Role of CLIC5A in Maintaining Glomerular Filtration Barrier Integrity Open Access
- Other title
chloride intracellular channel
- Type of item
- Degree grantor
University of Alberta
- Author or creator
- Supervisor and department
Ballermann, Barbara J. (Medicine)
- Examining committee member and department
baylis, Christine (physiology) ( External examiner)
Alexander, Todd (Pediatrics)
Hughes, Sarah C. (Medical Genetics)
McMurtry, Michael Sean (Medicine)
Department of Medicine
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
Renal glomeruli are specialized capillaries that produce a protein-free ultrafiltrate of plasma at an extremely high rate. Glomerular endothelial cells (EC) and podocytes sustain the integrity of filtration barrier, and glomerular mesangial cells provide intravascular support. Podocytes cover the exterior of renal glomerular capillaries and wrap regularly spaced, actin-based projections, the foot processes, around the capillary loops, giving strength to the capillary in the face of the high glomerular capillary pressure. Filtration slits between foot processes allow passage of water and small molecules, but not larger proteins. The lumen of the capillaries is lined by glomerular EC, which are perforated by fenestrae that make the capillary highly permeable to water and small solutes. A glycocalyx covers the EC surface and extends into the fenestrae, preventing large proteins from being filtered. To adapt to changes in mechanical forces, for instance glomerular capillary hypertension, glomerular cells undergo remodeling. Maladaptive remodeling can lead to proteinuria, and end stage renal disease. This thesis explores the function of CLIC5A (chloride intracellular channel 5A), which is selectively enriched in glomerular podocytes and EC. CLIC5A function is poorly understood, and it probably is not a chloride channel. CLIC5A co-localizes with ezrin in podocyte foot processes, and moesin in glomerular EC. Ezrin and moesin are ERM (ezrin, radixin, moesin) proteins that connect membrane-spanning proteins to cortical actin. Their activation requires docking on PI[4,5]P2 in lipid bilayers. CLIC5A stimulates clustered PI[4,5]P2 accumulation in the plasma membrane, facilitating ERM docking, unfolding and phosphorylation. Activated ezrin links the transmembrane glycoprotein podocalyxin to actin, controlling the architecture of podocytes. The molecular interactions between CLIC5A, ERM proteins and PI[4,5]P2 generating PIP5 kinases had not been worked out. Since it was previously shown that Rac1 stimulates the activity of PIP5 kinases and ERM protein activation, and since Rac1 participates in podocyte actin remodeling, I determined whether CLIC5A-dependent PI[4,5]P2 generation and ERM activation are mediated by Rac1. I also determined whether the podocyte response to DOCA/Salt hypertension is CLIC5A-dependent. In COS7 cells, null for CLIC5A at baseline, ectopic CLIC5A expression stimulated Rac1 activity and phosphorylation of the Rac1 effector Pak1. CLIC5A-induced PI[4,5]P2 generation, as well as Pak1 and ERM phosphorylation were all Rac1-dependent. In vivo, DOCA/Salt hypertension increased phosphorylated Pak1 in podocytes in CLIC5+/+, but not in CLIC5-/- mice. In DOCA/Salt hypertensive CLIC5-/- mice glomerular capillary microaneurysms and albuminuria were much greater than in hypertensive CLIC5+/+ mice. There also was a marked reduction in glomerular EC fenestrae in hypertensive CLIC5-/-, but not in CLIC5+/+ mice. Thus, CLIC5A stimulates Rac1-dependent PI[4,5]P2 generation, ERM and Pak1 phosphorylation, and the accumulation of phosphorylated Pak1 in DOCA/Salt hypertension requires CLIC5. My data suggest that augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ERM activation, perhaps reducing the tensile strength of podocytes. In CLIC5-/- mice, ERM phosphorylation is profoundly reduced in podocytes, but preserved in glomerular EC, even though CLIC5A is expressed in both cell types in wild-type mice. Since glomerular EC also express CLIC4, I reasoned that CLIC4 could potentially compensate for the CLIC5A loss in glomerular EC. In glomeruli of CLIC5-/- mice, CLIC4 expression was up-regulated in glomerular EC, but not in podocytes. In cultured glomerular EC, CLIC4 silencing reduced ERM activation, which was rescued by CLIC4 or CLIC5A. In mice lacking either CLIC4 or CLIC5, ERM phosphorylation was retained in glomerular EC, but in mice lacking both CLIC4 and CLIC5, glomerular EC ERM phosphorylation was profoundly reduced. Although glomerular EC fenestrae developed normally in dual CLIC4/CLIC5 deficient mice, the density of fenestrae declined substantially by 8 months of age. The dual CLIC4/CLIC5 deficient mice also developed spontaneous proteinuria and mesangial matrix expansion. Thus, CLIC4 stimulates ERM activation, and can compensate for CLIC5A in glomerular EC. My findings suggest that CLIC4/CLIC5A-mediated ERM activation is required for sustained maintenance of the glomerular capillary architecture. In summary, I found that CLIC5A activates Rac1, and that Rac1 activation is necessary for CLIC5A-dependent PI[4,5]P2 synthesis, as well as ERM and Pak1 activation. This mechanism is induced by DOCA/Salt hypertension in wild-type, but not CLIC5-/- mice. In glomerular EC, CLIC4 and CLIC5A both activate ERM proteins and compensate for each other. My work shows that these CLICs maintain the long-term integrity of glomerular capillaries.
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
- Citation for previous publication
Kidney International, 2016. Mahtab Tavasoli, Laiji Li, Abass Al-Momany, Lin-Fu Zhu, Benjamin A. Adam, Zhixiang Wang and Barbara J. Ballermann. , The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury. Kidney Int, 2016. 89(4): p. 833-47.Portions of Chapter 4 have been submitted in a peer-reviewed journal: Mahtab Tavasoli, Abass Al-Momany, Xin Wang, M, Laiji Li, John C. Edwards and Barbara J. Ballermann, Both CLIC4 and CLIC5A activate ERM Proteins in Glomerular Endothelium. Am. J. Physiol., Renal Physiology 2016.
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