Delineating the Metabolic Phenotype of Biopsy-Derived Kidney Cells

Summary

This protocol describes a unique method to conduct metabolic profiling of biopsy-derived kidney cells. The approach presented was optimized in healthy adult male pigs and has the potential to enhance the real-time assessment of kidney allograft viability. This method may also help uncover new biology across multiple metabolism-based kidney diseases.

Abstract

Kidney transplantation is the optimal treatment for end-stage kidney disease; however, transplanted kidneys are often lost prematurely, with up to 50% graft loss at 10 years post-transplant. One of the major causes of premature graft loss is the injury sustained by the graft at the time of transplantation, known as ischemia-reperfusion injury (IRI). Delayed graft function (DGF), defined as the need for dialysis in the first week post-transplant, is a manifestation of severe IRI that shows functional and histologic features of acute kidney injury (AKI). While the mechanisms driving AKI remain unclear, accumulating evidence suggests that altered metabolic function in the allograft mediates AKI and may be the reason for DGF. Thus, deciphering and monitoring the metabolic underpinnings of IRI will improve our capacity to diagnose and prevent AKI. This article describes a unique method to assess mitochondrial respiration (by means of oxygen consumption rate), glycolysis (extracellular acidification rate), and intracellular ATP levels in needle biopsy-derived kidney cell suspensions. The methodology has been optimized in healthy adult male pigs and validated in a porcine model of auto-transplantation. The approach presented has the potential to enhance the real-time assessment of kidney allograft viability in the clinic. Profiling metabolism in patient-derived biopsies may also uncover new biology in other metabolism-based kidney diseases.

Introduction

Kidney transplantation is the optimal treatment for end-stage kidney disease^1,2,3,4; however, transplanted kidneys are often lost prematurely, with up to 50% graft loss at 10 years post-transplant⁵. Affected patients have increased morbidity and mortality and pose a major economic burden on healthcare systems⁶. A major cause of premature graft loss is the injury sustained by the graft at the time of transplantation, known as ischemia-reperfusion injury (IRI). IRI is an unavoidable injury caused by diminished blood flow, followed by reperfusion⁷. Ischemia is characterized by tissue succinate accumulation, which drives reverse electron flow in the mitochondrial respiratory chain, leading to superoxide production and subsequent injury early after reperfusion⁸. IRI in transplanted organs increases the risk of primary non-function, delayed graft function (DGF), rejection, and inferior graft outcomes^8,9. DGF, defined as the need for dialysis in the first week post-transplant⁹, is a manifestation of severe IRI¹⁰. Histologically, IRI-associated DGF is manifested as acute tubular necrosis (ATN), and injury to the microvasculature. Functionally, there is a decrease in the glomerular filtration rate (GFR) and urine output, features of acute kidney injury (AKI) in the allograft¹¹. Despite its importance, there are currently no treatments for ischemia-reperfusion-associated post-transplant AKI. The lack of treatments stems from an incomplete understanding of disease mechanisms and a lack of markers to identify patients at the highest risk of graft dysfunction during the critical time points in which injury may be reversible¹².

Accumulating evidence suggests that altered energy metabolism in the allograft mediates AKI and may underpin DGF^13,14,15. Kidney cells generate energy via two major mechanisms: mitochondrial respiration and glycolysis¹⁶. In IRI, reperfusion results in mitochondrial dysfunction¹⁷. Consequently, glycolysis becomes the main energy source^13,18,19,20. The metabolic shift in IRI promotes tubular and microvascular endothelial cell death, leading to AKI^21,22,23,24. In a previous work, ATN was associated with increased expression of glycolytic enzymes and altered levels of mitochondrial proteins²⁵, in keeping with IRI in the graft^13,18. Moreover, in a porcine kidney auto-transplantation model, ischemia followed by cold storage led to reduced levels of kidney mitochondrial proteins and increased lactate excretion²⁶. Altogether, these findings solidify the clinical importance of monitoring the metabolic phenotype of kidney allografts, even before transplant, since basal metabolic features in the allograft can be predictive of allograft dysfunction²⁷.

Clinical prediction models of short-term risk of allograft rejection and loss have been developed^28,29. However, these models lack precision and do not consider the molecular features of the graft^30,31,32. Recent studies aimed to address this unmet need through the identification of molecular patterns at the tissue level that are associated with inferior graft outcomes, such as allograft dysfunction and rejection^{13,14,15,25,33,34}. These studies rely on steady state molecular measurements at the gene^33,34,35, protein^25,26, and metabolite level^13,14, leaving an incomplete picture of the metabolic phenotype of the allograft. This gap in knowledge is partly due to an inability to measure the metabolic function and the energy state of kidney-derived samples at a particular moment in time. The development of a standardized protocol to determine the metabolic phenotype of biopsy-derived kidney cells, which can potentially be translated into the study of patient samples, is warranted. This article describes a unique workflow to assess mitochondrial respiration (by means of oxygen consumption rate), glycolysis (extracellular acidification rate), and intracellular ATP levels in biopsy-derived kidney cell suspensions. The methodology has been optimized using kidney biopsy cores from healthy adult male pigs. To facilitate future clinical implementation, the biopsy cores were obtained using an 18 G needle, as in the standard clinical protocols of renal graft sampling^36,37. Finally, the utility of the protocol implementation in discriminating experimental conditions had been validated in a porcine model of auto-transplantation.

Protocol

This protocol has been approved by the Animal Care Committee of the Toronto General Research Institute (Animal Use Protocol number: 3652). Animals were cared for in accordance with the National Society of Medical Research and Guide for the care of laboratory animals, National Institute of Health (NIH), Ontario, Canada. Five 3-month-old male Yorkshire pigs, weighing approximately 30 kg each, were used in this study. The protocol for this methodology is subdivided into the following steps: (1) Dissociation of single kidney biopsy cores into cell suspensions; (2) Metabolic profiling of biopsy-derived kidney cells; and (3) Measurement of ATP content in biopsy-derived kidney cells. All biopsy samples collected for metabolic function or ATP content measurements were dissociated within 3 h of retrieval in a biosafety cabinet. The workflow of the methodology is shown in Figure 1. The reagents and the equipment used are listed in the Table of Materials.

1. Dissociation of single kidney biopsy cores into cell suspensions

1. Collect three biopsy cores of tissue from the renal cortex, using the coaxial needle (i.e., an outer guiding needle and an inner biopsy needle) gun system³⁸. For clinical translation, using an 18 G biopsy needle gun is recommended³⁹.
   NOTE: Be consistent with the angle of the biopsy needle gun (~45 °C) and depth of the puncture (~20 mm) when conducting biopsies of the kidney cortex.
2. Carefully place each biopsy core in a 15 mL tube containing 2 mL of RPMI 1640 on ice.
   1. Fragment biopsy tissue into small pieces (≤2 mm long) using a scalpel on a 6 cm Petri dish placed on ice. Using ultrathin fine-tip curved tweezers, place the tissue fragments from each biopsy into a different 1.5 mL tube containing 1 mL of dissociation media.
      NOTE: The dissociation media consists of RPMI 1640 medium containing 0.1 mg/mL DNase I, 10 mg/mL collagenase, and 2990 units/mL neutral protease from Bacillus polymyxa. Dissociation of human kidney biopsies using this media composition has proven to maximize cell viability and preserve representation of rare and fragile cell populations⁴⁰.
3. Allow dissociation to take place for 30 min at 37 °C (heating block) with intermittent agitation (vortexing the tubes every 5 min is recommended).
4. After dissociation, filter the cell suspensions through 35 µm cell strainer snap-cap tubes.
5. Rinse the strainer with 1:1 volume of 100% fetal bovine serum (FBS).
6. Pool the three biopsy-derived kidney cell suspensions into one 15 mL tube.
7. Pellet the cells by centrifugation (400 x g, 5 min, 4 °C), and resuspend them in 1 mL of RPMI 1640 containing 10% FBS. This cell suspension normally yields 0.8-1.2 × 10⁶ cells.

2. Metabolic profiling of biopsy-derived kidney cells

NOTE: The metabolic profiling step integrates the measurement of glycolysis (ECAR) and oxygen consumption rate (OCR) in a metabolic function analyzer, as well as the intracellular ATP levels using a commercial kit.

1. Cartridge utility plate hydration and calibration
   1. One day prior to sample retrieval, add 200 µL of ddH₂O to every well of the cartridge utility plate of the metabolic function analyzer.
   2. Incubate the cartridge utility plate in a CO₂-free incubator at 37 °C overnight.
   3. On the day of sample retrieval, remove the ddH₂O by aspiration using a Pasteur pipette connected to a vacuum system, and add 200 µL of calibrant solution (pH =7.4) to every well of the cartridge utility plate.
   4. Incubate the Cartridge utility plate in a CO₂-free incubator at 37 °C overnight.
2. Cell plating and preparation of assay media and stressor compounds for metabolic function measurements
   1. Add 100 µL of RPMI with 10% FBS in each well of a 96-well metabolic function assay plate.
   2. Separate a small aliquot (e.g., 10 µL) of the cell suspension (obtained in step 1) in a different tube, and stain the cells using a trypan blue exclusion viability dye.
   3. Using a counting chamber, determine the concentration of live cells (trypan blue negative)⁴¹ in the original suspension.
   4. Seed the cells in each of the study wells of the metabolic function assay plate, at a density of 1.0 x 10⁵ live cells/well. This cell number was determined after optimization (see Representative Results section).
      NOTE: Do not seed cells in any of the four corners of the metabolic function assay plate, as these are used as blanks.
   5. Let the cells adhere by incubating the metabolic function assay plate in a 37 °C, 5% CO₂ incubator overnight (~16 h).
   6. On the following day, confirm under the microscope that the cells are at a minimal confluence of 70%-80% (a healthy cell monolayer is important for optimal determination of metabolic function parameters).
   7. Thaw an aliquot (40-50 mL) of phenol red-free basal media at 37 °C, for at least 15 min.
   8. Prepare 35 mL of assay media by adding 2 mM glutamine (350 µL), 1 mM pyruvate (350 µL), and 5.55 mM glucose (385 µL) in 33.915 mL of pre-thawed basal media.
   9. Using a single or a multichannel pipette, gently remove the RPMI media from each study well of the metabolic function assay plate. Next, add 100 µL of basal media to wash the cells.
   10. Remove the basal media gently and add 150 µL of assay media to every well, including the wells without cells.
       NOTE: Leaving wells without assay media may affect the humidity conditions in the plate environment and lead to inconsistent measurements of metabolic function (Supplementary Figure 1).
   11. Place the metabolic function assay plate in a CO₂-free incubator at 37 °C for ~1 h (acclimation step). Take the cartridge utility plate (previously incubated in calibrant solution) back to the biosafety cabinet.
   12. During the acclimation, prepare the following four solutions of stressor compounds, in assay media: 7 µM oligomycin (7x, for a final concentration 1 µM oligomycin); 2.4 µM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (8x, for a final concentration 0.3 µM FCCP); 450 mM 2-deoxy-glucose (2-DG) (9x, for a final concentration 50 mM 2-DG); 10 µM Rotenone and 10 µM Antimycin A (Rot + AA) (10x, for a final concentration 1 µM Rot + 1 µM AA).
       NOTE: Oligomycin inhibits the activity of ATP synthase and therefore interrupts the electron transport chain (ETC). This lowers OCR and the ATP synthase-driven influx of protons into the mitochondria. As a result, glycolysis becomes the major source of ATP production⁴². FCCP is a cyanide drug (negative charge) that acts as an uncoupler of oxidative phosphorylation, by attracting protons (positive charge) and inducing a continuous proton influx into the mitochondria, which enables oxygen consumption by the ETC independently of ATP synthase activity⁴³. 2-DG is a synthetic structural analog of glucose in which the 2-hydroxyl group is replaced by hydrogen, and acts as a competitive inhibitor of glycolysis⁴⁴. Rot and AA are inhibitor drugs of the complexes I and III of the electron transport chain, respectively⁴⁵. The effects of injecting each drug at different steps of the metabolic function assay are depicted in Supplementary Figure 2.
   13. Load 25µL of each drug into the corresponding port on top of each well of the cartridge utility plate, including the wells without cells. Load the drugs according to the following sequence: 7x oligomycin (port A), 8x FCCP (port B), 9x 2-DG (port C), 10x Rot + AA (port D).
   14. Incubate the cartridge utility plate in the CO₂-free incubator until required to set up the metabolic function assay.
3. Determination of oxygen consumption and glycolytic rates in biopsy-derived kidney cells
   1. Verify that the metabolic function analyzer is turned on and connected to the controller software in an accessory computer.
   2. Launch the software.
   3. Click on the button Template > Blank.
   4. In the first tab, click on the button Injection strategy. Click on each button with letters A, B, C, or D that correspond to the four injection ports. Write down the name of the drug that will be injected through each port: oligomycin (port A), FCCP (port B), 2-DG (port C), Rot + AA (port D).
   5. In the second tab, define all the experimental conditions. Click on the button Add condition to introduce a new condition. The software will automatically assign a colour to each condition. Click on the button Plate map and assign the corresponding experimental condition to each well, which will be coloured accordingly.
      NOTE: The four corner wells need to remain assigned as blank (black colour), as these are used to subtract the background signal.
   6. In the third tab, click on the Injection button four times, so that the four injections (ports A, B, C, and D) will be added to the sequence. The assay can be run using the default injection sequence (A, B, C, D), number of measurements (3 basal measurements + 3 measurements after each injection), and time between measurements (3 min).
   7. In the fourth tab, fill in the boxes with the essential study information, and click on Run. The tray will open, and the software will ask the user to mount the cartridge utility plate. Ensure to remove the green lid before placing the plate on the tray.
   8. After calibration (~15 min), the tray will open again, and the software will ask the user to remove the cartridge utility plate and place the metabolic function assay plate onto the tray. Again, remove the plate lid before mounting the plate on the tray. The cartridge utility plate can be discarded.
   9. The assay will finish in approximately 2 h. After that time, save the corresponding '.asyr' file. Analyse the .asyr files computationally to examine the ECAR and OCR curves. Export data to the preferred format.
4. Measurement of DNA content for normalization of metabolic function data
   1. Determine the amount of DNA in the cells in each well after the metabolic function assay using the specified cell proliferation assay kit (see Table of Materials).
   2. Prepare the required volume of 1x cell-lysis buffer by diluting 20x cell lysis buffer in deionized water.
   3. To prepare a standard curve, prepare 100 µL samples of bacteriophage λ DNA standard (included in the cell proliferation assay kit) serially diluted in cell-lysis buffer to concentrations in the range of 0 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, 800 ng/mL. Load each dilution into a different well of a black 96-well microplate.
   4. Prepare 1x dye binding solution by diluting 400x dye reagent in 1x cell-lysis buffer. For each standard curve well and metabolic function assay study well, 100 µL will be required.
   5. Distribute 100µL of 1x dye solution into the first column, which already contains the standards. NOTE: The process up to this point can be started 30 min prior to the end of the metabolic function assay run.
   6. Remove the assay media from the metabolic function assay plate using a single or multichannel pipette.
   7. With a multichannel pipette, add 100 µL of 1x dye binding solution to each study well of the metabolic function assay plate. Pipette up and down a few times to lyse the cells, and transfer the contents to the black plate.
   8. Protect the microplate from the light and incubate at 37 °C for 2-5 min.
   9. Measure the fluorescence intensity of each sample using a fluorescence microplate reader with excitation at 480 nm and emission detection at 520 nm.

3. Measurement of ATP content in biopsy-derived kidney cells

NOTE: Measure the amount of ATP in biopsy-derived kidney cells using the specified ATP detection reagent and recombinant ATP (rATP) standard (see Table of Materials).

1. If frozen, thaw the ATP detection reagent at 4 °C overnight.
2. Following acquisition of biopsy-derived cell suspensions and cell counting, plate 1.0 x 10⁴ live cells/well into each well of a white 96-well microplate containing 100 µL of RPMI 1640 media with 10% FBS. Leave the first column of the microplate empty, as it will be used to load an rATP standard curve the following day.
3. Incubate the microplate at 37 °C in a 5% CO₂ incubator overnight.
4. On the following day, pre-warm the ATP detection reagent at room temperature.
5. Generate the rATP standard curve. Prepare 100 µL samples of rATP standard serially diluted in PBS to the following concentrations: 0 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM.
6. Load 50 µL of each rATP standard sample into the first column of the microplate.
7. Remove the RPMI 1640 media containing 10% FBS from each microplate study well, using a single or multichannel pipette.
8. Wash each study well with 100 µL of PBS (pre-warmed in a 37^{°C water bath).}
9. Remove the PBS and add 50µL of new PBS in each study well.
10. Add 50 µL of ATP detection reagent into both the standard and study wells.
11. Mix, incubate at 37 °C for 2 min, and subsequently at RT for 10 min.
12. Read and record luminescence in a microplate reader.

Results

The workflow was tested in biopsy-derived kidney cells from healthy 3-month-old male Yorkshire pigs. The protocol for tissue dissociation and cell plating was followed as outlined above. After letting the cells adhere for 16 h, OCR and ECAR were measured in real-time in the biopsy-derived porcine kidney cells using a metabolic function analyzer, as markers of mitochondrial respiration and glycolysis, respectively.

The first experiment aimed to determine the optimal seeding density for confiden...

Discussion

This article describes a unique method to assess the metabolic function of kidney cells obtained from biopsies. A protocol for obtaining single-cell suspensions from fresh core renal biopsies was coupled with an analysis of metabolic function and intracellular ATP levels. Glycolysis is assessed by measuring ECAR, and mitochondrial respiration by measuring OCR. Porine kidney biopsies were used to optimize the methodology.

To effectively study the bioenergetic function in cells obtained via<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Deoxy-D-glucose	Sigma	D6134	Competitive inhibitor of glycolysis
5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Falcon	352235	Dissociation tubes
Antimycin A from Streptomyces sp.	Sigma	A8674	Inhibitor of complex III of the respiratory chain
AOF BP Protease	VitaCyte	003-1000	Dissociation enzyme
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)	Sigma	C2920	Uncoupler of oxidative phosphorylation
CellTiter-Glo 2.0 Assay, 100mL	Promega	G9242	Measurement of intracellular ATP
Collagenase MA	VitaCyte	001-2030	Dissociation enzyme
CyQUANT Cell Proliferation Assay, for cells in culture	Invitrogen	C7026	Measurement of intracellular DNA
D-GLUCOSE, Anhydrous, Reagent Grade	Bioshop	GLU501	Metabolic substrate
DNase I	Stemcell	07469	Dissociation enzyme
Fetal Bovine Serum, Premium, US Origin, Heat Inactivated, 500 mL	Wisent	080-450	Dissociation media supplement
L-Glutamine-200 mM (100x) liquid (100 mL)	Gibco	25030081	Metabolic substrate
Oligomycin A	Sigma	75351	Inhibitor of the ATP synthase
PBS, -/-, 500 mL	Wisent	311-010-CL	Cell wash
rATP, 10 mM, 0?5 mL	Promega	P1132	Measurement of intracellular ATP
Rotenone	Sigma	R8875	Inhibitor of complex I of the respiratory chain
RPMI 1640	Gibco	11875119	Dissociation media
Seahorse Wave Controller Software 2.6	Agilent	N/A	Set up of metabolic function measurements
Seahorse Wave Desktop Software 2.6	Agilent	N/A	Analysis of metabolic function data
Seahorse XF base medium,  without phenol red, 500 mL	Agilent	103335-100	Measurement of metabolic function
Seahorse XFe96 FluxPak  (20 plates, 18 cartridges, and 500mL of calibrant solution)	Agilent	102416-100	Measurement of metabolic function
Seahorse XFe96 metabolic function analyzer	Agilent	101991-100	Measurement of metabolic function
Sodium Pyruvate Solution 100 mM (100X), liquid 100 mL	Gibco	11360070	Metabolic substrate