2.3 Assessment of renal injury
Mouse plasma creatinine levels were measured with ultra performance
liquid chromatography coupled with mass spectrometry (UPLC-MS). Ice-cold
acetonitrile (ACN) containing 50 µM creatinine-d3 (internal standard)
was added to plasma samples for protein precipitation (3:1 ratio).
Following the addition of ACN, all samples were vortexed, incubated at
-20°C for 20 minutes, and centrifuged at 14000 g for 10 minutes. The
resulting supernatant was isolated and used for creatinine
quantification. Chromatographic separation was achieved using a Waters
ACQUITY UPLC BEH Amide column (1.7 µm particle size, 2.1 mm x 100 mm)
maintained at 45°C in a Waters ACQUITY UPLC I-Class system. The mobile
phase consisted of A) water + 0.1% formic acid and B) ACN + 0.1%
formic acid. Mobile phase flow rate was set to 0.45 mL/min and the
following mobile phase gradient was used: 0–0.5 mins, 90% B; 0.5–1
min, 90–60% B; 1–1.5 min, 60% B; 1.5–1.51 min, 60–90%B; 1.51–2.5
min, 90% B. Creatinine was measured in positive electrospray ionization
mode using a Waters Xevo G2-S QTof mass spectrometer. The creatinine
signal was normalized to the creatinine-d3 internal standard and
concentrations were quantified using a creatinine standard curve ranging
from 0.78125 – 100 µM creatinine, using TargetLynx version 4.1
software.
Kidney samples were fixed in 10% formalin. All tissue processing,
sectioning, and staining were performed by the Department of Pathology
and Laboratory Medicine at Western University, Canada. Kidneys were
dehydrated, embedded in paraffin, and cut into 5 µm sections using a
microtome. Sections were then mounted on slides and subsequently stained
with hematoxylin and eosin (H&E). The extent of renal injury was
assessed using light microscopy by a trained pathologist who was blinded
to the treatment conditions. Tubular injury was graded on an arbitrary
scale of 0 – 5 (0, none; 1, <11%; 2, 11% to 25%; 3, 26%
to 45%; 4, 46% to 75%; 5, >75%), based on the degree of
observed proximal tubule dilation, brush-border damage, proteinaceous
casts, interstitial widening, and necrosis.