The Isolated Perfused Rat Kidney: A Technical Update

Abstract

Abstract:The isolated perfused kidney is commonly utilized as a screening tool for renal clearance and metabolism, and to correlate renal drug deposition to renal function. Here, we report on several aspects of this procedure that will facilitate a higher experimental success rate and lead to a reduction in animal use. First, we investigated the utility of inulin and creatinine as commonly used markers to measure glomerular filtration rate. For inulin, in the presence of either 20 mM glucose or 4.5% dextran in the buffer, significant interference was observed using an anthrone-based colorimetric assay. These findings suggest that caution needs to be exercised when using glucose or dextran and when inulin is quantitated using this method. Under these circumstances the use of alternative methods of inulin quantitation such as fluorescently tagged inulin is preferred. Second, we optimized bovine serum albumin (BSA) and BSA/dextran compositions that are routinely recommended as oncotic agents in the perfusion buffer and found that a 4% BSA/1.67% dextran composition had the best viability of kidney biomarkers in accordance with recommended threshold parameters. These considerations will be of particular relevance to researchers utilizing the isolated perfused kidney as a screening tool to measure renal biology and drug metabolism as well as applications to investigate diabetic nephropathy.

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

“…12 Nevertheless, we utilized creatinine in this instance as we found that high glucose concentrations (20 mM) interfered with inulin quantitation whereas creatinine measurement was unaffected. 10 The observed GFR values for non-diabetic kidneys utilizing creatinine as a clearance marker were similar to those reported by other studies using the isolated perfused kidney. 13,14 By comparison, GFR was decreased in diabetic kidneys by approximately 3-fold ( P < 0.001) showing an intrinsic impairment in renal function (Figure 1(d)).…”

“…Rats were anaesthetized with 4% isoflurane in 2 L/min oxygen and maintained on 2.5% isoflurane in 2 L/min oxygen The surgical technique was performed as described. 10 Following removal of the kidney, a 40-min stabilization period was included after which samples were collected for the next 20 min. Aliquots of both perfusate and urine were analyzed for sodium (blood-gas analyzer, Bayer), glucose (YSI 2300 STAT plus Glucose and lactose Analyzer, YSI Inc., USA), and creatinine (creatinine assay kit, Cayman, Ann Arbor, MI).…”

“…Perfused kidney viability was assessed using several criteria including glomerular filtration rate (GFR), glucose re-absorption, sodium ion re-absorption, urinary flow rate, and perfusion pressure. 10 The perfusion system comprised a single pass pump-driven forced perfusion (20-40 mL/min) where diabetic and non-diabetic kidneys were perfused with Krebs-Henseleit Buffer (118.5 mM NaCl, 1.18 mM MgSO 4 , 1.18 mM KH 2 PO 4 , 24.8 mM NaHCO 3 , 4.75 mM KCl, 2.54 mM CaCl 2 ), bovine serum albumin (4% w/v), dextran (1.67% w/v), 20 amino acids (13 mM), creatinine (200 mg/L), MI (50 mM), DCI (5 mM), and glucose (either 5 mM or 20 mM). GFR was calculated from the clearance of creatinine using the following equation: GFR ¼ (U Cr  UFR)/P Cr where UCr is the concentration of creatinine in the urine (mg/mL), UFR is the urinary flow rate (mL/min), and PCr is the concentration of creatinine in the perfusate (mg/mL).…”

Roles of glomerular endothelial hyaluronan in the development of proteinuria

“…In rats anesthetized with 0.5 mg/kg of pentobarbital, the right ureter was cannulated with a polyethylene tube (PE-10, outer diameter 0.61 mm, inner diameter 0.28 mm; Becton, Dickinson and Company), and a 24-gauge indwelling needle was positioned in the renal artery. Extracted right kidneys were connected to the isolated perfusion rat kidney (IPRK) system, as previously described (Chang et al, 2013; Figure S2). The renal artery was perfused at 37℃ with Krebs-Henseleit buffer containing 4% bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation), saturated with carbogen (95% O 2 , 5% CO 2 ) (Chang et al, 2013).…”

Estandarización de modelo de perfusión renal en ratones

“…El riñón juega un papel importante en la eliminación de toxinas y productos metabólicos de la sangre, así como también sobre la regulación de los metabolitos en el organismo. Si bien es cierto que la realización de investigación sobre la función renal es esencial para comprender el manejo y eliminación de metabolitos, suele ser complicada al utilizar animales completos debido a la influencia metabólica de los tejidos extrarrenales [2]; [3]. En este sentido, la técnica del riñón aislado y perfundido ofrece la ventaja de trabajar sobre el riñón permitiendo al investigador modificar las variables experimentales de manera controlada.…”