The role of mitochondrial calcium uptake in the cortical collecting duct Open Access

Galarza-Paez, Laura Isabel (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/9306sz56z?locale=en
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Abstract

Principal cells in the renal collecting duct (CCD) regulate total body salt and water. The principal cells respond differently depending on the origin of the signal. The release of ATP into the blood causes an increase in sodium reabsorption via the epithelial sodium channel (ENaC) whereas luminal ATP is inhibitory. Despite opposing effects, both basal and apical ATP increases intracellular calcium ([Ca2+]i). The cell compartmentalizes ([Ca2+]i) in the mitochondria and helps maintain the polarized effect calcium signals. Mitochondria act as dynamic buffers of intracellular calcium in epithelium by helping uptake calcium and help maintain intracellular homeostasis. Mitochondria can regulate local concentrations of [Ca2+]I in CCD by localizing near the apical and basal membrane to form mitochondrial bands. Mitochondria sequester [Ca2+]I and prevent Ca2+ from diffusing from one pole of the cell to the other; therefore, helping maintain cellular calcium homeostasis. Mitochondria in principal cells form mitochondrial ER associated membranes creating calcium microdomains for the regulation of cellular processes. The primary mechanism for Ca2+ uptake across the mitochondrial outer membrane is via the voltage dependent anion channel (VDAC). I hypothesize that when we inhibit the ability of the mitochondria to take up Ca2+ we disturb normal renal function and cell polarity. We obtained VDAC1 knock out (KO) mice and predict that inhibiting calcium uptake in CCD would prevent regulation of ENaC and animals would be unable to properly regulate total body salt and water. To test this hypothesis, VDAC1 knockout mice were given a nominally low salt diet, high-salt diet (8%) or high salt diet with daily IP injection of benzamil (ENaC inhibitor) would be physiologically challenged. VDAC1-/- blood pressure, urine concentration and electrolyte excretion was significantly different than the WT on the same diet. Kidney morphology, blood pressure and other parameters were measured. Overall, our data show that mitochondrial calcium plays an essential role in normal renal function.

Table of Contents

List of Figures ix

List of abbreviations x

I. Introduction 1

A. The Role of mitochondria in the regulation of Epithelial Sodium Channel in cortical collecting duct 2

B. The Importance of mitochondrial calcium in the regulation of water and salt homeostasis 3

C. The existence of Mitochondrial ER Associated membranes and their Role in the regulation of ENaC 5

D. Statement of Purpose 7

E. Significance 8

II. Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria 9

A. Abstract 10

B. Intro 11

C. Methods 12

D. Results 14

E. Discussion 19

III. The role of mitochondrial calcium uptake in Renal function 23

A. Intro 24

B. Methods 25

C. Results 27

D. Discussion 29

IV. Mitochondria Associated ER Membranes are Present in Cortical Collecting Duct 34

A. Intro 35

B. Methods 36

C. Results 38

D. Discussion 39

V. Discussion 42

VI. Figures 46

Schematic 1. Proposed Schema of ENaC regulation by intracellular Ca2+ 46

Schematic 2. Mitochondria-associated membrane components 47

Figure 1: Polarized effects of ionomycin on ENaC activity 48

Figure 2: Effect of inhibiting mitochondrial Ca2+ uptake on ENaC activity 49

Figure 3: Location of mitochondrial bands in CCD cells 50

Figure 4: Calcium localization following ionomycin treatment in mpkCCD cells 51

Figure 5: Mitochondrial calcium before and after apical application of ionomycin 52

Figure 3.0 VDAC1 methods of characterization of renal function 53

Figure 3.1. Histology of VDAC1 KO and WT mice 54

Figure 3.2. Effect of diet in renal cortical collecting duc 55

Figure 3.3. Systolic Blood Pressure Wild type and VDAC1 Knock Out mice 56

Figure 3.4. Urine Sodium in Wild type and VDAC1 Knock Out mice 56

Figure 3.5. Urine Osmolality of Wild type and VDAC1 Knock Out mice 57

Table 3.1. Metabolic parameters of VDAC1 KO mice 58

Figure 3.6. Aquaporin 2 and urea transporter (UT)-A1 protein abundance in VDAC1 KO mice on high salt diet 59

Table 3.2. Summary of VDAC1 knock out metabolic parameters 59

Figure 4.1. Mitochondria ER association 60

Figure 4.2. Voltage-dependent anion channel 1 co-localizes with IP3RIII in both mouse kidney cortex and cell culture 61

VII. References 62

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