is an important regulator of Mg(2+) homeostasis. In the kidney, insulin activates the renal Mg(2+) channel transient receptor potential melastatin type 6 that determines the final urinary Mg(2+) excretion. Consequently, patients with T2DM and hypomagnesemia enter a vicious circle in which hypomagnesemia causes insulin resistance and insulin resistance reduces serum Mg(2+) concentrations. This Perspective provides a systematic overview of the molecular mechanisms underlying the effects of Mg(2+) on insulin secretion and insulin signaling. In addition to providing a review of current knowledge, we provide novel directions for future research and identify previously neglected contributors to hypomagnesemia in T2DM.

Regulation of Mg2+ Reabsorption and Transient Receptor Potential Melastatin Type 6 Activity by cAMP Signaling.

The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg(2+) channels participate in transcellular Mg(2+) transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg(2+) reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na(+)/Cl(-) cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg(2+) reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal--and low--Mg(2+) diet. Increased urinary Mg(2+) wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg(2+) concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg(2+) diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels.

The Na+/Ca2+ Exchanger 1 (NCX1) Variant 3 as the Major Extrusion System in Renal Distal Tubular Transcellular Ca2+-Transport.

BACKGROUND/AIMS: Fine-tuning of renal calcium (Ca(2+)) reabsorption takes place in the late distal convoluted and connecting tubules (DCT2/CNT) of the kidney via transcellular Ca(2+) transport. Here, Ca(2+) enters the cell at the apical side via the epithelial Ca(2+) channel transient receptor potential vanilloid 5 and is subsequently extruded at the basolateral side by the concerted actions of the plasma membrane Ca(2+) ATPases and the Na(+)/Ca(2+) exchanger 1 (NCX1). NCX1 is responsible for ∼ 70% of basolateral Ca(2+) extrusion. The aim of this study was to determine the predominant NCX1 variant in the kidney and its role in Ca(2+) transport. METHODS: DCT2/CNT specific tubules were used to show the abundance of NCX1 specific isoforms. Renal NCX1 variants were cloned from mouse kidney tissue. Human Embryonic Kidney 293(T) cells were transiently transfected with NCX1.3, and Fura-2 measurements and 45Ca(2+) uptake assays were performed to determine several characteristics of NCX1.3 in the reverse mode. RESULTS: NCX1.3 was demonstrated to be the predominant NCX1 variant in the DCT2/CNT, next to NCX1.2 and NCX1.7. NCX1.3 could be inhibited by SN-6, an NCX-specific inhibitor, whereas stimulation of the cAMP/PKA or PKC-mediated pathway did not affect Ca(2+) influx as measured in the reverse mode. Lowering intracellular Ca(2+) concentrations resulted in a decreased Ca(2+) uptake. CONCLUSION: NCX1.3 is the predominant NCX variant in the DCT2/CNT tubules. Its function is dependent on intracellular Ca(2+) concentrations.

Shedding of klotho by ADAMs in the kidney.

The anti-aging gene klotho plays an important role in Ca(2+) and phosphate homeostasis. Membrane-bound klotho is an essential coreceptor for fibroblast growth factor-23 and can be cleaved by proteases, including a disintegrin and metalloproteinase (ADAM)10 and ADAM17. Cleavage of klotho occurs at a site directly above the plasma membrane (α-cut) or between the KL1 and KL2 domain (ß-cut), resulting in soluble full-length klotho or KL1 and KL2 fragments, respectively. The aim of the present study was to gain insights into the mechanisms behind klotho cleavage processes in the kidney. Klotho shedding was demonstrated using a Madin-Darby canine kidney cell line stably expressing klotho and human embryonic kidney-293 cells transiently transfected with klotho. Here, we report klotho expression on both the basolateral and apical membrane, with a higher abundance of klotho at the apical membrane and in the apical media. mRNA expression of ADAM17 and klotho were enriched in mouse distal convoluted and connecting tubules. In vitro ADAM/matrix metalloproteinase inhibition by TNF484 resulted in a concentration-dependent inhibition of the α-cut, with a less specific effect on ß-cut shedding. In vivo TNF484 treatment in wild-type mice did not change urinary klotho levels. However, ADAM/matrix metalloproteinase inhibition did increase renal and duodenal mRNA expression of phosphate transporters, whereas serum phosphate levels were significantly decreased. In conclusion, our data show that renal cells preferentially secrete klotho to the apical side and suggest that ADAMs are responsible for α-cut cleavage.

Recurrent FXYD2 p.Gly41Arg mutation in patients with isolated dominant hypomagnesaemia.

BACKGROUND: Magnesium (Mg(2+)) is an essential ion for cell growth, neuroplasticity and muscle contraction. Blood Mg(2+) levels <0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps and epilepsy and disturbances in K(+) and Ca(2+) homeostasis. Over the last decade, the genetic origin of several familial forms of hypomagnesaemia has been found. In 2000, mutations in FXYD2, encoding the γ-subunit of the Na(+)-K(+)-ATPase, were identified to cause isolated dominant hypomagnesaemia (IDH) in a large Dutch family suffering from hypomagnesaemia, hypocalciuria and chondrocalcinosis. However, no additional patients have been identified since then. METHODS: Here, two families with hypomagnesaemia and hypocalciuria were screened for mutations in the FXYD2 gene. Moreover, the patients were clinically and genetically characterized. RESULTS: We report a p.Gly41Arg FXYD2 mutation in two families with hypomagnesaemia and hypocalciuria. Interestingly, this is the same mutation as was described in the original study. As in the initial family, several patients suffered from muscle cramps, chondrocalcinosis and epilepsy. Haplotype analysis revealed an overlapping haplotype in all families, suggesting a founder effect. CONCLUSIONS: The recurrent p.Gly41Arg FXYD2 mutation in two new families with IDH confirms that FXYD2 mutation causes hypomagnesaemia. Until now, no other FXYD2 mutations have been reported which could indicate that other FXYD2 mutations will not cause hypomagnesaemia or are embryonically lethal.

TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.

Thrombin receptor deficiency leads to a high bone mass phenotype by decreasing the RANKL/OPG ratio.

Thrombin and its receptor (TR) are, respectively, expressed in osteoclasts and osteoblasts. However, their physiological roles on bone metabolism have not been fully elucidated. Here we investigated the bone microarchitecture by micro-computed tomography (µCT) and demonstrated increased trabecular and cortical bone mass in femurs of TR KO mice compared to WT littermates. Trabecular thickness and connectivity were significantly enhanced. The physiological role of TR on both inorganic and organic phases of bone is illustrated by a significant increase in BMD and a decrease in urinary deoxypyridinoline (DPD) crosslink concentration in TR KO mice. Moreover, TR KO cortical bone expanded and had a higher polar moment of inertia (J), implying stronger bone. Bone histomorphometry illustrated unaltered osteoblast and osteoclast number and surface in femoral metaphyses, indicating that thrombin/TR regulates osteoblasts and osteoclasts at functional levels. Serum analysis showed a decrease in RANKL and an increase in osteoprotegerin (OPG) levels and reflected a reduced RANKL/OPG ratio in the TR KO group. In vitro experiments using MC3T3 pre-osteoblasts demonstrated a TR-dependent stimulatory effect of thrombin on the RANKL/OPG ratio. This effect was blocked by TR antagonist and p42/p44-ERK inhibitor. In addition, thrombin also intensified p42/p44-ERK expression and phosphorylation. In conclusion, the thrombin/TR system maintains normal bone remodeling by activating RANKL and limiting OPG synthesis by osteoblasts through the p42/44-ERK signaling pathway. Consequently, TR deficiency inhibits osteoclastogenesis, resulting in a high bone mass phenotype.