The scientific premise of the proposed work is that defective flow-dependent proximal tubule transport may be instrumental in the development and progression of renal dysfunction, a notable example being polycystic kidney disease (PKD). Flow-dependent transport underlies glomerulotubular balance (GTB), whose physiologic importance has been appreciated in normal fluid and electrolyte balance. Whether impaired GTB predisposes to renal damage before GFR is reduced (such as renal cyst formation), has not been studied. Our hypothesis is that episodic increases in kidney tubule hydrostatic pressure promote the creation and growth of renal cysts. This is analogous to the role of arterial pressure in creating vascular aneurysms. There are two control mechanisms, which serve to mitigate the swings in tubule pressure that accompany increases in glomerular filtration (GFR), namely tubuloglomerular feedback and glomerulotubular balance. A mathematical model estimates that with a 50% increase in GFR, impaired GTB increases distal nephron flow, and provokes sharp increases pressure by about 40% in both proximal and distal tubule. This role of GTB, to mitigate GFR- dependent changes of tubule pressure, has never been examined experimentally. During the past 15 years, we have demonstrated that IP3 receptor-mediated intracellular Ca2+ signaling plays a critical role in GTB. Polycystin-2 (PC2) is a nonselective calcium permeable cation channel belonging to the Transient Receptor Potential (TRP) channel family and functions as a Ca2+ channel in the endoplasmic reticulum (ER). Mutations of PC2 abolished the IP3-induced calcium release from the ER. Our preliminary data show a) defective GTB in Pkd2 mutant mice before any renal cysts are formed; and b) Dopamine receptor (DA1) antagonist improves tubule sensitivity to flow and also reduces renal cyst formation in Pkd2 KO mouse, suggesting a new therapeutic method to treat renal disease. In the proposed work, mathematical modeling, renal clearance, microperfusion, immunocytochemistry, and measuring renal tubular pressure in vivo will be used. Hypotheses will be studied in novel nephron-specific conditional knockout PKD animal models, and in PKD null cells to test whether: 1) GTB stabilizes kidney tubule hydrostatic pressure during variations of GFR. This limits tubule distention during GFR elevation, and thus acts to slow cyst formation in Pkd2 KO mice. 2) Inhibition of Na/H- exchanger 3 (NHE3) endocytosis by a DA1 antagonist, increases proximal tubule transporter flow sensitivity, and prevents cyst formation in Pkd2 KO mice. 3) Flow-stimulated NHE3 and/or Na+/K-ATPase trafficking is abrogated in Pkd2 null cells and is similar to blocking the IP3 receptor in WT cells. Inhibition of NHE3 endocytosis by DA1 inhibitor and/or by increasing Ca2+ release from the ER restores the flow-sensing in Pkd2 null cells.
Flows and pressures within the kidney depend upon transport of sodium along the entire nephron, and about 2/3 of this occurs in proximal tubule. Defects in regulation of proximal sodium transport may underlie the development of cysts in patients who have the genetic mutations of polycystic kidney disease. The proposed studies will provide information on the regulation of this transport, and how defective regulation may impact renal tubule pressures and possible benefits of this work include identification of target molecules, which may be blocked or modified in order to modulate sodium transport, and thus preserve kidney architecture.
