Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C96
Renal Cyst Fluid From Human Polycystic Kidney Disease Patients Stimulates Cl- Secretion: Characterization of the Active Factor and Target Channels
B. L. Blazer-Yost1,4, B. J. Blacklock2, R. L. Bacallao3,4, V. H. Gattone1,4
1. Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, United States. 2. Chemistry and Chemical Biology, Indiana University Purdue University, Indianapolis, Indiana, United States. 3. Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, United States. 4. Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States.
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of epithelial lined, fluid filled cysts predominately in kidney tubules and liver bile ducts. Despite the formation of multiple, slowly growing cysts, renal function is usually not severely compromised until the 5th decade of life but then declines rather precipitously (1). Renal injury exacerbates cyst formation and expansion (2). Cyst expansion is thought to result from stimulation of ion secretory events that cause increased movement of electrolytes and, secondarily, water into the cyst lumen. In ADPKD renal tissue, several groups have observed increased intracellular cAMP that stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of epithelial cells lining the cysts. Interestingly, cyst fluid obtained from human patients can also stimulate secretory activity in renal epithelial cells. We used electrophysiological techniques to examine the effect of cyst fluid on the mpkCCDcl4 (mouse principal cells of kidney cortical collecting duct, clone 4) cell line. A 10% (V/V) addition of cyst fluid to the basolateral media stimulated two different Cl- channels, a GLyH101-inhibitable CFTR and a tannic acid inhibitable Ca2+-activated Cl- channel. We identified the active secretory component of the cyst fluid as lysophosphatic acid (LPA). A dose response for LPA showed that concentrations above 0.05 μM stimulated maximal secretory responses in mpkCCDcl4 cells. Tandem mass spectrometry was used to measure the concentration of LPA in the cyst fluid collected from one patient. The cyst fluid contained 4.0 + 0.4 μM 16:0 LPA and 1.4 + 0.04 μM 18:1 LPA (mean + SEM, quadruplicate), an amount sufficient to stimulate a maximal response. Pretreatment with N-palmitoyl-L-serine phosphoric acid, an LPA receptor antagonist, completely inhibited the secretory response to cyst fluid (n = 4). Pretreatment with 20 μM diacylglycerol pyrophosphate, an LPA1/LPA3 receptor antagonist substantially inhibited (72.8 + 4.5%; n=5) the Cl- secretory response. Molecular size separation of the cyst fluid indicated that the LPA activity is confined to a fraction containing molecular weight components larger than 100 kDa, suggesting that the LPA is bound to proteins. In conclusion, we hypothesize that LPA at concentrations found in cyst fluid or blood will exacerbate cyst growth. Under normal conditions, LPA is bound to proteins in the cyst fluid or blood and, therefore, unavailable for binding to the LPA receptors on the basolateral membrane of cystic epithelial cells. Under conditions such as renal injury or loss of cyst wall integrity due to ageing, the LPA-protein complexes would be released into the interstitial space where they would interact with specific receptors to exacerbate cyst growth.
Where applicable, experiments conform with Society ethical requirements