Annemiete W.C.M. van der Kemp

Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia.

Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

RACK1 Inhibits TRPM6 Activity via Phosphorylation of the Fused α-Kinase Domain

BACKGROUNDnThe maintenance of the bodys Mg(2+) balance is of great importance because of its involvement in numerous enzymatic systems and its intervention in neuromuscular excitability, protein synthesis, and nucleic acid stability. Recently, the transient receptor potential melastatin 6 (TRPM6) was identified as the gatekeeper of active Mg(2+) transport and therefore plays a crucial role in the regulation of Mg(2+) homeostasis. Remarkably, TRPM6 combines a Mg(2+) channel with an alpha-kinase domain whose function remains elusive.nnnRESULTSnHere, we identify the receptor for activated C-kinase 1 (RACK1) as the first regulatory protein of TRPM6 that associates with the alpha-kinase domain. RACK1 and TRPM6 are both present in renal Mg(2+)-transporting distal convoluted tubules. We demonstrate that RACK1 inhibits channel activity in an alpha-kinase activity-dependent manner, whereas small interference (si) RNA-mediated knockdown of RACK1 increases the current. Moreover, threonine(1851) in the alpha-kinase domain was identified as an autophosphorylation site of which the phosphorylation state is essential for the inhibitory effect of RACK1. Importantly, threonine(1851) was crucial for the Mg(2+) sensitivity of TRPM6 autophosphorylation and channel activity. TRPM6 channel activity was less sensitive to Mg(2+) when RACK1 was knocked down by siRNA. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity.nnnCONCLUSIONSnWe propose a unique mode of TRPM6 regulation in which the Mg(2+) influx is controlled by RACK1 through its interaction with the alpha-kinase and the phosphorylation state of the threonine(1851) residue.

Transient receptor potential melastatin 6 knockout mice are lethal whereas heterozygous deletion results in mild hypomagnesemia

Background: Hypomagnesemia with secondary hypocalcemia is due to disturbed renal and intestinal magnesium (Mg²⁺) (re)absorption. The underlying defect is a mutation in the transient receptor potential melastatin type 6 (TRPM6), a Mg²⁺-permeable ion channel expressed in the kidney and intestine. Our aim was to characterize homozygous (–/–) and heterozygous (+/–) TRPM6 knockout mice with respect to Mg²⁺ homeostasis. Methods: TRPM6^+/– mice were bred on a normal (0.19% wt/wt Mg²⁺) and high (0.48% wt/wt Mg²⁺) Mg²⁺ diet. In the offspring, 24-hour urinary Mg²⁺ and calcium excretion as well as serum concentrations of both were determined. TRPM6 mRNA expression in the kidney and colon was measured. Results: On the regular diet, 30% of the offspring were TRPM6 wild-type (^+/+), 70% were TRPM6^+/–, and none were TRPM6^–/–. The genotypic distribution of the litters remained the same on the 0.48% Mg²⁺ diet. In TRPM6^+/– mice on both diets, serum Mg²⁺ levels were significantly lower, and renal and intestinal TRPM6 mRNA expression was reduced. Urinary Mg²⁺ excretion was unaffected. Conclusions: Homozygous TRPM6 deletion is embryonic lethal in mice. Heterozygous deletion of TRPM6 results in a mild hypomagnesemia. The Mg²⁺-enriched diet could not compensate for either embryonic lethality or hypomagnesemia caused by TRPM6 deficiency.

Identification of BSPRY as a Novel Auxiliary Protein Inhibiting TRPV5 Activity

Transient receptor potential vallinoid 5 (TRPV5) and TRPV6 are the most Ca2+-selective members of the TRP superfamily and are essential for active Ca2+ (re)absorption in epithelia. However, little is known about intracellular proteins that regulate the activity of these channels. This study identified BSPRY (B-box and SPRY-domain containing protein) as a novel factor involved in the control of TRPV5. The interaction between BSPRY and TRPV5 by GST pull-down and co-immunoprecipitation assays was demonstrated. BSPRY showed co-localization with TRPV5 in mouse kidney. Expression of BSPRY resulted in a significant reduction of the Ca2+ influx in Madin-Darby Canine Kidney cells that stably express TRPV5 without affecting channel cell-surface abundance. Finally, BSPRY expression in kidney was increased in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice, suggesting an inverse regulation by vitamin D3. Together, these results demonstrate the physiologic role of the novel protein BSPRY in the regulation of epithelial Ca2+ transport via negative modulation of TRPV5 activity.

Cisplatin-induced injury of the renal distal convoluted tubule is associated with hypomagnesaemia in mice

BACKGROUNDnCisplatin is an effective anti-neoplastic drug, but its clinical use is limited due to dose-dependent nephrotoxicity. The majority of cisplatin-treated patients develop hypomagnesaemia, often associated with a reduced glomerular filtration rate (GFR), polyuria and other electrolyte disturbances. The aim of this study is to unravel the molecular mechanism responsible for these particular electrolyte disturbances.nnnMETHODSnTwo groups of 10 mice were injected intraperitoneally three times, once every 4 days, with cisplatin (5 mg/kg body weight,) or vehicle. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of distal convoluted tubule (DCT) genes epithelial Mg(2+) channel TRPM6, the Na(+)-Cl(-) cotransporter (NCC), and parvalbumin (PV), as well as marker genes for other tubular segments were measured by real-time qPCR. Subsequently, renal protein levels of NCC, PV, aquaporin 1 and aquaporin 2 were determined using immunoblotting and immunohistochemistry (IHC).nnnRESULTSnThe cisplatin-treated mice developed significant polyuria (2.5 ± 0.3 and 0.9 ± 0.1 mL/24 h, cisplatin versus control, P < 0.05), reduced creatinine clearance rate (CCr) (0.18 ± 0.02 and 0.26 ± 0.02 mL/min, cisplatin versus control, P < 0.05) and a substantially reduced serum level of Mg(2+) (1.23 ± 0.03 and 1.58 ± 0.03 mmol/L, cisplatin versus control, P < 0.05), whereas serum Ca(2+), Na(+) and K(+) values were not altered. Measurements of 24 h urinary excretion demonstrated markedly increased Mg(2+), Ca(2+), Na(+) and K(+) levels in the cisplatin-treated group, whereas Pi levels were not changed. The mRNA levels of TRPM6, NCC and PV were significantly reduced in the cisplatin group. The expression levels of the marker genes for other tubular segments were unaltered, except for claudin-16, which was significantly up-regulated by the cisplatin treatment. The observed DCT-specific down-regulation was confirmed at the protein level.nnnCONCLUSIONSnThe present study identified the DCT as an important cisplatin-affected renal segment, explaining the high prevalence of hypomagnesaemia following treatment.

Identification of Nipsnap1 as a novel auxiliary protein inhibiting TRPV6 activity

The transient receptor potential vanilloid channels 5 and 6 (TRPV5/6) are the most Ca2+-selective channels within the TRP superfamily of ion channels. These epithelial Ca2+ channels are regulated at different intra- and extracellular sites by the feedback response of Ca2+ itself, calciotropic hormones, and by TRPV5/6-associated proteins. In the present study, bioinformatics was used to search for novel TRPV5/6-associated genes. By including pull-down assays and functional analysis, Nipsnap1—a hitherto functionally uncharacterized globular protein—was identified as a novel factor involved in the regulation of TRPV6. Electrophysiological recordings revealed that Nipsnap1 abolishes TRPV6 currents. Subsequent biotinylation assays showed that TRPV6 plasma membrane expression did not change in the presence of Nipsnap1, suggesting that TRPV6 inhibition by Nipsnap1 is independently regulated from reduced cell surface channel expression. In addition, semi-quantitative reverse transcriptase PCR and immunohistochemical labeling of Nipsnap1 indicated that Nipsnap1 is expressed in mouse intestinal tissues—where TRPV6 is predominantly expressed—but that it does not co-localize with TRPV5 in the kidney. In conclusion, this study presents the first physiological function of Nipsnap1 as an associated protein inhibiting TRPV6 activity that possibly exerts its effect directly at the plasma membrane.

Characterization of vitamin D-deficient klotho−/− mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho−/− mice?

BACKGROUNDnKlotho(-/-) mice display disturbed Ca(2+) and vitamin D homeostasis. Renal cytochrome p450 27b1 (Cyp27b1), the enzyme that catalyzes the hydrolysis to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is increased in klotho(-/-) mice, and a 1,25(OH)(2)D(3)-deficient diet partially normalized Ca(2+) homeostasis in these klotho(-/-) mice. The aim of the present study was to further delineate the interplay between 1,25(OH)(2)D(3) and klotho and their relative contribution to the Ca(2+) homeostasis of klotho(-/-) mice.nnnMETHODSnDouble-klotho(-/-)/Cyp27b1(-/-) mice were generated and mice aged 8-12 weeks were housed in metabolic cages to collect 24-h urine. Blood samples were taken and the animals were sacrificed, and the kidney and duodenum tissues were sampled for RNA extraction. The bone was fixed in 10% v/v formalin and analysed by microcomputed tomography (μCT) scans.nnnRESULTSnKlotho(-/-)/Cyp27b1(-/-) mice, like Cyp27b1(-/-) mice, displayed significantly decreased serum total calcium concentrations compared with wild-type mice (1.44 ± 0.03 and 2.25 ± 0.02 mM) along with normal urinary total calcium excretion. Hyperphosphataemia of klotho(-/-) mice normalized to wild-type levels in klotho(-/-)/Cyp27b1(-/-) mice. The mRNA levels of duodenal transient receptor potential vanilloid subtype 6 (TRPV6) and calcium-binding protein-D(9K), and renal calbindin-D(28K) and NCX1 were significantly reduced in the double knockouts compared with wild-type or klotho(-/-) mice. Elevated TRPV5 protein levels in klotho(-/-) mice normalized to wild type in klotho(-/-)/Cyp27b1(-/-) mice, but were decreased in Cyp27b1(-/-) mice. μCT scans showed that klotho(-/-)/Cyp27b1(-/-) mice, as Cyp27b1(-/-) mice, display significant bone hypomineralization and severely decreased bone mass. Klotho(-/-) mice show a reduced bone mass and increased trabecular numbers.nnnCONCLUSIONSnKlotho(-/-)/Cyp27b1(-/-) mice resemble Cyp27b1(-/-) mice. Since 1,25(OH)(2)D(3) is absent in these mice, our results imply that Ca(2+) homeostasis in klotho(-/-) mice is affected by their excessive 1,25(OH)(2)D(3) levels.

Increased expression of renal TRPM6 compensates for Mg2+ wasting during furosemide treatment

Background Furosemide is a loop diuretic, which blocks the Na+, K+, 2Cl− cotransporter (NKCC2) in the thick ascending limb of Henle (TAL). By diminishing sodium (Na+) reabsorption, loop diuretics reduce the lumen-positive transepithelial voltage and consequently diminish paracellular transport of magnesium (Mg2+) and calcium (Ca2+) in TAL. Indeed, furosemide promotes urinary Mg2+ excretion; however, it is unclear whether this leads, especially during prolonged treatment, to hypomagnesaemia. The aim of the present study was, therefore, to determine the effect of chronic furosemide application on renal Mg2+ handling in mice. Methods Two groups of 10 mice received an osmotic minipump subcutaneously for 7 days with vehicle or 30 mg/kg/day furosemide. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of the epithelial Mg2+ channel (TRPM6), the Na+, Cl− cotransporter (NCC), the epithelial Ca2+ channel (TRPV5), the cytosolic Ca2+-binding protein calbindin-D28K, as well parvalbumin (PV), claudin-7 (CLDN7) and claudin-8 (CLDN8), the epithelial Na+ channel (ENaC) and the Na+–H+ exchanger 3 (NHE3) were determined by real-time quantitative polymerase chain reaction. Renal protein levels of NCC, TRPV5, calbindin-D28K and ENaC were also measured using semi-quantitative immunohistochemistry and immunoblotting. Results The mice chronically treated with 30 mg/kg/day furosemide displayed a significant polyuria (2.1 ± 0.3 and 1.3 ± 0.2 mL/24 h, furosemide versus control respectively, P < 0.05). Furosemide treatment resulted in increased serum concentrations of Na+ [158 ± 3 (treated) and 147 ± 1 mmol/L (control), P < 0.01], whereas serum K+, Ca2+ and Mg2+ values were not significantly altered in mice treated with furosemide. Urinary excretion of Na+, K+, Ca2+ and Mg2+ was not affected by chronic furosemide treatment. The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K. Conclusions During chronic furosemide treatment, enhanced active reabsorption of Mg2+ via the epithelial channel TRPM6 in DCT compensates for the reduced reabsorption of Mg2+ in TAL.

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner.

Tissue transglutaminase (tTG) is a multifunctional Ca2+-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier formation. In kidney, where active transcellular Ca2+ transport via TRPV5 predominates, the potential effect of tTG remains unknown. A multitude of factors regulate TRPV5, many secreted into the pro-urine and acting from the extracellular side. We detected tTG in mouse urine and in the apical medium of polarized cultures of rabbit connecting tubule and cortical collecting duct (CNT/CCD) cells. Extracellular application of tTG significantly reduced TRPV5 activity in human embryonic kidney cells transiently expressing the channel. Similarly, a strong inhibition of transepithelial Ca2+ transport was observed after apical application of purified tTG to polarized rabbit CNT/CCD cells. Furthermore, tTG promoted the aggregation of the plasma membrane-associated fraction of TRPV5. Using patch clamp analysis, we observed a reduction in the pore diameter after tTG treatment, suggesting distinct structural changes in TRPV5 upon crosslinking by tTG. As N-linked glycosylation of TRPV5 is a key step in regulating channel function, we determined the effect of tTG in the N-glycosylation-deficient TRPV5 mutant. In the absence of N-linked glycosylation, TRPV5 was insensitive to tTG. Taken together, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity.