Received March 30, 2006
Accepted on September 26, 2006

BASIC SCIENCE: Pathophysiology of Renal Disease and Progression

Early Embryonic Renal Tubules of Wild-Type and Polycystic Kidney Disease Kidneys Respond to cAMP Stimulation with Cystic Fibrosis Transmembrane Conductance Regulator/Na⁺,K⁺,2Cl^- Co-Transporter-Dependent Cystic Dilation

Brenda S. Magenheimer ^*, Patricia L. St. John , Kathryn S. Isom , Dale R. Abrahamson , Robert C. De Lisle , Darren P. Wallace , Robin L. Maser ^*, Jared J. Grantham ^*, and James P. Calvet ^*1

Departments of *Biochemistry and Molecular Biology, Anatomy and Cell Biology, Molecular and Integrative Physiology, and Internal Medicine and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas

¹ To whom correspondence should be addressed. E-mail: jcalvet{at}kumc.edu.

	Abstract

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR_inh172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl^- channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl^- transport was demonstrated by inhibiting the basolateral Na⁺,K⁺,2Cl^- co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1^m1Bei -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na⁺,K⁺,2Cl^- co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.