Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.
Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig’s shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.
A practical and quantitative evaluation of renal function is the glomerular filtration rate (GFR) measurement, which tells how well the kidneys filter blood based on the clearance principle1. An earlier method of measuring GFR entails the intravenous injection of exogenous compounds such as inulin or sinistrin, conducting serial measurements of plasma/urinary levels to detect their clearance2,3. This method is cumbersome, requiring the serial collection of plasma and urine samples4. An alternative is the measurement of endogenous metabolic end-products such as creatinine. However, this is time-consuming and, at times, inaccurate, as it is not only filtered by the glomerulus but also secreted by the tubules5,6. Furthermore, creatinine level is influenced by gender, age, diet, and muscle mass7,8,9.
A more precise, minimally invasive, and widely used measure of GFR is the use of transdermal GFR monitors, which measure real-time GFR in animals4,10. Sinistrin, a highly soluble and freely filtered exogenous renal marker, is labeled with fluorescein-isothiocyanate (FITC). This conjugated compound is injected intravenously, and real-time kidney function can be assessed without collecting blood and urine samples11. The use of transdermal GFR measurement has been validated in rodents12, dogs13, and cats14, but not in swine.
Porcine species share several anatomical and physiological characteristics with humans, making them ideal animals for studying various human diseases15. The use of pigs in translational biomedical research has become increasingly popular and preferred over rodent models because it mimics human physiology and pathophysiology16. Neonatal pigs are of interest in understanding the mechanisms of diseases unique to pediatric patients17. Moreover, the recent advancement in pig to human organ transplantation puts an urge to expand the diagnostic tools for preclinical and clinical trials18,19,20,21. This paper, for the first time, provides a guide for the use of the transdermal device in measuring GFR in neonatal pigs.
The procedures are written according to national standards for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee Health Science Center (UTHSC).
NOTE: Piglets in the experimental group are subjected to cecal ligation and puncture, while the sham group only undergoes opening of the abdomen without cecal ligation or puncture. Piglets in both groups are kept under anesthesia for 12 h post-procedure to allow enough time for sepsis and acute kidney injury (AKI) to ensue in the experimental group. Transdermal GFR measurement will ensue at 8 h post-procedure for a total of 12 h.
1. Piglet supply and housing
2. Pre-operative preparation
3. Anesthesia
4. Tracheostomy
NOTE: This experiment is non-survival, so a tracheotomy is performed to establish an airway for mechanical ventilation. Tracheostomy is a quick and easy procedure, as opposed to endotracheal intubation, which is challenging in piglets given their head and upper airway anatomy24,25. Additionally, laryngospasm is commonly reported during intubation, resulting in a prolonged period of hypoxia and hypercarbia that may compromise results26.
5. Femoral vessel cannulation
6. Maintenance of anesthesia, fluid and blood gas
7. Experiment group; cecal ligation and perforation (CLP) 25,28,29
NOTE: For piglets in the experiment group, perform CLP to induce polymicrobial sepsis28 and monitor the animal for 12 h post-surgery to allow enough time for severe sepsis to ensue. Transdermal GFR recording starts at 8 h post-cecal ligation to allow for 4 h of recording.
8. Sham group
9. Transdermal GFR device setup
10. FITC-sinistrin preparation and injection
11. Transdermal GFR recording
12. GFR measurement
13. Piglet euthanasia
In this section, we present for the first time, the representative data from the use of transdermal GFR in neonatal pigs. We used a cecal ligation and puncture model which has previously been shown to decrease kidney function28. Accordingly, we hypothesized that in our CLP pigs, there should be an acute drop in GFR corresponding to AKI, and this should be detected on the transdermal GFR device as increased clearance time (t1/2), thereby validating its use in pigs. Seven male piglets were included, three sham and four sepsis. The two groups had comparable weights (Figure 3A). As expected28, 12 h sepsis increased serum levels of C-reactive protein (CRP), a bacteremia and sepsis marker (Figure 3B). Representative FITC-sinistrin clearance curves in sham vs septic piglets are shown (Figure 4 A,B), with AKI shown by overlaying the sham and sepsis curves (Figure 4C). AKI is shown by an increased area under the curve for the CLP pigs. This can be visibly seen when the sham curve is layed on the CLP curve. The average half-life for FITC-sinistrin in the sham and sepsis groups were 114 and 537 minutes, respectively (Figure 5A). The average GFR in the sham group was 5.1 mL/min/100 gm of the body weight, while in the sepsis group, it was 1.06 mL/min/100 gm of the body weight (Figure 5B). An additional animal was excluded as the probe was displaced, which disturbed the clearance curve and time. Whereas 12 h serum creatinine (a biomarker of acute kidney injury) did not change in the sham group, it was increased from ~ 0.6 to 1.08 mg/dL in the septic pigs (Figure 6).
Figure 1: Cecum ligation surgery. (A) Cecum identified and brought to the exterior. (B) Cecum ligated at the base with a silk tie before puncturing with a needle. Please click here to view a larger version of this figure.
Figure 2: Attachment of transdermal device to the skin. (A) Skin shaved prior to attachment of adhesive patch. (B) Transdermal GFR device attached to the adhesive patch. Please click here to view a larger version of this figure.
Figure 3: Representative results. (A) Weight of the piglets used in this study and (B) Serum C-reactive protein (CRP) levels in mechanically ventilated sham and septic male piglets (unpaired t-test). Please click here to view a larger version of this figure.
Figure 4: Representative FITC-sinistrin clearance curves in mechanically ventilated sham and septic male piglets. (A) 12 h sham, (B) 12 h sepsis. Septic pigs present with impaired renal function as demonstrated by an increased area under the curve. Black data points represent raw data, blue lines the three-compartment fit, green lines the 95% confidence intervals, and red line the filtered data. (C) Overlay of representative curves to reflect the degree of divergence from baseline in septic pigs. The sepsis curve (red) showed minimal clearance of FITC-sinistrin, indicating AKI. Please click here to view a larger version of this figure.
Figure 5: Representative results. (A) FITC-sinistrin half-life and (B) GFR plots in mechanically ventilated sham and septic male piglets (unpaired t-test). Please click here to view a larger version of this figure.
Figure 6: Serum creatinine in mechanically ventilated sham and septic male piglets. (One-way ANOVA test). Please click here to view a larger version of this figure.
This paper describes practical steps to determining kidney function in pigs using the miniaturized transdermal GFR monitors and FITC-sinistrin in a mechanically ventilated, anesthetized neonatal pig model. Previous papers have established experimental transdermal GFR protocols in rodents11,12,14, but no protocols exist in pigs.
Recently, there has been a drive to explore alternative animal models to solve intractable diseases and ease the burden of kidney disease in humans. Unfortunately, many of these approaches have had translational limitations due to size, anatomical, and physiological differences. Rodents' renal anatomy and pathophysiology have major differences when compared to humans37. Since the human and pig systems share similar anatomical and functional characteristics, the porcine model may be a more realistic pathophysiological model of human diseases38,39. Pigs are now widely used to delineate pathophysiology and in drug development. With the publication of the pig genome, alongside successful transgenic production of disease-specific models, the porcine model stands to take a more critical role in translational research40,41.
Inulin clearance remains the most accepted means of GFR determination, but is impractical in large animal models due to the need for continuous infusion of inulin, catheterization of the bladder, and its time-consuming and cumbersome nature42. Serum creatinine and blood urea nitrogen (BUN) are commonly used to measure renal function in preclinical studies, but because creatinine is secreted in the tubules and urea is increasingly reabsorbed in dehydration, these markers have proved to be poor in estimating renal function5,43. Crucially, tubular creatinine secretion was found to cause overestimation of GFR when used as a marker of renal function in the pigs6. Also, due to their body habitus, a rise in creatinine is more likely to be seen in large animal models when compared to rodents. A study in mice revealed a 1.5-fold rise in serum creatinine 6 h post-cecal ligation44. Previously, we showed a rise in creatinine in neonatal pigs at 6 h post-CLP28. In this study, we kept the animals for a longer duration, ~12 hours post-cecal ligation to allow enough time for significant AKI and a subsequent rise in creatinine. As in our previous study, we confirmed the induction of sepsis by a rise in serum levels of CRP, an inflammation and sepsis marker. In this study, and as previous papers show, the severity of sepsis following CLP is dependent on the length of ligation and number of punctures44.
A protocol to measure GFR in pigs using Iohexol has previously been validated in pigs37, but in contrast, the transdermal GFR procedure is a marked improvement. It is less cumbersome, avoids repeated blood or urine sampling, and offers a real-time window into renal function and the possibility of repeated, serial measurements in the same animal45. This study provides practical guidelines for transdermal GFR determination in pigs.
As established by other groups, the most critical steps are the correct fixation of the device to the animal and the bolus injection of FITC-sinistrin. The measuring device must be well fixed to the skin surface to prevent movement artifacts on the trace. Because pigs are less hairy than rodents, using a depilatory cream is not required. A clean shave with a clipper might be all that is needed. This minimizes the depilation associated increase in the half-life of FITC-sinistrin, whose mechanism is unknown12. For proper fixation, a double-sided adhesive patch and tape are required to hold the device in place. The optimal device placement locations are the lateral thoracic wall and ventral abdominal region. These areas correlated with fewer movement artifacts.
When injecting the FITC-sinistrin, the correct and entire dose must be injected in one fluid motion into the vein. When the injection is interrupted and restarted, it creates multiple "mini-peaks" on the clearance curve. The tail vein is routinely used for small rodents, but the auricular ear vein offers a more accessible and prominent route in the pigs. A cannula can be placed in the ear vein for multiple measurements in conscious pigs. An important distinction to note in the sampling time is that, as opposed to rodents (~1-2 h), pigs lasts longer (~4 h), which approximates the time it takes for FITC-sinistrin to be cleared from the circulation. To the best of our knowledge, this is the first paper detailing transdermal GFR via FITC-sinistrin clearance in pigs. So, no citations exist for reference. The measuring time used ~4h was arrived at, via consultations with the manufacturer. This sampling time is comparable to a prior study validating transdermal GFR in other non-rodent mammals14.
In evaluating transdermal GFR in piglets, there are a few factors that must be considered. One-compartment models are known to overestimate GFR significantly46; we use the three-compartment kinetic model which is more accurate, providing three-way communication of the intravenously injected marker between the plasma, extracellular space, and deeper components46. Also, these are mechanically ventilated piglets under very deep anesthesia for ~12 h. Since anesthesia influences renal function47,48, it might be worth taking that into account in procedures that require long sedation or where experimental maneuvers require additional anesthesia alongside GFR monitoring. Finally, and perhaps most crucially, neonatal piglets have still-developing renal systems with immature nephrons that function at a fraction of the adult animal49. Hence, they demonstrate lower GFR and renal function50.
As previously indicated, transdermal GFR in pigs is not an absolute measure of sinistrin concentrations in the blood. Its only an estimation of decay in fluorescence over time12. The use of a conversion factor attempts to mitigate this, by expressing GFR in mL/min. However, because the conversion factor is dependent on extracellular space, which in turn relies on body weight34,35,36, it is possible for wide variations to exist if weight is not controlled for, or if the extracellular space is not accurately defined51,52.
Additionally, skin pigmentation appears to affect transdermal FITC-sinistrin clearance12,31. In our studies, we found that the pigmented pigs showed decreased signal. In one instance, we did not detect signal in an intensely dark colored pig. However, since background signal tends to be reduced in pigmented animals12, we found that GFR values were largely comparable. One solution to this is to opt for lighter colored areas of the skin when placing the device. Since these pigs were largely used in a surgical model of disease, with several forms of lighting and heat sources involved, one must account for potential movement artifacts on the GFR traces via reflected light absorbed from surrounding skin12. One solution to this might be to minimize infrared light during recording or covering the devices in foil.
In summary, this study offers a simple and reliable method for measuring glomerular filtration rate in neonatal pigs using the transdermal measurement of FITC-sinistrin clearance. Moreover, our data supports the utility of the system in evaluating kidney function in the settings of acute kidney injury.
The authors have nothing to disclose.
This study was supported by the National Institutes of Health grants R01 DK120595 and R01 DK127625 awarded to Dr. Adebiyi. The content of this paper is solely the authors' responsibility and does not necessarily represent the official views of the National Institutes of Health. Thanks to Dr. Daniel Schock-Kusch, site Director at MediBeacon GmbH, for his advice.
Alpha – Chloralose | Sigma-Aldrich | C0128-25G | Used for maintanining anesthesia |
Black braided silk 3-0 | Surgical Specialties | SP117 | Silk tie for blood vessel traction and ligation |
Centrifugation machine AccuSpin 8C | Fischer Scientific | 75-008-821 | Used to extract plasma from whole blood sample |
Endotracheal Tube 3.0 uncuffed | Progressive Medical International | 1109021995 | Inserted through tracheostomy |
FITC-Sinistrin 1.0 g | MediBeacon Inc. | FTCF S001 | Store at room temp and protect from light |
GEM Premier 3000 Blood gas analyzer | Instrumentation Laboratory | 5700 | For bedside blood gas analysis |
Heating Pad medium size 20 in x 29 in | Adroit Medical Systems | V029 | Connects to heat therapy pump |
HTP-Heat Therapy Pump | Adroit Medical Systems | HTP | Allows you to set temperature as needed. |
IDEXX Catalyst One | IDEXX Laboratories | 89-92525-00 | Plasma creatinine analysis |
Invasive blood pressure catheter 3.5Fr | Millar | SPR-524 | Inserted in femoral artery |
IV adminstration set with flow regulator | True Care | TCRTCBINF033G | Used to connect IV fluid bag to vein catheter |
Ketamine | Covetrus | 68317 | Used for induction of Anesthesia |
MediBeacon analysis software version 3.0 | MediBeacon Inc. | N/A | Software program used for analysing data to obtain sinistrin clearance half life and curve |
Millex-GV Syringe Filter Unit 0.22 µm | Millipore Sigma | SLGVR33RS | Syringe filter for chloralose injection |
Neonate/Infant Ventilator | Sechrist Millennium | 20409 | Connected to air supply to provide ventilation through endotracheal tube |
Phenobarbital Sodium + Phenytoin Sodium (Euthasol) | Covetrus | 72934 | Used for euthanasia |
Ringer Lactate 500 mL bag | Baxter | 2B2323Q | Maintanence fluid infusion |
Sterile Gloves | Henry Schein | 104-5920 | Used by operator during surgery |
Sterile Gown | Halyard Health | 95021 | Used by operator during surgery |
Steril Towel | Medline | 42131704 | Used as drape to maintaine sterile field when operating |
Suture 3-0 silk reverse cutting needle | Ethicon | NC1842168 | Used for suturing abdominal wall layers |
Transdermal Mini GFR Monitor | MediBeacon Inc. | TDM004 | Battery and USB connector included in package |
Transdermal monitor adhesive patch | MediBeacon Inc. | PTC-SM001 | Doubl sided adhesive patch for GFR probe |
Umbilical Tape 1/8 in x 20 yds | Fisher Scientific | NC9303017 | To secure endotracheal tube |
Venous Catheter size PE/5 | Micro medical tubing | BB31695 | For femoral vein cannulation |
Xylazine | Covetrus | 61035 | Used for induction of anesthesia |