Reversed polarity of Na(+) -K(+) -ATPase: mislocation to apical plasma membranes in polycystic kidney disease epithelia

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder in which renal tubules become enormously enlarged due to fluid accumulation. Na(+) -K(+) -ATPase was compared in normal and cystic regions of whole kidneys and in confluent primary cultures of microdissected renal tubule and cyst-lining epithelia. Immunostaining with antibodies directed against the Na(+) -K(+) -ATPase catalytic alpha-subunit was confined to apical, luminal plasma membranes of ADPKD epithelia, which was a complete reversal of the normal renal tubule polarized location in basolateral membranes. Mislocated Na(+) -K(+) -ATPase was shown to be functionally active, because identical intense apical staining was observed by use of a cytochemical assay. In addition, biochemical assays showed a significant increase in these ouabain-inhibitable Na(+) -K(+) -ATPase specific activity levels in ADPKD kidneys compared with age-matched normal kidneys. Specific binding of [3H] ouabain was not only increased but also confined to the apical membrane vesicles prepared from cystic regions of ADPKD kidneys compared with normal age-matched controls, in which binding was confined to basolateral membrane vesicles. Although steady-state levels of Na(+) -K(+) -ATPase alpha- and beta-subunit in mRNAs were increased somewhat in ADPKD kidneys, this alone was not sufficient to account for the observed activation. Confluent ADPKD epithelia grown on dual-chamber, permeable membrane supports also showed reversed polarity of 22NaCl vectorial transport, because this was from basal to apical media compartments. Because this transport could also be blocked by ouabain, this suggested apical Na(+) -K(+) -ATPase was responsible and implicated altered polarity of Na(+) -K(+) -ATPase and resultant Na+ secretion as a mechanism for cyst formation in ADPKD. Because no reversal of polarity of other basolateral or apical membrane proteins was detected, an intracellular sorting defect specific for Na(+) -K(+) -ATPase is proposed.

Collecting duct-specific deletion of peroxisome proliferator-activated receptor γ blocks thiazolidinedione-induced fluid retention

“…In contrast, the same RGZ treatment had no obvious effect on transepithelial resistance in the CD PPAR␥ KO cells. Similarly, the RGZ treatment induced a significant increase in transepithelial 22 Na flux in the control cells in an amiloride-sensitive manner. The RGZ-induced changes in 22 Na flux were significantly blocked in the PPAR␥ KO cells.…”

“…Therefore, primary cultures of CD cells derived from PPAR␥ f/f and CD PPAR␥ KO mice were established for parallel examination of sodium transport in response to RGZ treatment. In control cells, RGZ treatment had a direct stimulatory effect on sodium transport, as assessed by both transepithelial resistance and transepithelial 22 Na flux. In sharp contrast, the RGZ effect was almost completely blocked in the CD PPAR␥ KO cells.…”

“…Cells were kept on filters for at least 10 days until a confluent transporting cell monolayer had developed. This was evaluated by measurement of the [ 14 C]-inulin leak, as described (22). Transepithelial resistance was determined by using ''chopstick'' electrodes (EVOM, World Precision Instruments, Sarasota, FL).…”

“…Transepithelial resistance was determined by using ''chopstick'' electrodes (EVOM, World Precision Instruments, Sarasota, FL). Transepithelial transport of 22 Na was determined by adding 10 Ci (1 Ci ϭ 37 GBq) of 22 Na to the apical compartment followed by measurement of the radioactivity in the basal compartment. All studies described in this report were performed on cells between the third and fifth passages.…”

From Roscovitine to CYC202 to Seliciclib – from bench to bedside: discovery and development

“…Indeed in PKD cells PC-1 together with other normal desmosomal proteins are absent from intercellular junctions, remaining in the cytoplasm, despite unchanged levels of expression, thus suggesting that expression of normal PC-1 is required for correct assembly of desmosomal junctions [175]. There is also evidence that the expression of mutated PC-1 leads to a disruption in the polarity of renal epithelial cells [176] causing mislocalisation of ion channels and growth factor's receptors that in turn can reverse fluid flow and drive cellular proliferation in the kidney [177].…”