Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Summary

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.

Abstract

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.

Introduction

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 clearance^2,3. 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 cr....

Protocol

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-proc....

Results

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 (t_1/2), thereby validating its use in pigs. Seven male piglets wer.......

Discussion

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 rodents^11,12,14, but no protocols exist in pigs.

Recently, there has been a drive to explore alternative animal models to s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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