Shipman, Katherine E
(2023)
Modeling megalin traffic in the kidney proximal tubule to discern mechanisms of proteinuric disease.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein free urine. Proteins that escape the glomerular filtration barrier 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. Dent disease, a rare kidney disorder caused by the loss of function of the voltage-gated chloride/proton exchanger ClC-5, presents with low molecular weight proteinuria and frequently progresses to end-stage renal disease. ClC-5 knockout (KO) in animals results in a decrease in the expression of megalin and cubilin protein without concordant changes in mRNA, indicating a trafficking defect. Here, I 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. Using CRISPR/Cas9 technology, I developed ClC-5 KO OK cell culture and mouse models of Dent disease to apply the mathematical models of megalin traffic in order to pinpoint altered trafficking steps. The mathematical model predicts that under normal conditions megalin is rapidly internalized from the apical surface, resulting in a primarily intracellular distribution of the receptor at steady state. Moreover, our data show that apical early endosomes mature rapidly in PT cells and suggest that Rab11a is the primary mediator of apical recycling of megalin from maturing endocytic compartments. The rate of recycling to the apical surface represents the rate limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Loss of function of ClC-5 impaired maturation of early endosomes resulting in a decrease in megalin recycling. Additionally, chloride accumulation and acidification were revealed to be critical drivers of rapid early endosome maturation. Adaptation of our mathematical model to the S1 segment of mouse PT using colocalization data obtained in kidney sections from wild-type (WT) and ClC-5 KO mice suggests that the OK cell models largely recapitulate the membrane trafficking kinetics observed in this segment in vivo.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
3 January 2023 |
| Date Type: |
Publication |
| Defense Date: |
21 June 2022 |
| Approval Date: |
3 January 2023 |
| Submission Date: |
30 June 2022 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
170 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Integrative Systems Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
endocytosis, kidney function, receptor traffic, membrane traffic |
| Date Deposited: |
03 Jan 2023 16:24 |
| Last Modified: |
03 Jan 2023 16:24 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43216 |
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