Hypertonic stress increases claudin-4 expression and tight junction integrity in association with MUPP1 in IMCD3 cells

Abstract

We reported that the multiple PDZ protein 1 (MUPP1) is an osmotic response protein in kidney cells. This up-regulation was found to be necessary for the maintenance of tight epithelial properties in these cells. We investigated whether an interaction with one or more members of the claudin family is responsible for this observation. In response to hypertonicity, the up-regulation of claudin-4 (Cldn4) expression, and not other claudins, was initially identified in inner medullary collecting duct (IMCD3) cells by gene array and further verified by quantitative PCR and Western blotting. In kidney tissues, Cldn4 expression was substantial in the papilla and absent in the cortex. Furthermore, Cldn4 expression significantly increased in the papilla of mice after 36 h of thirsting. Cldn4 immunofluorescence in hypertonically stressed cells revealed colocalization with MUPP1 at the tight junctions. Interaction between Cldn4 and MUPP1 was also demonstrated by coimmunoprecipitation of both proteins from IMCD3 cells chronically adapted to hypertonicity. In IMCD3 cells stably silenced for MUPP1 expression under hypertonic conditions, a significant decrement in Cldn4 expression was observed that was restored after inhibition of lysosome activity. Immunofluorescence detection identified that in these MUPP1-silenced cells Cldn4 was mistargeted to the lysosomes. Functionally, silencing Cldn4 expression in IMCD3 cells resulted in a decrease in the transepithelial resistance to the same degree as observed when MUPP1 expression was silenced, suggesting that MUPP1 contributes to the maintenance of a tight epithelium in the medulla of the kidney under hypertonic stress by correctly localizing Cldn4 to the tight junctions.osmotic stress response ͉ kidney cells ͉ inner medulla

Claudins and the Kidney

“…It is predicted that the tight junction in cyst epithelia will be relatively impermeable to Cl − and highly permeable to Na + . Claudin-7 has been found to be disproportionately expressed in autosomal dominant polycystic kidney disease cyst epithelia (113). The role of claudins in other kidney disorders remains largely unexplored.…”

Claudin-4 Deficiency Results in Urothelial Hyperplasia and Lethal Hydronephrosis

“…In renal nephrons, for instance, Clds may regulate the reabsorption of selected cations or anions via a paracellular route between the tubular epithelial cells [11], [12], [13]. Among the Cld family members, Cld4 has been shown to act as selective paracellular channels for Cl – [14] and barriers against cations [15] as well as to play a role with MUPP1 in the maintenance of a tight epithelium under hypertonic stress [16], [17] at least in vitro . Clds may also be directly or indirectly involved in the regulation of cell proliferation.…”

Effects of Osmolality on Paracellular Transport in MDCK II Cells

“…However, there are not many examples of the acute regulation of paracellular transport by the physiologic inputs [9,38], and the mechanism of the acute regulation of the claudin channel property is poorly understood. In addition, osmotic changes have been reported to affect claudin expression pattern in euryhaline fishs and cultured cells [11–15], but it seems less likely that the changes in claudin expression pattern caused the rapid change in the cation selectivity by the osmotic changes in this study. Claudin-2 forms high conductive channels with cation selectivity in TJs and is responsible for the most of P Na and P Cl in MDCK II cells [16–18,22].…”

“…Epithelial cells express multiple different claudins, and the expression pattern of claudins provides a variety of TJ permeabilities [9,10]. After the identification of claudins in 1998, osmotic changes have been reported to affect claudin expression pattern in euryhaline fishs and cultured cells [11–15]. However, effects of osmotic changes on the permeaibility of claudin channels are poorly understood.…”