The Mouse Isolated Perfused Kidney Technique

Summary

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Abstract

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.

This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney's preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

Introduction

The isolated perfusion of organs has been the subject of an ongoing effort among physiologists for many decades¹. The technique enables the function of the organ, without systemic influences such as blood pressure, hormones, or nerves, to be studied. Carl Eduard Loebell is considered to be the first to have described the successful perfusion of an isolated kidney, in 1849². Since then, the perfusion apparatus has undergone significant refinement. Frey and Gruber introduced an artificial lung for oxygenation and pulsatile pumps for continuous perfusion². While early researchers mainly studied the kidneys of large mammals-namely, pigs

Protocol

All procedures involving animals described in this manuscript were conducted according to Swiss law and approved by the veterinary administration of the Canton of Zurich, Switzerland.

1. Buffer Preparation

1. Prepare solutions 1 - 4 and the antidiuretic hormone (ADH) solution (Table 1).
2. Prepare the dialysis buffer (Table 1).
   NOTE: This is the buffer used as the dialysis buffer during the perfusion. Later, the erythrocytes will be diluted in this buffer to form the final perfusate.
3. Erythrocyte preparation.
   1. Dilute 250 ml of human erythrocyte concentrate (tested ma....

Results

With the method described, isolated mouse kidneys can remain viable for at least 1 hr. We tested the tissue viability after 1 hr of continuous perfusion with functional (renal blood flow and vascular resistance, blood gas analysis of venous outflow, glomerular filtration rate, urinary fractional Na⁺ and K⁺ excretion, and urine osmolality) and morphological (transmission electron microscopy, TEM) methods in four kidneys of wildtype C57Bl/6 mice. Additionally, western .......

Discussion

The mouse isolated perfused kidney is a tool for studying kidney function in a controlled environment ex vivo for 1 hr, bridging the gap between in vivo experiments in intact animals, which may be flawed by the impact of numerous systemic factors, and in vitro experiments in isolated nephron segments or cultured cells, which necessarily neglect the impact of intact organ structure on function. There is, to the authors' knowledge, no alternative technique with which to perform this specific .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Perfusion Circuit:
Moist chamber 834/8	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2901
Cannular with basket and side port	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2947
Thermostat TC120-ST5	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-4544
ISM 827/230V Roller Pump Reglo Analogue	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-0114
Reservoir jacketed for buffer solution 1 L	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3438
Reservoir jacketed for buffer solution 0.5 L	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3436
Pressure Transducer APT300	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3862
TAM-D Plugsys Transducer	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-1793
SCP Plugsys servo controller	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-2806
Windkessel	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3717
HSE-USB data acquisition	Harvard Apparatus/Hugo Sachs Elektronik GmbH	73-3330
Low-Flux Dialysator Diacap Polysulfone	B.Braun	7203525
PE-Tubing for aorta cannulation 1.19 mm I.D. x 1.70 mm O.D.	Scientific Commodities Inc.	BB31695-PE/8
Name	Company	Catalog Number	Comments
Buffer reagents:
Aminoplasmal 10%	B.Braun	134518064
Sodium pyruvate	Sigma-Aldrich	P2256-25G
L-Glutamic acid monosodium salt hydrate	Sigma-Aldrich	G1626-100G
L-(-)-Malic acid sodium salt	Sigma-Aldrich	M1125-25G
Sodium-L-Lactate	Sigma-Aldrich	L7022-10G
alpha-Ketoglutaric acid sodium salt	Sigma-Aldrich	K1875-25G
NaCl	Sigma-Aldrich	31434-1KG-R
NaHCO3	Sigma-Aldrich	S5761-5KG
KCl	Sigma-Aldrich	60130-1KG
Urea	Sigma-Aldrich	U5378-500G
Creatinine	Sigma-Aldrich	C4255-10G
Ampicillin	Roche	10835242001
MgCl2 * 6H2O	Sigma-Aldrich	M2393-500G
D-Glucose	Sigma-Aldrich	G8270-1KG
CaCl2 * 6H2O	Riedel-de-Haën	12074
NaH2PO4	Sigma-Aldrich	S9638-500G
Na2HPO4	Sigma-Aldrich	S0876-500G
Antidiuretic Hormone dDAVP	Sigma-Aldrich	V2013-1MG
FITC-Inulin	Sigma-Aldrich
Filter used for erythrocyte filtration	Macherey-Nagel	MN 615
BGA Analysis:
ABL 80 flex	Radiometer Medical ApS
Electron Microscope:
Philips CM100 TEM	FEI