Kidney proximal tubule cells cultured under shear stress become remarkably well differentiated and endocytic capacity is rapidly tuned in response to acute changes in shear stress. The results have implications for understanding how proximal tubule function is regulated acutely by daily variations in glomerular filtration rate.
BackgroundLowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn’t clear.MethodsWe examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish.ResultsOCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment.ConclusionsOur data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.
The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly-differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically-quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.
Dent disease is a rare kidney disorder caused by mutations in the Cl-/H+ exchanger ClC-5. Extensive physiologic characterization of the transporter has begun to illuminate its role in endosomal ion homeostasis. Nevertheless, we have yet to understand how loss of ClC-5 function in the kidney proximal tubule impairs membrane traffic of megalin and cubilin receptors to cause the low molecular weight proteinuria characteristic of Dent disease. This review identifies open questions that remain to be answered, evaluates the current literature addressing these questions, and suggests new testable models that may link loss of ClC-5 function to tubular proteinuria in Dent disease.
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