Venkatesan Raghavan

The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). The majority of filtered water, Na+, proteins, and other solutes are reabsorbed in the proximal tubule (PT), which plays a critical role in blood volume homeostasis. Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the PT multiligand receptors megalin and cubilin.  Microvillar bending in response to increases in GFR and the accompanying fluid shear stress (FSS) has been shown to modulate acute changes in PT ion transport capacity. However, whether fluid shear stress affects megalin/cubilin mediated endocytosis in PT cells is unknown. We found that subjecting immortalized PT cell lines to physiologically relevant levels of FSS (1dynes/cm2) causes a dramatic increase in the clathrin-dependent uptake of both the megalin/cubilin ligand albumin and of fluid phase markers. Flow stimulated endocytosis is initiated between 15 and 30 min post induction of the FSS and is rapidly reversed upon removing the FSS. Strikingly, this flow dependent increase in endocytosis was completely blocked by prior deciliation of the cells. Exposure of PT cells to FSS also caused a rapid elevation in intracellular Ca2+ levels, which was not observed in deciliated cells. We also observed that calcium mediated calcium release through ER calcium channels was requisite for the FSS stimulated rapid elevation in cytosolic Ca2+ levels. Interestingly, we also observe that the depletion of extracellular calcium and ATP levels ablate the FSS stimulated elevation of intracellular Ca2+ levels. We hypothesize that ATP triggered purinergic signaling amplifies the FSS induced cytosolic calcium increase in PT cells. We also hypothesize that changes in intracellular Ca2+ in response to flow-dependent ciliary bending triggers the observed increase in PT apical endocytosis. Experiments are underway to dissect the signaling cascade that leads to flow stimulated endocytosis.