Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells

Long, Kimberly R.; Rbaibi, Youssef; Bondi, Corry D.; Ford, B. Rhodes; Poholek, Amanda C.; Boyd‐Shiwarski, Cary R.; Tan, Roderick J.; Locker, Joseph; Weisz, Ora A.

doi:10.1152/ajprenal.00259.2021

Cited by 22 publications

(30 citation statements)

References 68 publications

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“…Megalin, cubilin, NHE3, and SGLT2 were detected with anti-megalin antibody generously provided by Dr. Daniel Biemesderfer and Dr. Peter Aronson (Yale University, MC-220, 1:20,000, 21 ), anticubilin antibody (No. 27445, validated in 20,22 , 1:5000), and anti-SGLT2 antibody (Abcam, ab85626, 1:500), respectively.…”

Section: Western Blottingmentioning

confidence: 99%

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models

Shipman

Baty

Long

et al. 2023

JASN

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Significance Statement Loss of function of the 2Cl−/H+ antiporter ClC-5 in Dent disease causes an unknown impairment in endocytic traffic, leading to tubular proteinuria. The authors integrated data from biochemical and quantitative imaging studies in proximal tubule cells into a mathematical model to determine that loss of ClC-5 impairs endosome acidification and delays early endosome maturation in proximal tubule cells, resulting in reduced megalin recycling, surface expression, and half-life. Studies in a Dent mouse model also revealed subsegment-specific differences in the effects of ClC-5 knockout on proximal tubule subsegments. The approach provides a template to dissect the effects of mutations or perturbations that alter tubular recovery of filtered proteins from the level of individual cells to the entire proximal tubule axis. Background Loss of function of the 2Cl−/H+ antiporter ClC-5 in Dent disease impairs the uptake of filtered proteins by the kidney proximal tubule, resulting in tubular proteinuria. Reduced posttranslational stability of megalin and cubilin, the receptors that bind to and recover filtered proteins, is believed to underlie the tubular defect. How loss of ClC-5 leads to reduced receptor expression remains unknown. Methods We used biochemical and quantitative imaging data to adapt a mathematical model of megalin traffic in ClC-5 knockout and control cells. Studies in ClC-5 knockout mice were performed to describe the effect of ClC-5 knockout on megalin traffic in the S1 segment and along the proximal tubule axis. Results The model predicts that ClC-5 knockout cells have reduced rates of exit from early endosomes, resulting in decreased megalin recycling, surface expression, and half-life. Early endosomes had lower [Cl−] and higher pH. We observed more profound effects in ClC-5 knockout cells expressing the pathogenic ClC-5E211G mutant. Alterations in the cellular distribution of megalin in ClC-5 knockout mice were consistent with delayed endosome maturation and reduced recycling. Greater reductions in megalin expression were observed in the proximal tubule S2 cells compared with S1, with consequences to the profile of protein retrieval along the proximal tubule axis. Conclusions Delayed early endosome maturation due to impaired acidification and reduced [Cl−] accumulation is the primary mediator of reduced proximal tubule receptor expression and tubular proteinuria in Dent disease. Rapid endosome maturation in proximal tubule cells is critical for the efficient recovery of filtered proteins.

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Section: Western Blottingmentioning

confidence: 99%

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models

Shipman

Baty

Long

et al. 2023

JASN

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“…A7030) to compete for receptor-mediated uptake. Studies in CRISPR/Cas9 Cubn KO versus control cells were performed to determine the role of cubilin in uptake of both ligands (Long et al 2022).…”

Section: Impact Of Igg and β 2 -Microglobulin On Albumin Uptakementioning

confidence: 99%

Modelling normal and nephrotic axial uptake of albumin and other filtered proteins along the proximal tubule

Edwards

Long

Baty

et al. 2022

The Journal of Physiology

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“… 27–30 Indeed, in stark contrast to the much weaker megalin expression in other cell culture models of PT cells, the Lrp2 mRNA that encodes megalin is the most abundant transcript in OK cells cultured under these conditions. 31 , 32 …”

Section: Introductionmentioning

confidence: 99%

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule

et al. 2022

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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.

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Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells

Cited by 22 publications

References 68 publications

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models

Modelling normal and nephrotic axial uptake of albumin and other filtered proteins along the proximal tubule

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule

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