Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Summary

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Abstract

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.

Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F₀F₁-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F₀F₁-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.

These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

Protocol

1. Isolation of Renal Proximal Tubules for Primary Culture

1. Anesthetize the rabbit, excise both kidneys and place them in a Petri dish filled with sterile prep buffer (DMEM/F12 medium) in a laminar flow hood.
2. Perfuse each kidney with 50 ml of prep buffer followed by 50 ml of sterile phosphate-buffered saline, pH 7.4 (PBS), and PBS-iron oxide solution (45 ml + 5 ml). De-capsulate kidneys and transfer them to the prep buffer containing deferoxamine.
3. Harvest the cortex of each kidney.
4. Homogenize the tissue using a 15 ml Dounce homogenizer. Pour the homogenate over 2 sterile mesh sieves (85 μm on the bottom and 235 μm on top)....

Results

Figure 1 shows that adenoviral delivery of cDNA coding the constitutively active (caPKC-ε) and inactive (dnPKC-ε) mutants of PKC-ε results in significantly increased protein levels of PKC-ε in RPTC and in mitochondria. Cells infected with cDNA carrying the caPKC-ε vector overexpressed the phosphorylated (active) form of PKC-ε whereas cells infected with cDNA coding dnPKC-ε overexpressed PKC-ε that was inactive (not phosphorylated) (Figure 1). The presence of active PKC-ε decreased .......

Discussion

The approach presented here allows for overexpression of individual isozymes of PKC in the primary culture of renal proximal tubular cells. There are several strengths of this approach: 1. It allows for investigating regulatory mechanisms in a homogenous population of cells (renal proximal tubular cells) that are the primary target for various insults (ischemia, hypoxia, oxidative stress), drugs, and nephrotoxicants within the kidney. 2. Mitochondrial functions in this in vitro model of RPTC grown in pri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Laminar flow hood	Thermo Electron Corporation	FORMA 1104
2 ml and 15 ml Dounce tissue grinder	WHEATON	989-24607, 357544
85 and 235 micron nylon mesh	Small Parts	CMN - 0085 - 10YD CMN - 0250 - 10YD
50 ml sterile centrifuge tube	BIOLOGIX	BCT-P 50BS
1.5 ml micro tube	Sarstedt	72.690.001
35 x 10 mm sterile culture dishes	Corning	430165
Jouan Centrifuge	Jouan	Jouan CR3 11175704 Rotors: Jouan T40
Adjustable micro-centrifuge	SIGMA	Model 1 - 15
Biological Oxygen Monitor	YSI Incorporated	YSI Model 5300A
Single Chamber Micro Oxygen System	YSI Incorporated	5356S
Oxygen Probe	YSI Incorporated	5331A
Circulating Bath	YSI Incorporated	5310
KCl and Standard Membrane Kit	YSI Incorporated	5775
Magnetic Stirrer	YSI Incorporated	5222
Flatbed Recorder	Kipp Zonen	BD 11E
48-well and 96-well transparent plates	Costar	3548, 3679
Thermomixer R	Eppendorf	5355 21919
Orbital shaker MAXQ 2000	Thermo Scientific	SHKA 2000
Spectra FLUOR Plus (absorbance/fluorescence/luminescence reader)	Tecan	F129005
Water-Jacketed US Autoflow Automatic CO2 Incubator	NUAIRE	NU 4850
12x75 mm polystyrene culture test tubes for flow cytometry	Fisher Brand	14-961-20
Axioskop Water immersion objective 63x / 0,90W	Carl Zeiss	114846 ACHROPLAN 44 00 67
DMEM / F12	Cellgro	99 - 830 - PB
DMEM / F12 Ham	Sigma	D 2906 - 1L
Deferoxamine Mesylate	Hospira	D110
Collagenase Type I	Worthington	4196
Trypsin inhibitor	Sigma	T 6522 - 500mg
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1)	Invitrogen	T3168
Mitotracker Red	Invitrogen	M22425
ATP Bioluminescence Assay Kit HS II	Roche	11 699 709 001
Annexin V - FITC solution	BioVision	1001 - 200
Flow cytometer	BD Biosciences	BD FACSCalibur