Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.
Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.
Kidneys maintain homeostatic balance for various substances and regulate blood volume in a way that determines total blood pressure. Disturbances in the renal filtration, reabsorption or secretion lead to or accompany pathological states, ranging from hyper- or hypotension to end stage renal disease that eventually requires kidney transplantation. The renal filtering unit (glomerulus) consists of three layers – the capillary endothelium, basement membrane and a single-cell layer of epithelial cells – podocytes, which play a major role in the maintenance of the slit-diaphragm integrity and function1. Dysfunction in the permselective glomerular filter causes urinary loss of macromolecules, such as proteinuria. Various agents may affect the structure of the podocytes and their foot processes, which determine the integrity of the glomeruli filtration barrier.
The podocytes are involved in the maintenance of the glomeruli filtration function. It has been established that improper calcium handling by the podocyte leads to cell injury and plays an important role in the progression of various forms of nephropathies2,3. Therefore, development of a model which allows for direct measuring of intracellular calcium concentration changes will be instrumental for studies of podocyte function. Isolated glomeruli were previously used in a numerous studies including measurement of albumin reflection coefficient changes4 and assessment of integral cellular currents in the whole-cell electrophysiological patch-clamp measurements5,6. In the present paper we describe the protocol that allows the researcher to measure intracellular calcium concentration changes in response to applications of pharmacological agents, estimate basal levels of calcium within the cells, and assess individual calcium channels activity. Ratometric calcium concentration measurements and patch-clamp electrophysiology were used to determine changes in the intracellular calcium concentration within the podocyte and channel activity, respectively.
Animal use and welfare should adhere to the NIH Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Kidney Flush
2. Isolation of the Rat Glomeruli
3. Single-channel Patch-clamp Electrophysiology
4. Ratiometric Confocal Fluorescence Measurements of Intracellular Calcium Concentration in the Podocytes
5. Image Analysis for the Calcium Measurements
6. Intracellular Calcium Concentration Calculations Using Fluo-4 Fluorescence Signal
Here we addressed the problem of measuring acute changes in the calcium levels in the podocytes. Figure 1 shows a schematic representation of the experimental protocol designed in order to perform high resolution live fluorescence confocal imaging and single ion channel activity recordings in the podocytes of the freshly isolated rodent glomeruli. Briefly, after the rat is anaesthetized, the kidneys should be flushed with PBS to clear them of blood. Then, the kidneys are excised and decapsulated, and glomeruli are isolated from the kidney cortex by differential sieving. Part of the sample can be taken for patch-clamp analysis and the rest can be loaded with fluorescent calcium dyes Fluo-4, AM and Fura Red, AM in order to perform confocal ratiometric calcium imaging.
Electrophysiological measurements of the single ion channel activity can be performed right away after the isolation of the glomeruli. Activity of ion channels can be measured both in cell-attached and whole-cell configurations of the patch-clamp method. To demonstrate the feasibility of the approach shown is the recording of the single TRPC-like calcium-conducting ion channel activity (Figure 2). Additionally, it is possible to apply cell-permeable or receptor-binding drugs to test their effects on the calcium influx in the podocytes at the level of single ion channels.
An approach that allows single ion channel activity assessment produces a variety of data that can characterize podocyte function. However, it can be greatly supplemented by measuring the calcium concentration changes at the level of the whole cells. To perform such studies, freshly isolated glomeruli were loaded with fluorescent dyes for the high throughput confocal imaging. With a high optical resolution it is possible to monitor calcium levels in several podocytes at a time. Figure 3 demonstrates the time-course for the typical experiment designed to evaluate the changes of calcium concentration within the podocyte. The basal calcium concentration within the podocyte was evaluated based on the Fluo-4 fluorescence. After recording the basal signal intensity for 1 min (F), Ionomycin is applied and causes peak fluorescence (Fmax) to be reached, which is then quenched by MnCl2 (Fmin is noted). According to the formula in 6.3, intensity of Fluo-4 signal at each time point of the experiment can be then translated into the actual concentration of the calcium ions in the cell. As seen from the graph, mean fluorescence in the background (approximately 400 arbitrary units) translates into 140 nM, which is within a normal concentration range for intracellular calcium in healthy non-apoptotic cells.
The importance and usefulness of the described approach is justified by another application, which allows for measuring acute calcium transients in podocytes in response to various drugs. Figure 4A illustrates representative images of the rat glomerulus stained with Fluo-4 and Fura Red in the 2 mM calcium-containing solution (described in 4.5) before and after addition of 10 µm ATP. Note a dramatic increase in the green (Fluo-4 AM) fluorescence of the podocytes (marked with arrows), and a drop in Fura Red fluorescence (red pseudocolor). Fluo4 and FuraRed reflect an increase in calcium concentration at an excitation wavelength of 488 nm in opposite way i.e., when calcium concentration within the cell increases Fluo 4 intensity goes up, whereas FuraRed goes down. That is possible due to dual excitation nature of FuraRed fluorophore. Depending on excitation wavelength it can behave like calcium-bound (420 nm) or calcium-free (488 nm) fluorophore with corresponding increase or decrease in fluorescence. Therefore, the use of FuraRed alone in a ratiometric imaging mode is possible, however it requires two excitation wavelengths – 420 nm and 488 nm. This will decrease the frequency of the imaging at least twice (compared to the use of one excitation wavelength); if the researcher would want to keep the fast pace of imaging, the image resolution should be decreased. However, Fluo 4 and FuraRed can be used together in a ratiometric mode without loss of resolution or decrease in imaging frequency, as these fluorophores can be excited by the same 488 nm laser, and provide a good differentiation of the emission spectra. A typical transient summarized from the ROIs marked with arrows in Figure 4A is shown in Figure 4B; the graph clearly demonstrates an acute increase in Fluo4/FuraRed intensity ratio after addition of 10 µM ATP, which decays within several minutes. Images were taken every 4 sec, and hence, the technique allows observing fast changes in the calcium concentration within the cell.
Figure 1. Schematic representation of the experimental protocol. After the animal is properly anaesthetized and prepared for the surgery, the kidneys are flushed with PBS to remove blood. Then, the kidneys are excised and decapsulated, and glomeruli are isolated from the kidney cortex by differential sieving. Here, part of the sample is taken for patch-clamp analysis, and the rest is loaded with fluorescent calcium dyes and confocal ratiometric calcium imaging is performed. Please click here to view a larger version of this figure.
Figure 2. Representative recording showing the activity of the TRPC calcium channels in the podocytes of the freshly isolated rat glomeruli. Left panel demonstrates the patch-clamp pipette attached to the podocyte body on the surface of the glomerulus during an electrophysiological recording in a cell-attached configuration; a part of a capsulated glomerulus can be seen in the right lower corner of the image. Representative current trace of the TRPC-like channels activity from a cell-attached patch made on the podocyte of the rat glomerulus at a -40 mV holding potential is shown on the right. The c and oi denote closed and open channel levels, respectively. Please click here to view a larger version of this figure.
Figure 3. Representative experiment designed to assess the intracellular calcium level in the podocytes. To measure intracellular calcium concentration, glomeruli are loaded with Fluo-4, AM, fluorescence intensity is recorded in the baseline and after addition of ionomycin and MnCl2. The graph demonstrates the fluorescence signal changes in response to ionomycin (producing the maximum of the Fluo-4 fluorescence, Fmax) and MnCl2, which quenches the dye and results in the lowest fluorescence intensity (Fmin). Intensity of fluorescence (left abscissa) for each time point can be translated into the actual calcium concentration in nanomoles (right abscissa) according to the formula in 6.3. Note the insets with the images showing podocytes at the time points when F, Fmax and Fmin were calculated. The graph shown here reflects fluorescence intensity of the podocyte marked by a circle (ROI); images were taken every 4 sec. Please click here to view a larger version of this figure.
Figure 4. Measurement of the calcium transients in the podocytes in response to ATP. (A) Representative images illustrating the wild type rat glomerulus stained with Fluo-4 (green pseudocolor) and Fura Red (red pseudocolor) in the 2 mM calcium-containing solution before and after addition of 10 µM ATP. Lower intensity green-colored fluorescence before application of the drug should be noted. (B) Summarized example of the intracellular calcium transient evoked in podocytes by application of 10 µm ATP. Note a strong and fast increase in Fluo-4/Fura Red fluorescence intensity ratio following addition of the drug, which accounts for the stimulation and depletion of the calcium depot and calcium influx from the outside in the cell, resulting in the elevation of the intracellular calcium concentration. Error bars represent the standard error of means calculated for 11 podocytes of the same glomerulus. Please click here to view a larger version of this figure.
The approach described here allows for the analysis of calcium handling by the podocytes of the rodent glomeruli. This technique allows application of patch-clamp single channel electrophysiology and fluorescence ratiometric confocal imaging. However, both approaches can be used separately, on their own. The proposed protocol has several relatively simple steps, including 1) kidney flush; 2) isolation of the glomeruli by differential sieving; 3) performing patch-clamp electrophysiological experiments, or incubation of the glomeruli with fluorescent calcium labeling dyes for ratiometric confocal imaging of changes in intracellular calcium.
To isolate the glomeruli, a modified protocol based on Gloy et al.5 was used. The main advantage of the current technique of differential sieving is that it produces glomeruli stripped of the Bowman’s capsule and thus do not require the sophisticated and time-consuming step of manual decapsulation. The majority of the glomeruli in the isolated fraction are decapsulated, however the researcher must be aware that there are capsulated glomeruli, too. It is important to make sure that the glomeruli stripped off of the Bowman’s capsule are imaged, as capsule prevents the drugs from reaching the podocytes. As seen in Figure 2, decapsulated glomeruli have a rough surface with distinct capillaries and podocyte bodies, whereas capsulated glomeruli appear smooth and round-shaped. Furthermore, encapsulated Fluo4 and FuraRed loaded glomeruli emit less intense fluorescence signal compared to decapsulated ones.
The fraction of rat glomeruli obtained is 95% pure (with traces of proximal tubules) and can be easily used for western blotting or other molecular biology applications if needed. It is exceptionally important that in this preparation podocytes are attached to the capillaries, retain intact foot processes and function as a part of the native glomerulus filtration apparatus machinery. The mechanisms of calcium handling by the podocytes serve an essential regulatory role in the development and prevention of renal complications in such diseases as diabetes or hypertension9-12. Podocytes contribute significantly to the permeability barriers, as evidenced by the fact that acute injury alters podocyte morphology and structure and may cause proteinuria2,3,13-15. Therefore, it is of great importance to observe the responsiveness of calcium influx in these cells to drugs, which can be easily screened in this preparation. It should be understood that if the researcher is probing podocytes in a pathological state, for instance, hypertension or diabetes, which usually result in proteinuria and glomeruli damage, the yield of glomeruli might be less pure than that of the healthy animals. It is recommended to always monitor the glomeruli fractions at each stage of the isolation process to avoid loss of the tissue. In some cases, heavily diseased glomeruli or glomeruli from aged or too young animals might require individual adjustment of isolation process, including the selection of larger/smaller mesh sieves.
We have applied this approach initially to determine the efficacy of non-steroid anti-inflammatory drugs in the inhibition of the calcium TRPC channels in podocytes16. Following these studies7,16-22 we employed the described techniques to establish several critical mechanisms controlling podocytes function. For instance we have described the profile of the P2 (purinergic) receptors in the podocytes of the rats and defined the regulation of calcium influx by Angiotensin II in the mouse glomeruli. As described in a later manuscript20, the protocol can be adapted to perform similar studies not only in rats, but also in other species, such as mice. Ratiometric calcium fluorescence imaging (with the use of two fluorescent dyes – Fluo-4 and Fura Red) ensures additional reliability of the data, and if needed calcium imaging can be combined with other fluorescent dyes to monitor changes in other substances within the podocytes. For instance, we have used this approach to measure nitric oxide with the help of the DAF-FM dye. Patch-clamp electrophysiology can be used alone or (setup permitting) in combination with calcium imaging. Apart from calcium channels, the technique allows recording the activity of other ion channels. Furthermore, the use of transfection and siRNA may expand the area of application even more, as has recently been shown by Dr. Dryer’s group23.
Conventional epifluorescence microscopy can be used here instead of the less affordable confocal imaging. However, the researcher should be aware that the effective sensitivity of wide field fluorescence microscopy is generally limited by out-of-focus light. This (in comparison to confocal imaging) causes such limitations as inability to produce high image resolution of single ROI (podocyte) or its foot processes. Furthermore, wide-field microscopy complicates identification of podocytes as it is hard to differentiate between surface of the glomerulus and deep tissue. As of now commercial availability of various fluorophores for confocal imaging is much better and rapidly expanding. Alternatively, epifluorescence studies could be easily combined with an electrophysiological setup which can provide simultaneous intracellular Ca2+ and single channel activity recording. Fura2-AM loading of glomeruli podocytes with filter-wheel monochromator and neutral density filters setup is widely used, but is not always suitable for quality high resolution imaging.
It should also be noted that the isolated, functional glomerulus is able to change its volume in response to physiological stimuli. The researcher should be aware of this while performing the experiments, and carefully choose the focal plane during confocal studies to avoid the loss of focus, and perform control experiments upon selection of novel drugs in order to test unintended contraction or relaxation.
This technique offers a unique opportunity to monitor changes in calcium influx at the single ion channel and individual cells levels after pharmacological treatments or other manipulations, in a variety of rodent backgrounds. This technique can be also used in human renal disease studies as a modified protocol can be applied to tissues obtained from kidney biopsies, which will undoubtedly provide exceptionally valuable information. Additionally, calcium concentration measurements could be paired with the electrophysiological patch-clamp experiments in real time, which may provide more in-depth and mechanistic insight into the regulation of calcium handling within podocytes in normal and pathological conditions.
The authors have nothing to disclose.
The authors would like to thank Glen Slocum (Medical College of Wisconsin) and Colleen A. Lavin (Nikon Instruments, Inc.) for excellent technical assistance with microscopy experiments. Gregory Blass is acknowledged for critical proofreading of the manuscript. This research was supported by the National Institutes of Health grant HL108880 and American Diabetes Association grant 1-15-BS-172 (to AS), and the Ben J. Lipps Research Fellowship from the American Society of Nephrology (to DVI).
Fluo4 AM | Life Technologies | F14217 | 500µl in DMSO |
FuraRed AM | Life Technologies | F-3020 | |
Poly-L-lysine | Sigma-Aldrich | P4707 | |
Pluronic acid | Sigma-Aldrich | F-68 | solution |
Ionomycin | Sigma-Aldrich | I3909-1ML | |
Tube rotator | Miltenyi Biotec GmbH | 130-090-753 | Germany |
Nikon confocal microscope (inverted) | Nikon | Nikon A1R | Laser exitation 488nm. Emission filters 500-550nm and 570-620nm |
Objective | Nikon | Plan Apo 60x/NA 1.4 Oil | |
Cover Glass | Thermo Scientific | 6661B52 | |
High vacuum grease | Dow Corning | Silicone Compound | |
Software | Nikon | Nikon NIS-Elements | |
Recording/perfusion chamber | Warner Instruments | RC-26 | |
Patch Clamp amplifier | Molecular Devices | MultiClamp 700B | |
Data Acquisition System | Molecular Devices | Digidata 1440A | Axon Digidata® System |
Low Pass Filter | Warner Instruments | LPF-8 | 8 pole Bessel |
Borosilicate glass capillaries | World Precision Instruments | 1B150F-4 | |
Micropipette Puller | Sutter Instrument Co | P-97 | Flaming/Brown type micropipette puller |
Microforge | Narishige | MF-830 | Japan |
Motorized Micromanipulator | Sutter Instrument Co | MP-225 | |
Inverted microscope | Nikon | Eclipse Ti | |
Microvibration isolation table | TMC | equipped with Faraday cage | |
Multichannel valve perfusion system | AutoMake Scientific | Valve Bank II | |
Recording/perfusion chamber | Warner Instruments | RC-26 | |
Software | Molecular Devices | pClamp 10 . 2 | |
Nicardipine | Sigma-Aldrich | N7510 | |
Iberiotoxin | Sigma | I5904-5UG | |
Niflumic acid | Sigma-Aldrich | N0630 | |
DIDS | Sigma-Aldrich | D3514-25MG | |
TEA chloride | Tocris | T2265 | |
RPMI 1640 | Life Technologies | 11835030 | without antibiotics |
BSA | Sigma-Aldrich | A8327 | 30% albumin solution |
Temperature controlled surgical table | MCW core | for rodents | |
Steel sieves: | #100 (150 μm), 140 (106 μm) | ||
Gilson, Inc SIEVE 3 SS FH NO200 | Fisher Sci | 50-871-316 | |
Gilson, Inc SIEVE 3 SS FH NO270 | Fisher Sci | 50-871-318 | |
Gilson, Inc SIEVE 3 SS FH NO400 | Fisher Sci | 50-871-320 | |
mesh 200 | Sigma-Aldrich | s4145 | screen for CD-1 |
Binocular microscope | Nikon | Eclipse TS100 | |
Binocular microscope | Nikon | SMZ745 | |
Syringe pump-based perfusion system | Harvard Apparatus | ||
polyethylene tubing | Sigma-Aldrich | PE50 | |
Isofluorane anesthesia | http://www.vetequip.com/ | 911103 | |
Other basic reagents | Sigma-Aldrich |