Transgenic RNAi Depletion of Claudin-16 and the Renal Handling of Magnesium

Abstract

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 ...

Biology of claudins

“…This is supported by recent analysis of claudin-16 RNAi knockdown mice, which reproduce the human phenotype of renal Ca 2ϩ and Mg 2ϩ wasting and nephrocalcinosis (46). In studies using in vitro perfused TALH tubules from these mice, the P Na /P Cl ratio was found to be decreased twofold with no change in P Na /P Mg or transtubular resistance, suggesting that the major defect is a decrease in P Na in the TALH.…”

“…The second model, put forward by Goodenough and colleagues (46), is that claudin-16 really functions primarily as a paracellular Na ϩ pore. This is supported by recent analysis of claudin-16 RNAi knockdown mice, which reproduce the human phenotype of renal Ca 2ϩ and Mg 2ϩ wasting and nephrocalcinosis (46).…”

Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium

“…We previously described a CLDN16 KD animal model that recapitulates human FHHNC phenotypes (17). Human mutations in CLDN19 also result in FHHNC (15).…”

“…S2). These sequences were then cloned into the pUG-U6 vector (17), upstream of the red fluorescent protein (''cherry'') driven by the ubiquitin-C promoter. After testing of the lentivirus in cultured mouse TAL cells, transgenic mouse lines were generated by perivitelline injection of virus to fertilized mouse embryos.…”

“…The expression of CLDN16 is restricted to the thick ascending limb (TAL) of the nephron (16). CLDN16-deficient mice exhibit defects in paracellular cation selectivity and develop severe renal wasting of magnesium and calcium (17) similar to that seen in the human disease. In the kidney, CLDN19 is also exclusive to the TAL (15).…”