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.
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.
1. Isolation of Renal Proximal Tubules for Primary Culture
2. Renal Proximal Tubule Cell Culture
3. Adenoviral Infection of RPTC
4. Measurement of RPTC Respiration
5. Analysis of Mitochondrial Membrane Potential (ΔΨm)
6. Isolation of Mitochondria
7. Measurement of Activity of NADH-ubiquinone Oxidoreductase (Complex I)
8. Measurement of Activity of Succinate-ubiquinone Oxidoreductase (Complex II)
9. Measurement of Activity of Ubiquinol-cytochrome c Oxidoreductase (Complex III)
10. Measurement of Activity of Cytochrome Oxidase (Complex IV)
11. Measurement of Activity of F0F1-ATPase (ATP synthase)
12. Measurement of Intracellular ATP Content
13. Visualization of Mitochondrial Morphology
14. Analysis of Cell Viability
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 mitochondrial respiration in RPTC regardless of the substrate used to energize mitochondria whereas the inactive PKC-ε had no effect (Figure 2). In order to determine the targets of the active PKC-ε within the respiratory chain, we determined the activities of all enzymatic complexes of the respiratory chain. As shown in Figure 3, activation of PKC-ε reduced activity of complex I and complex IV in RPTC but had no effect on the activities of complex II and complex III (data not shown). The decrease in RPTC respiration induced by PKC-ε activation was associated with increases in the mitochondrial membrane potential (mitochondrial hyperpolarization) (Figure 4). These changes were accompanied by decreases in the activity of F0F1-ATPase (ATP synthase) in mitochondria isolated from RPTC overexpressing the active PKC-ε (Figure 5A). As a result, the ATP content of RPTC overexpressing the active PKC-ε decreased (Figure 5B). Sustained activation of PKC-ε had profound effects on mitochondrial morphology by inducing mitochondrial fragmentation (fission) (Figure 6B). PKC-ε activation, but not inhibition, also resulted in changes in RPTC morphology causing cell shrinkage and elongation of surviving cells. Mitochondrial dysfunction and alterations in mitochondrial morphology in RPTC overexpressing the active PKC-ε were accompanied by cell death by both oncosis and apoptosis (Figure 7). At 48 hr after the adenoviral vector delivery, approximately 50% of RPTC overexpressing the active PKC-ε were not viable. Overexpression of the inactive form of PKC-ε had no effect on mitochondrial function and morphology, and on RPTC viability.
Thus, adenoviral delivery of cDNA coding different isozymes of PKC is an effective tool enabling selective overexpression of individual PKC isozymes and their active or inactive mutants in primary cultures of renal cells. It allows for efficient transfection of cells grown in primary culture and for studying the regulation of different cellular functions and for the assessment of cell morphology and viability by protein kinases.
Figure 1. Protein levels of phosphorylated (active) PKC-ε (p-PKC-ε) and total PKC-ε in cell homogenates (left panel) and mitochondria (right panel) isolated from primary cultures of RPTC infected with adenoviral vector carrying the constitutively active (caPKC-ε) or inactive mutants (dnPKC-ε) of PKC-ε at different time points after adenoviral vector delivery. The levels of β-actin and F0F1-ATPase are used as gel loading controls for cell homogenates and mitochondria, respectively. Figure modified from Nowak et al.5
Figure 2. State 3 and uncoupled respirations in RPTC expressing the active and inactive mutants of PKC-ε at 48 h after adenoviral delivery. For state 3 respiration, mitochondria in digitonin-permeabilized cells were energized using 5 mM glutamate + 5 mM malate (substrates oxidized through complex I), 10 mM succinate + 0.1 μM rotenone (substrate oxidized through complex II), or 1 mM ascorbate + 1 mM N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) (substrates oxidized through complex IV). The results are average ± SE. * – P<0.05.
Figure 3. Activities of complex I and complex IV in mitochondria isolated from RPTC expressing the active and inactive mutants of PKC-ε at 48 h after adenoviral vector delivery. The results are average ± SE. * – P<0.05.
Figure 4. Time-dependent changes in mitochondrial membrane potential (ΔΨm) in RPTC following infection with adenoviral vector carrying the constitutively active (caPKC-ε) and inactive mutants (dnPKC-ε) of PKC-ε. The results are expressed as the ratio of aggregate to monomeric forms of JC-1. The results are average ± SE. * – P<0.05.
Figure 5. Activity of F0F1-ATPase in isolated mitochondria (A) and ATP content (B) in RPTC expressing the active and inactive mutants of PKC-ε at 48 h after adenoviral vector delivery. The results are average ± SE. * – P<0.05.
Figure 6. Mitochondrial morphology in live RPTC expressing the active and inactive mutants of PKC-ε at 48 h after adenoviral vector delivery. A. Non-infected controls. B. RPTC expressing caPKC-ε. C. RPTC expressing dnPKC-ε. Representative images of live cells examined under a fluorescent microscope. Original magnification, x630.
Figure 7. Time-dependent changes in RPTC oncosis (A) and apoptosis (B) following infection with adenoviral vector carrying the constitutively active (caPKC-ε) or inactive mutants (dnPKC-ε) of PKC-ε. Infection with adenoviral particles encoding an empty pShuttle vector (MOI = 50) resulted in 5.2 ± 2.3% oncosis and 5.5 ± 1.0% apoptosis at 48 h after infection. The results are average ± SE. * – P<0.05. C. Representative dot plots demonstrating annexin V and propidium iodide fluorescence in RPTC overexpressing caPKC-ε and dnPKC-ε at 48 h after adenoviral vector delivery.
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 primary culture in the improved conditions resemble mitochondrial functions of renal proximal tubules in vivo.1,2 3. Response of mitochondria to different toxicants in this in vitro model is similar to the response of renal proximal tubules in vivo. 4. This approach allows for an efficient transfection and delivery of genes coding specific proteins including proteins involved in signal transduction mechanisms that regulate mitochondrial functions. Thus, this model allows for selective expression of constitutively active and inactive isozymes of kinases and phosphatases and replaces the need for pharmacological inhibitors and activators that are not as selective and often have toxic side effects. The limitations of this model are the following: 1. using cultured RPTC in an in vitro environment does not allow for studying endocrine and paracrine signals that contribute to the regulation of mitochondrial function in renal proximal tubules in vivo, and 2. this model does not allow for studying chronic conditions.
The authors have nothing to disclose.
This work was supported by a grant from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2R01DK59558 (to G.N.). UAMS Translational Research Institute supported by the National Institutes of Health National Center for Research Resources grant UL1 RR029884 provided partial funding for Flow Cytometry Core at UAMS. We thank Dr. Peipei Ping (University of California at Los Angeles; Los Angeles, CA) for providing an aliquot of adenovirus carrying cDNA coding the dominant negative (inactive) mutant of PKC-ε and Dr. Allen Samarel (Loyola University Medical Center; Maywood, IL) for providing an aliquot of adenoviral vector coding the constitutively active mutant of PKC-ε. We also thank Drs. Peter Parker and Peter Sugden (Imperial College London, London, UK) for providing cDNA coding constitutively active PKC-ε.
Name of the reagent | Company | Catalogue number |
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 |
12×75 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 |