Even after recovery from acute kidney injury, glomeruli remain vulnerable to further injury by way of interstitial fibrosis. This study is aimed at elucidating the effects of post ischemia-reperfusion (I/R) treatment with trimetazidine on the progression to renal fibrosis as well as short- and intermediate-term aspects. Trimetazidine 3 mg/kg or 0.9% saline was given intraperitoneally once upon reperfusion or daily thereafter for 5 d or 8 w. Renal histologic changes and related signaling proteins were assessed. After 24 h, post I/R treatment with trimetazidine significantly reduced serum blood urea nitrogen and creatinine levels and tubular injury accompanied with upregulation of hypoxia-inducible factor- (HIF-) 1
Acute kidney injury (AKI) is one of the most frequent complication in hospitalized patients, which is believed to be the consequence of ischemia-reperfusion (I/R) injury [
As an innate response to renal I/R injury and AKI, activation of hypoxia-inducible factor- (HIF-) 1
Trimetazidine (TMZ, 1-[2,3,4-trimethoxybenzyl]piperazine dihydrochloride), an anti-ischemic agent in current use, has been shown to reduce myocardial and renal I/R injury at both the cellular and mitochondrial levels [
Thus, this study is aimed at elucidating the effects of post I/R treatment with TMZ on the progression to renal fibrosis as well as short- and intermediate-term aspects and at investigating relevant signaling pathways.
All experiments were approved by the committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and were performed conforming to the
Male Sprague-Dawley rats (10–12 w old, 250–300 g) were anesthetized with Rompun (vial Korea, 10 mg/kg, intraperitoneally (ip)) plus Zoletil 50 (Virbac Korea, 30 mg/kg, ip). The rats were intubated with a 16-gauge catheter and artificially ventilated (Harvard Apparatus 683, Holliston, MA) at 30–35 cycles/min. The body temperature was continuously monitored throughout the experiment and maintained around 37°C using a heating pad.
We employed an established rodent model of renal I/R injury having the longest period of ischemia to induce an appropriate degree of renal injury while allowing long-term survival [
The animals were randomly assigned to four groups: (1) sham (
To examine the effect of TMZ on renoprotection, TMZ (3 mg/kg, Sigma, St. Louis, MO) was administered to the TMZ-treated groups while the sham and IRC groups received equivalent amounts of 0.9% saline via ip. The dose of TMZ was chosen based on our pilot study that investigated the optimal dose of TMZ in terms of attenuating apoptotic cell death after renal I/R injury (3 mg/kg versus 1 and 5 mg/kg, data not shown).
Serum samples were obtained at 24 h, 5 d, and 8 w after reperfusion and assayed for BUN and creatinine levels using the picric acid and diacetyl monoxime methods [
Detection of apoptosis on paraffin sections from each group was examined using the terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) [
Paraffin-embedded kidney tissues were cross sectioned (5-6 specimens per group) through the midpoint to measure histologic damage. Periodic acid-Schiff (PAS) staining was performed and the degree of tubular damage was graded on a scale from 1 to 4. Grading and definitions for cell degradation, necrosis, and neutrophil infiltration are listed in Table
Parameters for semiquantitative assessment of histopathology.
Cell degradation | Necrosis | Neutrophil infiltration | |
---|---|---|---|
Grade 1 | No change from normal | Nil | Nil |
Grade 2 | <5% of total field | Some single-cell necrosis | 1–3 cells/field |
Grade 3 | 5–30% of total field | Dispersed focal necrotic tubules | 4–6 cells/field |
Grade 4 | >30% of total field | Confluent necrosis in most tubules | Heavy infiltration |
After processing, equal amounts of protein from each group underwent immunoblot assay as described previously [
All data were expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) or Student’s
Renal I/R injury produced a significant increase in serum BUN and creatinine levels, which were significantly attenuated in the TMZS group at 24 h after reperfusion. The elevated serum BUN and creatinine levels returned to their respective baseline values in all groups at 5 d after renal I/R injury and were at similar levels at 8 w after renal I/R injury without any intergroup differences (Figures
Effects of postischemia-reperfusion (I/R) treatment with TMZ on renal function at different time points. Renal I/R injury produced a significant increase in serum BUN (a) and creatinine levels (b), which were significantly attenuated in the TMZS group at 24 h after reperfusion. The elevated serum BUN and creatinine levels returned to their respective baseline values in all groups at 5 d after renal I/R injury and were at similar levels at 8 w after renal I/R injury without any intergroup differences. TMZ = trimetazidine; BUN = blood urea nitrogen. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (24 h, 5 d, and 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
Compared to the sham groups, I/R injury resulted in significant apoptotic cell death (Figures
Effects of postischemia-reperfusion (I/R) treatment with TMZ on renal tubular apoptotic cell death. Representative histology of renal tubules after TUNEL assay (a) and a percentage of apoptotic cells (apoptotic cells/total cells) (b). Compared to the sham groups, I/R injury resulted in significant apoptotic cell death in the IRC groups at 24 h and 5 d after I/R injury. TMZ significantly attenuated apoptotic cell death compared to the IRC groups at 24 h and 5 d after I/R injury. At 8 w after I/R injury, TUNEL assay of renal tissues demonstrated almost full recovery of histopathologic findings in the IRC and the TMZ-treated groups, without intergroup differences. TMZ = trimetazidine; TUNEL = terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (24 h, 5 d, and 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
Effects of postischemia-reperfusion (I/R) treatment with TMZ on renal tubular cell necrosis. Representative histology of renal tubules after and PAS staining. Compared to the sham groups, I/R injury resulted in significant necrosis in the IRC groups at 24 h and 5 d after I/R injury. TMZ significantly attenuated cell necrosis compared to the IRC groups at 24 h and 5 d after I/R injury. At 8 w after I/R injury, PAS staining of renal tissues demonstrated almost full recovery of histopathologic findings in the IRC and the TMZ-treated groups, without intergroup differences. TMZ = trimetazidine; PAS = periodic acid-Schiff. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (24 h, 5 d, and 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
Renal histopathology assessed with PAS staining.
Group | Cell degradation | Necrosis | Neutrophil infiltration | |
---|---|---|---|---|
24 h | Sham | |||
IRC | 2.6 ± 0.8 |
3.6 ± 0.5 |
1.3 ± 0.5 | |
TMZS | 1.7 ± 0.8 |
1.9 ± 0.7 |
1.1 ± 0.4 | |
5 d | Sham | |||
IRC | 3.1 ± 0.9 |
2.9 ± 0.9 |
1.9 ± 1.2 | |
TMZS | 2.1 ± 0.7 | 1.3 ± 0.5# | 1.1 ± 0.4 | |
TMZD | 2.1 ± 0.7 | 1.6 ± 0.8# | ||
8 w | Sham | |||
IRC | 1.9 ± 0.4 | 1.1 ± 0.4 | ||
TMZS | 1.4 ± 0.5 | |||
TMZD | 1.1 ± 0.4 |
PAS = periodic acid-Schiff; TMZ = trimetazidine. Sham = rats not underwent ischemia-reperfusion; IRC = rats underwent ischemia (45 min)-reperfusion (24 h, 5 d, or 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
Bcl-2 level was significantly decreased, and Bax level was significantly increased in the IRC groups compared to that in the sham groups at 24 h and 5 d after I/R injury (Figures
Effects of postischemia-reperfusion (I/R) treatment with TMZ on the expressions of Bcl-2 and Bax. Western blot analysis (a) and densitometric analysis (b, c) of the Western blot for Bcl-2 and Bax at 24 h, 5 d, and 8 w after I/R injury, respectively. Bcl-2 level was significantly decreased, and Bax level was significantly increased in the IRC groups compared to that in the sham groups at 24 h and 5 d after I/R injury. Bcl-2 level was significantly higher, and Bax level was significantly lower in the TMZ-treated groups than that in the IRC groups. At 8 w after renal I/R injury, Bcl-2 and Bax levels were significantly greater in the IRC and the TMZ-treated groups than those in the sham groups, without any intergroup differences between the IRC and the TMZ-treated groups. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (24 h, 5 d, and 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
Renal I/R injury induced increases in HIF-1
Effects of postischemia-reperfusion (I/R) treatment with TMZ on the expressions of HIF-1
In Masson’s trichrome staining, renal fibrosis was significantly more prominent in the IRC and TMZ-treated groups compared to that in the sham group at 8 w after renal I/R injury, without any intergroup differences between the IRC and TMZ-treated groups (Figures
Effect of postischemia-reperfusion (I/R) treatment with TMZ on renal tubulointerstitial fibrosis. Representative histology (a) of renal tubules after Masson’s trichrome staining and quantitative analysis (b) of tubulointerstitial fibrosis. Renal fibrosis was significantly more prominent in the IRC and TMZ-treated groups compared to that in the sham group at 8 w after renal I/R injury, without any intergroup differences between the IRC and TMZ-treated groups. TMZ = trimetazidine. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 8 w starting upon reperfusion.
Post I/R treatment with TMZ reduced the degree of I/R-induced increases in MMP-2 and MMP-9 levels (Figures
Effects of postischemia-reperfusion (I/R) treatment with TMZ on the expressions of MMPs and TIMPs. Western blot analysis (a) and densitometric analysis (b–e) of the Western blot for MMP-2, -9 and TIMP-1, -2 at 5 d, and 8 w after I/R injury, respectively. Post I/R treatment with TMZ reduced the degree of I/R-induced increases in MMP-2 and MMP-9 levels and further increased the levels of TIMP-1 and TIMP-2 at 5 d after renal I/R injury compared to the IRC group. At 8 w after I/R injury, MMP levels in the TMZ-treated groups were similar to those of the IRC groups, which were all significantly greater than those of the sham groups. TIMP levels in the TMZ-treated groups became also comparable to those of the IRC groups, which were significantly greater than those of the sham groups. TMZ = trimetazidine. Sham = rats not underwent I/R; IRC = rats underwent ischemia (45 min)-reperfusion (5 d or 8 w); TMZS = rats treated with TMZ (3 mg/kg) upon reperfusion; TMZD = rats treated with TMZ (3 mg/kg) once daily for 5 d or 8 w starting upon reperfusion.
In the current study investigating the impact of post I/R treatment with TMZ on AKI and its progression to renal fibrosis against renal I/R injury in a rat model, we observed significant attenuation of renal injury by TMZ at short (24 h) as well as intermediate (5 d) period after renal I/R injury. Contrary to its early effects, however, post I/R treatment with TMZ, either given once or daily for 8 w, could not further suppress the progression to renal fibrosis and expressions of the related signaling pathways compared to those of the IRC group.
AKI is a major risk factor increasing morbidity and mortality in hospitalized patients. Notably, renal deterioration is gradually continued even after complete clinical recovery from AKI [
TMZ is a commonly used antianginal drug with well-known cardioprotective effect and hemodynamic stability [
In the current study, post I/R treatment with TMZ conferred renoprotective effects at 24 h and 5 d after I/R injury. Post I/R treatment with TMZ significantly mitigated apoptotic cell death and necrosis that were associated with inhibition of apoptotic pathways, enhanced expression of HIF-1
Contrary to the protective effects observed in the short- and intermediate-term, however, TMZ could not prevent renal fibrosis at 8 w after renal I/R injury in the present study. At 8 w, all protein expressions including VEGF, MMPs, and TIMPs as well as Bcl-2 and Bax were enhanced compared to those in the sham group. TMZ-induced early modulation of these signal protein expressions disappeared, resulting in comparable expression levels of all measured signal proteins among the IRC and TMZ-treated groups. These results are discordant with the results of previous reports showing that TMZ could reduce fibrosis development after I/R injury in pigs [
A complex interplay of multiple mechanisms contributes to I/R-induced organ damage. In the hypoxic kidney, HIF-1
In the present study, TMZ attenuated expressions of MMPs and enhanced expressions of TIMPs at 5 d after I/R injury. Early after renal I/R injury, the influx of inflammatory mediators together with the degradation of necrotic cells induce upregulation of degradative enzymes in the renal interstitium [
Contrary to our expectations, although a single dose was sufficient to exert early- and intermediate-term renoprotection, daily TMZ treatment did not exert additional effect over a single dose in the current study. Yet, considering that the elimination half-life of TMZ is approximately 6 h [
In conclusion, post I/R treatment with TMZ might allow ischemic kidneys to regain renal function and structure more rapidly compared to nontreated kidneys but not enough to resolute renal fibrosis in long-term aspect. Despite that, the early and intermediate renoprotective effects of TMZ treatment after I/R injury as affirmed by upregulation of HIF-1
Data will be made available on request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Jin Ha Park and Ji Hae Jun contributed equally to this work and are co-first author.
This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2015-0071).