2047
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 pre-treatment with HA also protected 20-month-old mice from ischaemic AKI despite the presence ofinterstitial fibrosis at baseline (Renal Association 2012).
Objective:This study aimed to determine whetherthe 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 injuryseverity 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 Ki-67+ 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 dramaticHO-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±7.9vs38±8.4 Ki67+ cells/hpf; PBS vs HA; p>0.05 and Medulla 34±13.6vs46±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 4following 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.