Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg 2؉ /Ca 2؉ channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.The human renal disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC, 3 OMIM 248250), is characterized by progressive renal Mg 2ϩ and Ca 2ϩ wasting, leading to impaired renal function and chronic renal failure. FHHNC is genetically linked to mutations in the gene of claudin-16 (CLDN16, also known as paracellin-1; Ref. 1), which is expressed exclusively in the kidney (2). The claudins comprise a 22 gene family that encodes essential structural components of the tight junction, the principal regulator of paracellular permeability. In vitro studies have shown that ion selectivity of the paracellular conductance (see review: Ref.3) is a complex function of claudin subtype and cellular context (4, 5). Thus, in vivo models of FNNHC are essential to our understanding of its pathogenesis.To develop an in vivo model of FNNHC, we have employed transgenic RNA interference (RNAi), which is in theory more rapid and flexible than a conventional knock-out approach. While the use of transgenic RNAi has been limited thus far, it has been shown that an RNAi knockdown of Rasa1 recapitulates a null phenotype in mice (6). In addition, transgenic RNAi has been used to establish a role for Ryk in axon guidance (7) and a role for Nramp1 in controlling susceptibility to Type 1 diabetes (8). We used lentiviral transgenesis because it is more resistant than onco-retroviral transgenesis to epigenetic silencing during embryonic development (9 -11).In this study, we report the generation of CLDN16-deficient transgenic mouse lines using RNAi and have established physiological functions of CLDN16. We observed homeostatic changes of Mg 2ϩ , Ca 2ϩ , Na ϩ , and K ϩ resulting from RNAimediated knockdown. The lumen-positive transepithelial potential in the thick ascending limb (TAL) of ...

Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca(2+) and Mg(2+) in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca(2+) and Mg(2+) homeostasis and the pathological mechanisms underlying a human disease associated with CLDN16 dysfunction [familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), OMIM 248250], we generated a mouse model of CLDN16 deficiency. Similar to patients, CLDN16-deficient mice displayed hypercalciuria and hypomagnesemia. Contrary to FHHNC patients, nephrocalcinosis was absent in our model, indicating the existence of compensatory pathways in ion handling in this model. In line with the renal loss of Ca(2+), compensatory mechanisms like parathyroid hormone and 1,25(OH)(2)D(3) were significantly elevated. Also, gene expression profiling revealed transcriptional upregulation of several Ca(2+) and Mg(2+) transport systems including Trpv5, Trpm6, and calbindin-D9k. Induced gene expression was also seen for the transcripts of two putative Mg(2+) transport proteins, Cnnm2 and Atp13a4. Moreover, urinary pH was significantly lower when compared with wild-type mice. Taken together, our findings demonstrate that loss of CLDN16 activity leads to specific alterations in Ca(2+) and Mg(2+) homeostasis and that CLDN16-deficient mice represent a useful model to further elucidate pathways involved in renal Ca(2+) and Mg(2+) handling.

Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V 2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na ϩ -Cl Ϫ cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH 2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V 2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11␤-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V 2 receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT. antidiuretic hormone; distal convoluted tubule; phosphorylation; sodium-chloride cotransporter trafficking ANTIDIURETIC HORMONE [arginine vasopressin (AVP)] serves to control extracellular fluid homeostasis. The principal effect of AVP is found in the collecting duct where AVP increases water reabsorption. The prerequisite to ensure this function is the creation of a hypertonic interstitium via action of the thick ascending limb (TAL), which is also promoted by AVP. These epithelial effects of AVP are mediated by type 2 AVP receptors (V 2 R) (2, 29), whereas type 1a receptor signaling rather serves to limit the antidiuretic effects of AVP (31). Mapping receptorspecific probes to the tubular segments has revealed subtypeselective distribution along TAL, macula densa, distal convolutions, and collecting ducts (3, 28). In TAL, strong V 2 R expression has been observed, which agreed with increased abundance and phosphorylation of the furosemide-sensitive NaϪ cotransporter (NKCC2) and enhanced NaCl reabsorption in TAL in response to AVP (8, 13, 28). V 2 R signaling also activates epithelial Na ϩ channel (ENaC)-dependent Na ϩ reabsorption in collecting ducts (16); AVP-induced antinatriuresis in this segment has therefore been considered as a causal element in the context of salt-sensitive hype...

Claudin-16 is defective in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Claudin-16 knockdown (CLDN16 KD) mice show reduced cation selectivity in the thick ascending limb. The defect leads to a collapse of the lumen-positive diffusion voltage, which drives Ca(2+) and Mg(2+) absorption. Because of the reduced tight junction permeability ratio for Na(+) over Cl(-), we proposed a backleak of NaCl into the lumen. Systemic analysis had revealed lower blood pressure and a moderately increased plasma aldosterone concentration. In this study, we measured the amiloride-sensitive equivalent short-circuit current in isolated, perfused collecting ducts and found it increased by fivefold in CLDN16 KD mice compared with wild-type (WT) mice. Amiloride treatment unmasked renal Na(+) loss in the thick ascending limb of the nephron. Under amiloride treatment, CLDN16 KD mice developed hyponatremia and the renal fractional excretion of Na(+) was twofold higher in CLDN16 KD compared with WT mice. The loss of claudin-16 also resulted in increased urinary flow, reduced HCO(3)(-) excretion, and lower urine pH. We conclude that perturbation in salt and acid-base metabolism in CLDN16 KD mice has its origin in the defective cation permselectivity of the thick ascending limb of the nephron. This study has contributed to the still incomplete understanding of the symptoms of FHHNC patients.

Background. To evaluate serum chemerin levels in patients with osteoporosis and healthy controls and to investigate the relationship between serum chemerin levels and bone mineral density (BMD). Methods. An age- and gender-matched case-control study was conducted. Pearson's correlation test was performed to investigate the relationship between serum chemerin levels and BMD. Results. There were 93 patients included in the osteoporosis group and 93 matched controls. Serum chemerin level was significantly higher in patients with osteoporosis (87.27±5.80 ng/mL) than patients in control (71.13±5.12 ng/mL) (P < 0.01). There was a negative correlation between femoral bone mineral density and chemerin in both groups (R = −0.395, P < 0.01 in osteoporosis group; R = −0.680, P < 0.01 in control) and also a negative correlation between lumbar bone mineral density with chemerin in both groups (R = −0.306, P < 0.01 in osteoporosis group; R = −0.362, P < 0.01 in control). Conclusions. Patients with osteoporosis presented a higher level of serum chemerin, which witnessed an inverse correlation with BMD. Further studies are needed to explore the role of chemerin in the pathophysiology of osteoporosis.