O51
ROBUST INDUCTION OF HEMEOXYGENASE-1 EXPRESSION DOES NOT
AUGMENT TUBULAR REGENERATION IN 20-MONTH-OLD MICE FOLLOWING
ACUTE KIDNEY INJURY
Peng Ding, David Ferenbach, Gary Borthwick, Spike Clay, David Kluth and Jeremy Hughes
MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of
Edinburgh
Introduction: Elderly individuals are more prone to acute kidney injury (AKI). Our previous
work indicated that ‘middle-aged’ 12-month old female FVB/nj mice developed more severe
AKI compared to young 8-week old mice following renal ischaemia-reperfusion injury (IRI).
Compared to young mice, 12-month old mice exhibited reduced medullary upregulation of the
anti-inflammatory enzyme hemeoxygenase-1 (HO-1) whilst pre-treatment of 12-month old mice
with the potent HO-1 inducer hemearginate (HA) strongly upregulated HO-1 and afforded
marked protection from AKI (KI 2011). We extended this work and demonstrated that pretreatment with HA also protected 20-month-old mice from ischaemic AKI despite the presence
of interstitial fibrosis at baseline (Renal Association 2012).
Objective: This study aimed to determine whether the administration of HA to 20-month-old
mice after the induction of AKI augmented tubular cell regeneration.
Methods: Initial studies involved 20-month-old female FVB/nj mice undergoing a right
nephrectomy (to provide baseline tissue) and IRI of the left kidney induced by clamping the left
renal pedicle for 20 minutes. However this functional AKI model resulted in an excessively
high mortality and we thus opted to leave the right kidney in situ and focus on histological
parameters. Mice received IV HA (30mg/kg) or PBS at days 1 and 4 following the induction of
AKI (4-7 mice/group). Tissue was harvested at day 1 to determine injury severity and day 5 to
assess tubular regeneration. Readouts included HO-1 expression (immunostaining quantified by
computer image analysis), acute tubular necrosis (ATN) score (determined on PAS stained
sections) and tubular cell proliferation (Ki-67 immunostaining). Data is expressed as % total
area stained (HO-1), % of tubules exhibiting cell necrosis (ATN score) and the number of Ki67+ cells per high power field (hpf). Data is expressed as mean±SE.
Results: In the absence of HA treatment, 20-month-old mice exhibited minimal HO-1
expression at day 1 (cortex 0.9±0.4% total area, medulla 0.2±0.1%) or day 5 (cortex 0.3±0.1%
total area, medulla 0.2±0.1%). HA administration at days 1 and 4 following IRI induced
dramatic HO-1 expression at day 5 (cortex 10.2±3.2% total area, medulla 4.7±1.7%). The ATN
score at day 1 was 40±17% indicating significant injury. The ATN score fell to 12±8% at day 5
in the PBS treated group indicating significant tubular repair. However, HA treated mice
exhibited a comparable ATN score of 12±6% at day 5. Limited tubular cell proliferation was
evident at day 1 (Cortex 4.5±0.8, Medulla 6.3±3.6 Ki67+ cells/hpf). Although there was a trend
towards increased tubular cell proliferation in HA treated mice in both the renal cortex and
medulla this did not reach statistical significance (Cortex 25±7.9 vs 38±8.4 Ki67+ cells/hpf; PBS
vs HA; p>0.05 and Medulla 34±13.6 vs 46±14.6 Ki67+ cells/hpf; PBS vs HA; p>0.05).
Conclusion: 20-month old mice exhibit very limited HO-1 expression at days 1 or 5 following
AKI consistent with our previous work. Treatment with HA at days 1 and 4 following injury
strongly upregulated HO-1 expression in the renal cortex and medulla. The ATN score
exhibited a comparable reduction between days 1 and 5 in HA and PBS treated groups. HA
administration and subsequent HO-1 induction was associated with a trend to increased tubular
cell proliferation but this was not statistically significant. Although the pretreatment of aged
mice with HA is markedly renoprotective, the administration of HA after injury is established
does not significantly promote tubular regeneration.