The renal proximal tubule (PT) plays a central role in maintaining glomerulotubular balance by reabsorbing the majority of filtered water, ions, and low molecular weight (LMW) proteins. This function is critically dependent on the activity of multiligand receptors megalin and cubilin, which mediate the internalization of solutes such as albumin, vitamins, and hormones from the glomerular ultrafiltrate. The efficiency of this process must remain stable across a wide range of glomerular filtration rates (GFRs), yet the mechanisms regulating apical endocytic capacity in response to physiological fluctuations in fluid flow are poorly understood. Recent evidence suggests that changes in GFR induce fluid shear stress (FSS) on PT cells, which may modulate ion transport through microvillar bending. Here, we demonstrate that FSS also acutely regulates apical endocytosis via a primary cilia-dependent mechanism.
Exposure of immortalized PT cell lines—OK and LLC-PK1—to physiologically relevant levels of FSS (1 dyne/cm²) led to a significant, twofold increase in the uptake of both the megalin-cubilin ligand Alexa Fluor 647-albumin and the fluid phase marker rhodamine-dextran. This enhancement was observed within 30 minutes of flow initiation and persisted throughout a 3-hour incubation period. Notably, the response was rapidly reversible: upon cessation of flow, endocytic activity returned to baseline within 15 minutes. Furthermore, increasing FSS within the normal human range (0.7–1.5 dyne/cm²) resulted in a dose-dependent increase in albumin internalization, with maximal stimulation occurring at 1.0 dyne/cm², suggesting a finely tuned adaptive response.
Mechanistically, FSS-stimulated endocytosis required clathrin-coated pit formation and dynamin activity. Inhibition of clathrin assembly with chlorpromazine or blockade of dynamin with Dyngo-4a significantly reduced both basal and flow-induced endocytosis. These findings indicate that FSS enhances the same clathrin- and dynamin-dependent pathway responsible for constitutive endocytosis in PT cells.
A key mediator of this response was intracellular Ca²⁺ ([Ca²⁺]i). FSS triggered an immediate three- to fourfold rise in [Ca²⁺]i, which was abolished in deciliated cells, when extracellular Ca²⁺ was omitted, or when chelating cytosolic Ca²⁺ with BAPTA-AM. Moreover, depletion of extracellular ATP using apyrase or inhibition of P2Y purinergic receptors with suramin blocked the Ca²⁺ response and abrogated the FSS-induced increase in endocytosis. This indicates that FSS-induced ciliary bending leads to ATP release, activating P2YRs and initiating a Ca²⁺ signaling cascade.
Importantly, primary cilia were essential for this entire pathway. Deciliation eliminated the FSS-triggered Ca²⁺ spike and prevented endocytic stimulation without affecting basal uptake.Didox Cell Cycle/DNA Damage Conversely, addition of ATP alone under static conditions was sufficient to mimic the FSS effect, inducing a transient increase in [Ca²⁺]i and enhancing endocytosis by ~50%.2,6-Dichloro-4-nitroaniline manufacturer This confirms that Ca²⁺ elevation is not only necessary but also sufficient to activate the endocytic machinery.PMID:35184717
We propose a model in which FSS induces bending of primary cilia, triggering mechanosensitive Ca²⁺ entry and ATP release. This activates P2YRs and promotes Ca²⁺-induced Ca²⁺ release from ER stores via ryanodine receptors. Elevated [Ca²⁺]i then modulates actin dynamics at the apical surface, likely increasing the size and number of clathrin-coated pits to enhance endocytic capacity. This rapid, reversible adaptation ensures efficient retrieval of filtered proteins during fluctuating GFRs.
These findings reveal a novel cilia-mediated mechanism by which PT cells dynamically adjust their endocytic function in response to hemodynamic changes. Disruption of this pathway may contribute to tubular proteinuria in diseases involving ciliary dysfunction, such as Lowe syndrome and Dent disease. Understanding this regulatory axis offers new insights into kidney physiology and potential therapeutic targets for proteinuric disorders.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com