This work was supported by the following grants to Lina A. Shehadeh: the National Institute of Health (R56HL132209&#160;and 1R01HL140468) and the Miami Heart Research Institute.

All experiments involving animals were approved by the Institutional Animal Care and Use Committee at the University of Miami, conforming to NIH guidelines.
1. Plate Coating and Preparation of Reagents
<ol>
	<li>Prepare collagen coating:
	<ol>
		<li>Add 35 &#956;L of collagen I to 2 mL of a pre-filtered 20 mM acetic acid solution onto a single 60-mm Petri dish. Incubate it at room temperature for 1 h, air-dry it, and expose it to UV.</li>
		<li>Wash the coating 3x with PBS to remove any acid residue and save it in a 37 &#176;C CO2-free cell culture incubator until the cells are ready for seeding. The final concentration of the collagen coating is 5 &#956;g/cm2.</li>
	</ol>
	</li>
	<li>Prepare perfusion buffer: add 300 &#956;L of penicillin-streptomycin (P/S) to 30 mL of PBS and warm the mixture up in a 37 &#176;C water bath until the isolation begins.</li>
	<li>Prepare digestion buffer: dissolve 3.9 mg of collagenase type 2 into 30 mL of PBS, filter the solution through a 0.2-&#956;m bottle-top filter and warm it up in a water bath at 37 &#176;C until the isolation begins.</li>
	<li>Prepare cell culture media:
	<ol>
		<li>Bring the supplements to room temperature. Without filtration, add the supplement (0.05 mL of fetal calf serum, 10 ng/mL of epidermal growth factor, 5 &#956;g/mL of insulin, 0.5 &#956;g/mL of epinephrine, 36 ng/mL of hydrocortisone, 5 &#956;g/mL of transferrin, and 4 pg/mL of triiodo-L-thyronine) to the 500 mL of renal epithelial cell growth basal medium 2.</li>
		<li>Warm up the media in a 37 &#176;C water bath until it is ready to use.</li>
	</ol>
	</li>
	<li>Prepare compounds: prepare 50 mM FCCP, 10 mM rotenone, 10 mM oligomycin, 10 mM antimycin A, 50 mM L-carnitine, and 50 mM etomoxir stock solutions all in DMSO, aliquot them, and store the compounds at -20 &#176;C.</li>
	<li>Prepare 2.5 mM sodium palmitate in 220 mL of a 150-mM NaCl solution and warm the solution up in a 75 &#176;C water bath until the palmitate is fully dissolved.</li>
	<li>Prepare bovine serum albumin (BSA): prepare 0.34 mM fat-free BSA in 250 mL of 150 mM NaCl. The control BSA serves as a negative control for the palm-BSA solution that can be prepared by following step 1.8.</li>
	<li>Conjugate the palmitate to BSA (Palm-BSA):
	<ol>
		<li>Add the palmitate solution gradually into the BSA solution while it is still hot. Then, adjust the pH to 7.4 and mix them at 37 &#176;C for at least 1 h to complete the conjugation.</li>
		<li>When the conjugation is completed, add another 150 mL of 150 mM NaCl to the solution, mix it well, and save the aliquots at -20 &#176;C. The final solution contains 1 mM sodium palmitate and 0.17 mM BSA and will be used as a fatty acid substrate for cells in a fatty acid-based extracellular flux assay.</li>
	</ol>
	</li>
	<li>Prepare extracellular flux assay basal media:
	<ol>
		<li>Add 1 bag of DMEM powder and 20 mL of 200 mM L-glutamine (4 mM final) to 1 L of autoclaved dH2O and mix them gently.</li>
		<li>On the day of the bioenergetics experiment, add 100 &#181;M sodium pyruvate to the prepared basal media for subsequent preparations of extracellular flux assay media to be used in a glucose- or fatty acid- based respiration assay.</li>
	</ol>
	</li>
	<li>Prepare glucose-based media:
	<ol>
		<li>To measure cellular respiration capacity through glycolysis, add 17.5 mM glucose powder, 100 &#181;M control BSA (as described in step 1.7), and 20 &#181;M etomoxir (to inhibit the fatty acid oxidation) to the basal media described above in step 1.9.</li>
		<li>Warm up the media at 37 &#176;C, adjust the pH to 7.4 and keep it in the 37 &#176;C water bath until it is used in an extracellular flux assay.</li>
	</ol>
	</li>
	<li>Prepare fatty acid-based media:
	<ol>
		<li>To measure cellular respiration capacity through fatty acid oxidation, add 10 mM 2D-glucose powder (glucose analog to inhibit glycolysis), 100 &#181;M Palm-BSA (as described in step 1.8), and 100 &#181;M L-carnitine to the basal media described above in step 1.9.</li>
		<li>Warm up the media at 37 &#176;C, adjust the pH to 7.4, and keep it in the 37 &#176;C water bath until it is used in the extracellular flux assay.</li>
	</ol>
	</li>
</ol>
2. Perfusion, Digestion and Harvesting Kidneys from Mice
<ol>
	<li>Anesthetize the mouse with an isoflurane flow and fix it in a supine position. Make sure the isolation only begins after the animal loses its righting reflexes and the anesthetic depth is monitored by pinch assessments using atraumatic forceps before and during the procedure22.</li>
	<li>Remove fur, using a depilatory cream, from the mouse&#8217;s chest to its abdominal area, disinfect it with iodine, and wipe the iodine residue.</li>
	<li>Make an incision in the chest, cut the skin to open the whole abdominal area, and expose the heart and kidneys.</li>
	<li>Set up a perfusion pump at 32 mL/min and remove any bubbles in the tubing before starting the perfusion.</li>
	<li>Insert a 27-G needle into the left ventricle through the heart apex as soon as the buffer fills up the heart, and poke the right atrium to create an exit so the perfusion buffer circulates as the heart pumps and eventually gets removed from the right atrium exit.</li>
	<li>After the perfusion, switch the pump speed to 30 mL/min for digestion.</li>
	<li>After 20 mL of digestion buffer is perfused through the apex, remove both kidneys for the tubular cell isolation.</li>
</ol>
3. Tissue Processing and Primary Tubular Cells Isolation
<ol>
	<li>Remove the renal capsules and medulla, mince both kidneys into tiny pieces, and incubate them in 10 mL of a digestion buffer in a 37 &#176;C oven with gentle rotation for 5 min.</li>
	<li>Remove any undigested kidney tissues by passing the buffer through a 70-&#956;m filter. Add 10 mL of culture media to stop the digestion.</li>
	<li>To collect tubular cells, centrifuge the filtered cell suspension at 50 x g for 5 min to collect the first pellet. Transfer the supernatant to a new tube and add 5 mL of culture media, centrifuge it at 50 x g for 5 min to ensure all tubular cells are collected into the second pellet. 
	The centrifugation is at a lower speed to primarily pellet heavy tubules. Later, after the cells recover from the isolation, the pure tubular culture is centrifuged at a higher speed during the sub-cultures.</li>
	<li>Resuspend the first pellet in 20 mL of culture media and centrifuge it at 50 x g for 5 min to collect the third pellet.</li>
	<li>Resuspend the second and third pellets in 1 mL of culture media. Mix 10 &#181;L of the cell suspension with 10 &#181;L of Trypan blue, load the mixture into chamber A of a counting slide, and record the cell viability from the automatic cell counter (see Table of Materials).</li>
	<li>Seed up to 107 cells (a heterogeneous population) onto a single 60-mm dish pre-coated with collagen and let the tubular cells attach overnight.</li>
</ol>
4. Primary Tubular Cells Sub-culture and Characterization
<ol>
	<li>On day 1 after the isolation, collect culture media and centrifuge it at 50 x g for 5 min to pellet any floating tubules. Remove the supernatant and resuspend the cell pellet in 4 mL of fresh culture media and plate it back to the same culture dish.</li>
	<li>On day 4 after the isolation, remove the old culture media and add fresh media.</li>
	<li>On day 7 after the isolation, detach the cells by incubating them at 37 &#176;C in 2 mL of 0.25% trypsin-EDTA for 5 min. Add 3 mL of culture media to stop the reaction and collect the cells by centrifugation at 250 x g for 5 min.</li>
	<li>To sub-culture and characterize the cells from P0 to P1, seed 5,000 cells per well onto a 24-well plate coated with collagen I as described above.</li>
	<li>24 h after step 4.4, fix the cells at P1 with 4% PFA for 10 min, permeabilize them with 0.2% Triton X-100 for 3 min, and block them with 10% donkey serum (DS) for 1 h at room temperature.</li>
	<li>Dilute, to 1:100 in 10% DS, each of the following proteins: the proximal tubular markers angiotensinogen (AGT) and aquaporin 1 (AQP1); the distal tubular marker E-cadherin; the mesangial marker CD90/Thy1; and the macrophage markers EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80) and cluster of differentiation 68 (CD68), and incubate them with cells overnight at 4 &#176;C.</li>
	<li>The next day, detect any staining using 1:200 anti-Rabbit, anti-Mouse, or anti-Rat fluorescent secondary antibodies for 45 min. Take images under confocal microscopy to confirm the expression of the markers, as shown in Figure 2A.</li>
	<li>On day 3 after the sub-culture of P1, detach the cells for a sub-culture and characterization at P2 by a staining of the tubular, mesangial, and macrophage markers described in step 4.5. Image the staining under confocal microscopy to confirm the expression of the markers, as shown in Figure 2A.</li>
	<li>On day 3 after the sub-culture of P2, detach the cells for a sub-culture and characterization at P3 by a staining of the tubular, mesangial, and macrophage markers described in step 4.5. Image the staining under confocal microscopy to confirm the expression of the markers, as shown in Figure 2A.</li>
	<li>Prepare the tissue staining:
	<ol>
		<li>Air-dry frozen wild-type and Col4a3-/- kidney slides for 1 h at room temperature and fix them with 4% PFA for 10 min. Permeabilize them with 0.2% Triton X-100 for 10 min and block them with 10% donkey serum (DS) for 1 h at room temperature. Add antibodies against the marker proteins described in step 4.5 at 1:200 and incubate them at 4 &#176;C overnight.</li>
		<li>The next day, detect any staining with 1:200 anti-Rabbit, anti-Mouse, or anti-Rat fluorescent secondary antibodies for 45 min. Take images under a confocal microscope to confirm the expression of the markers, as shown in Figures 2B and 2C.</li>
	</ol>
	</li>
</ol>
5. Mitochondria Bioenergetics Assay
<ol>
	<li>Seed P1 tubular cells at 20,000, 30,000, or 40,000 cells per well in 100 &#956;L of culture media onto a 96-well microplate pre-coated with 5 &#956;g/cm2 collagen I the day before the extracellular flux assays.</li>
	<li>For the hydration of the sensor cartridge, lift the sensor cartridge and fill each well of the plate with 200 &#956;L of a calibration solution. Carefully load the cartridge back to submerge the sensors in the calibration solution. Place the cartridge in a 37 &#176;C oven without CO2 for at least 7 h prior to use. 
	For the best results, overnight cartridge hydration is recommended.</li>
	<li>Prepare the compounds: prepare 8 &#181;M oligomycin, 9 &#181;M FCCP, and 20 &#181;M rotenone/antimycin A mixture in both glucose (described in step 1.10) and fatty acid (described in step 1.11) extracellular flux assay media.</li>
	<li>Change the media: aspirate the cell culture media, add 175 &#181;L of glucose or fatty acid assay media (dependent on the compound that is being worked in, see step 5.3), and incubate them for 1 h in a 37 &#176;C CO2-free incubator.</li>
	<li>Load the cartridge ports with 25 &#181;L of the following compounds: 8 &#956;M oligomycin for port A to achieve a final concentration of 1 &#181;M (note: as each well will contain 175 &#181;L of media, the compound will get diluted 8x), 9 &#956;M FCCP in port B to achieve a final concentration of 1 &#181;M (note: as each well will contain 175 &#181;L of media plus 25 &#181;L of the solution injected from port A, the compound will get diluted 9x), and 20 &#181;M rotenone/antimycin A in port C to achieve a final concentration of 2 &#181;M for each compound (note: as each well will contain 175 &#181;L of media plus 50 &#181;L of the solution injected from ports A and B, the compound will get diluted 10x).</li>
	<li>Add water to all wells in port D and all other ports of the background wells (no cells). Incubate the cartridge in a 37 &#176;C CO2-free incubator for 10 min.</li>
	<li>Turn on the extracellular flux analyzer and the controller.</li>
	<li>Open the Analyzer Software and input the following protocol:
	<ol>
		<li>Choose Standard Assay. Press Assay Wizard. Using the Compounds tab, assign the compound layout and use the Groups and Labels tab to label the experimental groups. Remember to assign the empty wells (without cells) as background.</li>
		<li>Under the Protocol tab, set the following mix and measure cycles using the available commands as indicated in Table 1: calibrate, mix for 2 min, wait for 2 min, and measure for 3 min (repeat this cycle 2 - 3x); inject port A, mix for 2 min, wait for 2 min, measure for 3 min (repeat this cycle 2 - 3x); inject port B, mix for 2 min, wait for 2 min, measure for 3 min (repeat this cycle 2 - 3x); inject port C, mix for 2min, wait for 2 min, measure for 3 min (repeat this cycle 2 - 3x). Press End Wizard. It is also possible to save the current template for future use.</li>
		<li>Press Start to begin the calibration. The analyzer then automatically ejects the plate holder and asks for the cartridge plate to be inserted.</li>
	</ol>
	</li>
	<li>When the calibration step is done (usually in 20 - 25 min), press the prompt command to change the cartridge plate to the cell plate and continue the run.</li>
	<li>When the run is completed, transfer the data, and remove the 96-well plate. Add Hoechst (1:1,000) to each of the assay wells and incubate them for 5 min at 37 &#176;C. Normalize the OCR data to cell count by a measurement of the Hoechst fluorescence reading at a 355-nm excitation and a 460-nm emission.</li>
</ol>

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<li>Barros, M. H., Hauck, F., Dreyer, J. H., Kempkes, B., Niedobitek, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+polarisation%3A+An+immunohistochemical+approach+for+identifying+M1+and+M2+macrophages%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Macrophage polarisation: An immunohistochemical approach for identifying M1 and M2 macrophages.</a> PLoS One. 8 (11), e80908(2013).</li>
<li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progression+of+Alport+kidney+disease+in+Col4a3+knock+out+mice+is+independent+of+sex+or+macrophage+depletion+by+clodronate+treatment%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Progression of Alport kidney disease in Col4a3 knock out mice is independent of sex or macrophage depletion by clodronate treatment.</a> PLoS One. 10 (11), e0141231(2015).</li>
<li>Linnett, P. E., Beechey, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitors+of+the+ATP+synthethase+system%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Inhibitors of the ATP synthethase system.</a> Methods in Enzymology. 55, 472-518 (1979).</li>
<li>Bockenhauer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+phenotype+in+Lowe+Syndrome%3A+a+selective+proximal+tubular+dysfunction%5BTitle%5D)+AND+%22Clinical+Journal+of+the+American+Society+of+Nephrology%22%5BJournal%5D)">Renal phenotype in Lowe Syndrome: a selective proximal tubular dysfunction.</a> Clinical Journal of the American Society of Nephrology. 3 (5), 1430-1436 (2008).</li>
<li>Ranawaka, R., Dayasiri, K., Gamage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+child+with+distal+%28type+1%29+renal+tubular+acidosis+presenting+with+progressive+gross+motor+developmental+regression+and+acute+paralysis%5BTitle%5D)+AND+%22BMC+Research+Notes%22%5BJournal%5D)">A child with distal (type 1) renal tubular acidosis presenting with progressive gross motor developmental regression and acute paralysis.</a> BMC Research Notes. 10 (1), 618(2017).</li>
</ol>

The authors have nothing to declare.

We optimized a protocol that allows for the efficient isolation of mouse renal tubular epithelial cells (TECs) and showed that the cells can be sub-cultured for an extracellular flux analysis to evaluate the mitochondrial respiration in the presence of fatty acid- and/or glucose-based substrates. This protocol is designed for studies focusing on both proximal and distal tubular cells and serves as a framework with which to build more complex experiments for the understanding of TEC pathology-associated kidney diseases. Compared to previously published protocols9,10,19, this method does not require gradient separations with long centrifugation times or an ample antibody usage for sorting and, therefore, offers a more efficient and optimized guide for researchers working in the renal tubular metabolic field. There are several critical steps in this protocol, including the digestion, re-collection, and plating density and compound optimization for the extracellular flux assay.
Choosing the correct type of collagenase and optimal concentration is the key to a successful digestion and dissociation of the tubular cells from renal tissues. Compared to other types of collagenases, type 2 collagenase contains relatively higher levels of protease activity, capable of dissociating compact renal structures. To minimize the chances of contamination as a result of a prolonged perfusion and digestion time, 0.013% type 2 collagenase was perfused at 30 mL/min. The renal capsule was only removed after both kidneys were harvested from the animal and transferred into a sterilized cell culture hood. The kidneys were minced into tiny pieces and continued their incubation with 10 mL of a digestion buffer for another 5 min for a complete digestion and maximal release of the tubular cells.
Although, after the digestion, the tissue suspension is passed through a 70-&#181;m filter to remove very large tissue pieces, there will still be undigested tubules that pass through the filter and stay within the cell suspension and get plated onto the culture dish. It takes a longer time than normal for these tubules to release tubular cells and attach firmly to the culture dish. Therefore, it is rather important to collect the cell suspension and centrifuge it to pellet the unattached tubules and cells the second day after the cell plating. This low-speed centrifugation step further removes other cell types that are lighter than tubular cells and allows for unattached tubules and tubular cells to settle.
The identification of proper cell density is the first and key step for a successful extracellular flux assay. The results showed that 40,000 cells per well on an XF96 microplate is ideal for primary tubular cells in both a fatty acid and a glucose-based respiration assay (Figure 3C). In this protocol, isolated tubular cells were used for the extracellular flux assay at passages 1 and 2. The cells sub-cultured to passage 3, although they maintained an expression of the tubular markers (Figure 2) and a decent performance in the bioenergetics assays (Figure 3A), and showed decreased basal respiration levels compared to passage 2 (shown by comparing OCR in the rightmost panels of Figure 3A to Figure 3C). This decrease may not affect substantially healthy tubular cells (for example, ones isolated from young wild-type mice). However, for studies on cells isolated from CKD mouse models that already have a diminished mitochondrial respiration, higher passages of the cells may cause a further decrease in the basal respiration which would affect the results of the extracellular flux assay. In the studies conducted here, the cells from both passage 1 and passage 2 showed high basal respiration levels. Therefore, following this protocol, we recommend using these two early passages for mitochondrial respiration studies with cells isolated from both healthy and diseased animals. Cells from passage 2 should still be taken into consideration if the passage 1 sub-culture does not yield sufficient cells for the flux assay. In addition to bioenergetics studies, our previous research shows that primary TECs at passage 3 can be extremely useful for treatments with compounds followed by protein and RNA studies (data not shown). That being said, we suggest that investigators using this protocol to isolate tubular cells should carefully choose the optimal passage for different research applications.
The working principle of the extracellular flux analysis is based on the interactions between the injected compounds and the respiration chain complexes and the effect of the uncoupler. Oligomycin is an inhibitor of complex V (ATP synthase) and is used to distinguish ATP-linked oxygen consumption and the oxygen consumption that is required to overcome the regular proton leak across a mitochondrial inner membrane32. FCCP uncouples oxygen consumption from the ATP production by disrupting the mitochondrial membrane potential. Thus, it provides a measurement of the maximal respiration capacity as it circumvents the limited capacity of a proton ion efflux by ATP synthase by allowing a proton transport through the membrane. Antimycin-A, a complex III inhibitor, and rotenone, a complex I blocker, are used in combination to shut down the entire mitochondrial respiration allowing a differentiation between the mitochondrial vs. the non-mitochondrial oxygen consumption in the cells. These compounds should always be titrated for a specific cell type before the extracellular flux assay to determine the optimal concentrations that yield the optimal OCR curves. Here, we recommend 1 &#181;M of oligomycin, 1 &#181;M of FCCP, and 2 &#181;M of rotenone/antimycin A for the extracellular flux assay on primary TECs.
In conclusion, this protocol provides a simple and cost-effective way to isolate renal primary proximal and distal tubular epithelial cells that can be used for assessing mitochondrial bioenergetics ex vivo. While this protocol can be useful in a wide range of molecular biology studies exploring the biological function of renal tubular epithelial cells, we acknowledge its limitations when applying it to studies needing pure proximal or distal tubules. For example, studies on the Lowe Syndrome, a selective proximal tubular dysfunction33, or studies on distal renal tubular acidosis, a distal tubular dysfunction34, would require a more sophisticated protocol for cell isolation and purification. However, for the majority of the studies that compare tubules vs. glomeruli, and for studies to screen potential mitochondrial respiration regulators in tubular cells in general, the protocol provides a feasible high throughput approach. Therefore, this protocol may have broad applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or target validation purposes.

Renal tubular epithelial cell (TEC) function is strongly associated with the overall health condition of the kidney. Pathological signaling in the kidney causes the dedifferentiation of TECs, which plays a major role in kidney fibrosis and chronic kidney disease (CKD)1,2. As a highly energetic organ, the kidney is second only to the heart in oxygen consumption, primarily through mitochondrial oxidative phosphorylation3. Electron microscopy studies have revealed a positive correlation of mitochondrial morphological changes to pathological events in the renal tubules4. Mitochondrial dysfunction in TECs causes renal fibrosis through epithelial to mesenchymal transition5 and defective fatty acid oxidation6. Fibrosis is a progressive renal pathology that results in CKD. Therefore, understanding the energetic status of renal TECs is a necessity to uncover the pathophysiology of CKD.
There are &#62; 20 cell types in the adult kidney7. To study the function of TECs, a primary culture of the renal epithelial cells is needed as a platform for molecular biology applications such as chemical treatments and genetic manipulations. Importantly, genetic manipulations can be done in vivo in mice via transgenesis or by using AAV gene delivery techniques8 so that the isolated primary cells would already be genetically manipulated. The isolation of primary renal tubular cells from mice9,10, rats11,12,13, canines14, rabbits15,16, and humans17,18 has been reported with purification steps to yield pure proximal tubular cells. In these previously published protocols that focus on the isolation of proximal tubular cells, gradient centrifugation and sorting experiments were performed for purification purposes19. While these protocols are valuable for studying proximal tubules, they are not sufficient when both proximal and distal tubules are needed to be studied. For example, our study on the Alport syndrome has revealed that both proximal and distal renal tubules play important roles in the disease progression20, and therefore both kinds of the renal tubules should be investigated in culture. A recent study on renal fluoride toxicity also showed that pathological changes took place in both the proximal and distal tubules21. Therefore, this isolation protocol is designed and optimized for both proximal and distal tubular cells from mouse kidneys with a minimal cost of reagents and simple procedures. Alternatively, investigators can still follow the protocol until step 3.1 and add purification steps9 from this point forward for the isolation of pure proximal tubular cells.
The isolated cells present high energetic levels and maintain renal epithelial characteristics after the sub-cultures to 4 passages. Using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate, which leads to further insights into cell density optimization. These observations suggest that this protocol can be applied to renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. An added significance of this protocol is its feasible usage as a high throughput tool for the ex vivo characterization of mitochondrial bioenergetics in renal proximal and distal tubular cells. Therefore, it can serve as a platform for drug discovery or drug characterization purposes of renal disorders.

<table><tbody><tr><td>Collagen I Rat Protein, Tail</td><td>ThermoFisher Scientific</td><td>A1048301</td><td></td></tr><tr><td>Acetic Acid</td><td>J.T.Baker</td><td>9508</td><td></td></tr><tr><td>Collagenase Type II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Renal Epithelial Cell Growth Medium 2 Kit</td><td>PromoCell</td><td>C-26130</td><td></td></tr><tr><td>2-Deoxy glucose</td><td>Sigma</td><td>D6134</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C0283</td><td></td></tr><tr><td>Etomoxir</td><td>Sigma</td><td>E-1905</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>75351</td><td></td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td><td>Sigma</td><td>C2920</td><td></td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R8875</td><td></td></tr><tr><td>Antimycin-A</td><td>Sigma</td><td>A8674</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Sodium palmitate</td><td>Sigma</td><td>P9767</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P5280</td><td></td></tr><tr><td>L-Glutamine 200mM solution</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030-1L</td><td></td></tr><tr><td>Hoechst 33342</td><td>LifeTechnologies</td><td>H3570</td><td></td></tr><tr><td>Trypan Blue Staining (0.4%)</td><td>LifeTechnologies</td><td>T10282</td><td></td></tr><tr><td>Counting Slides</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Roboz</td><td>RS-5101</td><td></td></tr><tr><td>TC10 automated cell counter</td><td>Bio-Rad</td><td>506BR2119</td><td></td></tr><tr><td>MINIPULS 3 Peristaltic Pump</td><td>Gilson Inc.</td><td>GM3P</td><td></td></tr><tr><td>Seahorse XFe96 Analyzer</td><td>Seahorse Bioscience</td><td>S7800A</td><td></td></tr><tr><td>Seahorse XFe96 FluxPack (includes sensor cartridges, microplates, and calibrant)</td><td>Seahorse Bioscience</td><td>10260-100</td><td></td></tr></tbody></table>

isolation, characterization, and high throughput extracellular flux analysis of mouse primary renal tubular epithelial cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

Kidney Perfusion and Digestion Yield Highly Viable Tubular Epithelial Cells: 
Mouse renal tubular epithelial cells were isolated following the steps outlined in sections 1 - 3 of the protocol described above.
After the digestions, a heterogeneous population of kidney cells, incompletely digested tubules, and other tissue debris that is smaller than 70 &#181;m were plated onto the culture dish on isolation day. Changes from day 0 to day 1 after the isolation are usually expected to be seen only in the cell attachment rather than in the cell growth. Looking through the floating heterogenous population, only a few tubular cells were attached at day 1 (Figure 1A). A re-collection of the cells at day 1, a centrifugation, and a re-plating helped to remove light debris and settle the small tubule pieces for the tubular cell release. From day 1 to day 3, changes were expected not only in a better cell attachment but also in a remarkably improved cell growth rate that was observed with a tripled cell density as compared to day 1 (Figure 1A). In the initial growth phase, the cells formed several colonies and populated around the colonies. From this point forward, the isolated cells were fully recovered and displayed a healthy proliferation. By day 5, the cells were at an 80 - 90% confluency in a 60-mm Petri dish with some spaces between cell to cell and colony to colony (Figure 1A).
Sub-culture and Characterization of the Isolated Tubular Cells: 
The isolated renal tubular epithelial cells were sub-cultured for a characterization following the steps outlined in section 4 of the protocol described above.
From day 5, the cells fully recovered from the isolation and started to proliferate vigorously. One week after the isolation, the cells grew to confluency in a 60-mm Petri dish. After 1 week in a culture at passage 0, the cells were ready to be sub-cultured to passage 1 and, subsequently, for 2 more passages. Similar growth patterns were observed in passage 1 and passage 2. Usually, it takes less than a week for cells at passage 1 and 2 to grow to confluency for further sub-cultures (Figure 1B). The continuous culture of confluent cells from passage 0 to passage 2 showed an extensive dome formation23,24 (Figure 1B), suggesting that the isolated cells maintained a healthy status where they excreted liquids similar to an in vivo status. This caused the monolayer of the cells to lift off the plate but stay connected via tight junctions.
To characterize the cells in the culture, we performed immunofluorescent staining in the cultured cells from passage 1 through passage 4, as well as in a control cell line-human proximal renal epithelial HK-2 cells. Proximal tubular markers, aquaporin 1 (AQP1)25 and angiotensinogen (AGT)9, the distal tubular marker E-cadherin25, the epithelial marker smooth muscle actin (SMA)26,27,28, macrophage markers F4/8029 and CD6830, and the mesangial marker thymocyte differentiation antigen 1 (Thy1/CD90)9 were used for the characterization studies. Both proximal tubular proteins AQP1 and AGT were consistently highly expressed in the isolated tubular cells from passage 1 to passage 4 as well as in the positive control HK-2 proximal epithelial cells (Figure 2A). The distal tubular protein E-cadherin was expressed in the isolated tubular cells through passage 4 and was also observed in the HK-2 cells (Figure 2A). SMA was expressed abundantly in the isolated tubular cells and in the control HK-2 cells, consistent with published reports26,27. On the other hand, the mesangial protein Thy1 and macrophage protein F4/80 were absent in both the isolated tubular cells and the control HK-2 cells (Figure 2A). CD68 showed a minimal expression in the HK-2 cells and in the isolated tubular cells at passage 1 and passage 2, and then its expression became undetectable from passage 3 through passage 4 (Figure 2A). The results suggest that the cells isolated following this protocol are a mix of proximal and distal tubular cells. To compare the expressions of these marker proteins in vivo, we performed staining in frozen kidney tissues. Tubular markers, including AQP1, AGT, and E-cadherin, and mesangial protein Thy1 were found highly expressed in kidneys harvested from a wild-type healthy mouse (Figure 2B). Low expressions of F4/80 and CD68 were observed in wild-type kidneys but extensively expressed in kidneys harvested from a Col4a3-/- mouse that developed renal failure with a macrophage infiltration20,31 (Figure 2C).
Mitochondrial Bioenergetics Assay on Isolated Primary Tubular Cells: 
The mitochondrial respiration assay steps are outlined in section 5 of the protocol described above.
The mitochondrial respiration of isolated primary tubular cells is measured by an extracellular flux analysis of the oxygen consumption rate (OCR) at different plating densities. To titrate the plating density, 20,000, 30,000, and 40,000 primary TECs per well were seeded onto a 96-well XF96 microplate the day before (approximately 20 h before) the extracellular flux assay (Figure 3A). Following the extracellular flux analysis, the OCR measurements were then normalized to the cell counts by a quantification of Hoechst staining. Plating the TECs at 20,000, 30,000, or 40,000 cells/well resulted in an average basal OCR of 25, 45, or 50 pmol/min, respectively (Figure 3A). Moreover, microscopic images of the plated cells revealed that 40,000 cells/well covered the entire surface of the bottom of the microplate wells better than the other plating densities (Figure 3B). Even though the maximal OCR did not increase using 40,000 cells compared to the density of 30,000 cells, we recommend a cell density of around 40,000 cells/well for optimal interactions between cells and compounds.
Moreover, in our experiments, the optimal concentration of the inhibitory/uncoupler compounds was shown to be 1 &#181;M oligomycin, 1 &#181;M FCCP, and 2 &#181;M rotenone/antimycin-A (the protocols of the extracellular flux assay and the port injections are listed in Table 1; the optimization experiments are not shown). However, it is recommended for all users to run a preliminary test with various concentrations of these compounds, ideally lower and higher than the published values, to warrant the best results.
An extracellular flux assay yields a number of important parameters for assessing the bioenergetics of renal TECs. For instance, as fatty acid oxidation is shown to be specifically defective in TECs, the use of media containing fatty acid substrate (palmitate) along with the inhibitor of glycolysis (2DG) can serve as a useful tool to directly evaluate fatty acid oxidation in TECs at passages 1 and 2 (Figure 3C). In the case of renal fibrosis, the overall respiration capacity of the cells is expected to be lower than that of a healthy kidney even though the glucose-based media may not reveal any differences.
Taken together, the extracellular flux assay, especially to assess the fatty acid oxidation capacity, can be utilized as an informative measure to evaluate the energetic status of renal TECs in which pathological changes affecting the bioenergetics profile play a major role in renal fibrosis and the progression to kidney failure.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Steps</td>
			<td>Time (min)</td>
		</tr>
		<tr>
			<td>Calibrate</td>
			<td></td>
		</tr>
		<tr>
			<td>Equilibrate:</td>
			<td>00:12:00</td>
		</tr>
		<tr>
			<td>Measurement 1</td>
			<td>3 loops</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Inject Port A (1 &#956;M Oligomycin)</td>
			<td>2 loops</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Inject Port B (1 &#956;M FCCP)</td>
			<td>2 loops</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Inject Port C (2 &#956;M Rotenon/Antimycin)</td>
			<td>2 loops</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>00:02:00</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>00:03:00</td>
		</tr>
	</tbody>
</table>
Table 1. Standardized extracellular flux analysis running protocol for optimal OCR measurements in primary tubular cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57718/57718fig1.jpg" /> 
Figure 1. Cell culture of isolated primary tubular cells. (A) The isolated primary tubular cells attach early at day 1 and grow robustly from day 3 to day 5. The images are taken under 10X and 20X objectives. (B) This panel shows a sub-culture of isolated primary tubular cells from passage 0 to passage 3. The images are taken under 10X and 20X objectives for visions of the cell domes, and under a 40X objective for a vision of the morphological changes through the passages. The scalebar = 100 &#181;m. <a href="/files/ftp_upload/57718/57718fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57718/57718fig2.jpg" /> 
Figure 2. Characterization of isolated primary tubular cells. (A) Immunostaining with antibodies against proximal tubular proteins (AQP1, AGT), a distal tubular protein (E-cadherin), an epithelial protein (SMA), a mesangial protein (Thy1), and macrophage proteins (F4/80, CD68) show that the isolated primary tubular cells and subsequent sub-cultures are pure proximal and distal tubular cells. (B) This panel shows a staining of the proximal tubule, distal tubule, and mesangial proteins in kidney tissues from a healthy wild-type mouse as a positive control and a no-primary negative control. (C) This panel shows a staining of the macrophage proteins F4/80 and CD68 in kidney tissue collected from a Col4a3-/- mouse as a positive control. Scalebar = 20 &#181;m. The DAPI is shown in blue. The marker proteins are shown in green. <a href="/files/ftp_upload/57718/57718fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57718/57718fig3.jpg" /> 
Figure 3. Extracellular flux assay in isolated primary tubular cells at different plating densities. (A) Increasing cellular plating density enhances the basal respiration levels in primary tubular cells, tested at passage 3. (B) This panel shows microscopic images of primary tubular cells cultured on an XF96 microplate seeded at 20,000, 30,000, and 40,000 cells/well densities. Scalebar = 100 &#181;m. (C) This panel shows a fatty acid- or glucose-based extracellular flux assay in primary tubular cells at passages 2 and 1. The data is mean &#177; SEM. <a href="/files/ftp_upload/57718/57718fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

isolation-characterization-high-throughput-extracellular-flux

Nanyang Technological University

Research

JoVE Journal

Biology

17.4K Views.  University of Miami Leonard M. Miller School of Medicine. Dit protocol biedt een rendabele aanpak om te isoleren en karakteriseren van muis primaire renale tubulaire cellen die vervolgens sub kunnen worden gekweekt om te beoordelen renale biologische functies- ex vivo, met inbegrip van de mitochondriale Bioenergetica.

Video: Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioengineering

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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.

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 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Immunology and Infection

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include environmental influences, genetic predispositions, co-existing medical conditions, and a history of acute kidney injury. These factors often take months or years to develop, complicating longitudinal studies of disease etiology and pathophysiology. Advanced kidney models are needed to improve our understanding of disease mechanisms and enhance nephrotoxicity prediction in drug development. Proximal tubule epithelial cells (PTECs) in the kidney play a critical role in xenobiotic and toxin clearance as well as the reabsorption of essential nutrients. We have previously demonstrated that three-dimensional (3D) microphysiological system (MPS) platforms, populated with isolated primary PTECs, can be used to investigate renal drug interactions, assess nephrotoxicity of compounds, and predict drug clearance. Here, we present protocols for isolating and culturing primary PTECs from whole human kidneys and for seeding them into a 3D MPS platform that mimics in vivo renal physiology. This protocol enables long-term studies supporting PTEC viability, physiological morphology, and functional polarization of key transporter proteins in MPS devices for up to 6 months.

We present an optimized protocol for processing whole human kidneys to isolate and culture primary renal proximal tubule epithelial cells and the application of these cells in a three-dimensional, microfluidic, microphysiological platform to recapitulate the renal proximal tubule.

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include ...

Medicine

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

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