Cholesterol-dependent microvilli mediate the interaction of ENaC and PIP2 Público

Linck, Valerie (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/vh53wv78c?locale=es
Published

Abstract

Epithelial sodium channels (ENaC) in the distal nephron play an important role in the regulation of total body Na+ homeostasis. Gain-of-function mutations of ENaC, as seen in Liddle’s syndrome, and hypercholesterolemia are both associated with hypertension. However, it is not clear how cholesterol can induce ENaC activation to result in hypertension. We know from previous studies that cholesterol stimulates ENaC in cultured distal nephron cells. In addition, we have shown that anionic phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2), can bind and stimulate ENaC in cultured distal nephron cells. We have also previously shown through atomic force microscopy studies that ENaC is mainly located in microvilli, which are cholesterol-dependent structures on the apical membrane of epithelial nephron cells. Likewise, we have also shown in the cortical collecting duct (CCD) that PIP2 is predominantly located in microvilli and that PIP2 distribution between microvilli and planar region depends on cholesterol synthesis. In the inner leaflet, it has been suggested that cholesterol may also interact with PIPto form PIP2 microdomains or to localize PIP2 in lipid rafts. However, it is unclear whether ENaC and cholesterol are also co-localized in microvilli and, whether cholesterol affects the microvillar localization of PIP2 and ENaC. In vivo results show that increasing cholesterol, through the blockade of the cholesterol regulating proteins, ABC-binding cassette transporter A1 (ABCA1) or apolipoprotein E (ApoE), induces hypertension. Furthermore, this hypertension may be alleviated through cholesterol synthesis inhibition. The blockade of ABCA1 also results in increased ENaC activity, which can be alleviated by cholesterol synthesis inhibition. The mechanism that mediates this hypertension is further elucidated by our in vitro studies. First, we show that ENaC activity is dependent on both cholesterol and PIP2. Next, we show that the structure of microvilli are dependent on cholesterol, and extraction of cholesterol or synthesis inhibition destroys microvillar structure. Furthermore, ENaC and PIP2 both localize to the microvilli and each other in a cholesterol-dependent manner. Collectively, these results indicate that microvilli are cholesterol-dependent structures in which ENaC and PIP2 colocalize to promote ENaC activation. An excess of cholesterol may result in ENaC-mediated hypertension by allowing an increase in microvillar domains and increased ENaC activation. 

Table of Contents

Introduction.......................................................................................................................1 – 6

The renal system maintains ion homeostasis.......................................................................1

ENaC is responsible for Na+ homeostasis...........................................................................2

Cholesterol stimulates ENaC...............................................................................................2

Cholesterol can be regulated through ABCA1 and ApoE...................................................3

Microvilli are cholesterol dependent...................................................................................4

PIP2 is necessary for ENaC activation.................................................................................4

Study Aims..........................................................................................................................5

Materials and Methods.....................................................................................................7 – 15 Animals................................................................................................................................7

Single-channel Patch Clamp ex vivo....................................................................................8 Immunohistochemistry........................................................................................................9

Western Blotting................................................................................................................10 Antibodies..........................................................................................................................11

Systolic blood pressure measurements..............................................................................11

Cell Culture........................................................................................................................12

Patch-clamp recordings in vitro.........................................................................................12

Transfection of mpkCCDc14 Cells......................................................................................13

Confocal and Atomic Force Microscopy Imaging.............................................................14 Chemicals...........................................................................................................................14

Statistical Analysis.............................................................................................................15

Results...........................................................................................................................16 – 20

Modulating intracellular cholesterol alters systolic blood pressure...................................16

Deletion of ABCA1 in CCD principal cells elevates ENaC activity.................................17

Cholesterol stimulates ENaC in a PIP2-dependent manner...............................................17

PIP2 and α-ENaC are localized in microvilli.....................................................................18

MβCD and lovastatin reduce α-ENaC and PIP2 in the microvilli.....................................19

MβCD and lovastatin reduce cholesterol and PIP2 in the microvilli.................................19

Discussion..................................................................................................................21 – 25

Increased intracellular cholesterol induced hypertension via ENaC.................................21

ENaC and PIP2 are localized to the microvilli...................................................................22

Microvilli are cholesterol-dependent and allow ENaC and PIP2 to associate...................24

Summary: Cholesterol mediates the interaction of ENaC and

PIP2 via microvilli..............................................................................................................25 Figures..........................................................................................................................26 – 47

Figure 1. ENaC structure and anatomy of the human nephron..........................................26

Figure 2. Pharmacological blockade of ABCA1 induces hypertension and can be alleviated by inhibiting intracellular cholesterol synthesis..............................27

Figure 3. Deletion of ApoE induces faster onset of CsA-mediated hypertension.............28

Figure 4. ABCA1 was specifically deleted in the principal cells of mouse cortical collecting duct...........................................................................................30

Figure 5. Lovastatin corrects hypertension caused by loss of ABCA1 function...............31

Figure 6. Lovastatin reduces both ENaC PO and the number of active ENaC in the patch of ABCA1 KO mice.......................................................................................32

Figure 7. Cholesterol in the inner (cytoplasmic) leaflet of the apical membrane is required for ENaC PO ....................................................................................................34

Figure 8. Sequestration of PIP2 with its antibody abolishes the elevation of ENaC PO by cholesterol.....................................................................................................35

Figure 9. PIP2 is localized in cholesterol-dependent microvilli.........................................36

 

Figure 10. α-ENaC is localized in cholesterol-dependent microvilli.................................37

Figure 11. Microvilli structure is dependent on cholesterol..............................................38

Figure 12. Extraction of cholesterol with MβCD decreases α-ENaC and PIP2 in the apical microvillar membrane...........................................................................39

Figure 13. Inhibition of cholesterol synthesis with lovastatin decreases

α-ENaC and PIP2 in the apical microvillar membrane......................................................41

Figure 14. Cholesterol is co-localized with PIP2 in microvilli and reduced by MβCD...........................................................................................................................43

Figure 15. Cholesterol is co-localized with PIP2 in microvilli and reduced

by lovastatin.......................................................................................................................45

Figure 16. Proposed Schema..............................................................................................47 References....................................................................................................................48 – 55 

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