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-&epsilon; and Dr. Allen Samarel (Loyola University Medical Center; Maywood, IL) for providing an aliquot of adenoviral vector coding the constitutively active mutant of PKC-&epsilon;. We also thank Drs. Peter Parker and Peter Sugden (Imperial College London, London, UK) for providing cDNA coding constitutively active PKC-&epsilon;.

1. Isolation of Renal Proximal Tubules for Primary Culture
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	<li>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.</li>
	<li>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.</li>
	<li>Harvest the cortex of each kidney.</li>
	<l...

<ol>
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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...

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

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

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-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; 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-&epsilon; 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-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. 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.

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

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

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

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 ...

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18.1K vistas.  University of Arkansas for Medical Sciences. Los efectos de la activación de la proteína quinasa C (PKC) las isoenzimas en las funciones mitocondriales asociadas con la respiración y la fosforilación oxidativa y en la viabilidad celular se describen. El enfoque se adapta técnica adenoviral para sobreexpresar selectivamente las isozimas de la PKC en cultivo de células primarias y una diversidad de ensayos para determinar las funciones mitocondriales y el estado de energía de la célula.