Attenuation of Skeletal Muscle and Renal Injury to the Lower Limb following Ischemia-Reperfusion Using mPTP Inhibitor NIM-811.

INTRODUCTION: Operation on the infrarenal aorta and large arteries of the lower extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury, by inhibiting mitochondrial permeability transition pores (mPTP). OBJECTIVES: Our aim was to reduce damages in the skeletal muscle and the kidney after IR of the lower limb with NIM-811. MATERIALS AND METHODS: Wistar rats underwent 180 minutes of bilateral lower limb ischemia and 240 minutes of reperfusion. Four animal groups were formed called Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and surgery). Serum, urine and histological samples were taken at the end of reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations, laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were performed. Renal peroxynitrite concentration, serum TNF-alpha and IL-6 levels were measured. RESULTS: Less significant histopathological changes were observable in the NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK: p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be significantly higher (p = 0.001) and renal function parameters were significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group in comparison to the IR group. Serum TNF-alpha and IL-6 levels were significantly lower (TNF-alpha: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group than in the IR group. CONCLUSION: NIM-811 could have the potential of reducing rhabdomyolysis and impairment of the kidney after lower limb IR injury

Levosimendan: a cardiovascular drug to prevent liver ischemia-reperfusion injury?

INTRODUCTION: Temporary occlusion of the hepatoduodenal ligament leads to an ischemic-reperfusion (IR) injury in the liver. Levosimendan is a new positive inotropic drug, which induces preconditioning-like adaptive mechanisms due to opening of mitochondrial KATP channels. The aim of this study was to examine possible protective effects of levosimendan in a rat model of hepatic IR injury. MATERIAL AND METHODS: Levosimendan was administered to male Wistar rats 1 hour (early pretreatment) or 24 hours (late pretreatment) before induction of 60-minute segmental liver ischemia. Microcirculation of the liver was monitored by laser Doppler flowmeter. After 24 hours of reperfusion, liver and blood samples were taken for histology, immuno- and enzyme-histochemistry (TUNEL; PARP; NADH-TR) as well as for laboratory tests. Furthermore, liver antioxidant status was assessed and HSP72 expression was measured. RESULTS: In both groups pretreated with levosimendan, significantly better hepatic microcirculation was observed compared to respective IR control groups. Similarly, histological damage was also reduced after levosimendan administration. This observation was supported by significantly lower activities of serum ALT (pearly = 0.02; plate = 0.005), AST (pearly = 0.02; plate = 0.004) and less DNA damage by TUNEL test (pearly = 0.05; plate = 0.034) and PAR positivity (pearly = 0.02; plate = 0.04). Levosimendan pretreatment resulted in significant improvement of liver redox homeostasis. Further, significantly better mitochondrial function was detected in animals receiving late pretreatment. Finally, HSP72 expression was increased by IR injury, but it was not affected by levosimendan pretreatment. CONCLUSION: Levosimendan pretreatment can be hepatoprotective and it could be useful before extensive liver resection

Serum TNF-α and peroxynitrite concentration in the kidney.

<p>(<b>A</b>)<b>:</b> Serum TNF-α concentration was significantly elevated in the IR group, compared with the Sham and NIM-Sham groups. A significantly lower level was detected in the NIM-IR group compared with the IR group, leading to the conclusion that there may be a lower extent of systemic inflammation (# p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.01 vs. IR; ‡ p>0.05 vs. Sham; & p>0.05 vs. NIM-Sham; pg/ml – pictogram/mililitre). (<b>B</b>)<b>:</b> A significantly elevated peroxynitrite concentration can be observed in homogenized kidney samples in the IR group, compared with the Sham and NIM-Sham groups. In the NIM-IR group, a significantly lower value was measured compared with the IR group (# p<0.01 vs. Sham; † p<0.05 vs. NIM-Sham; § p<0.01 vs. IR).</p

Renal function and calculated renal parameters.

<p>(<b>A</b>)<b>:</b> Based on the measured urine output during reperfusion, diuresis is significantly lower in the IR group compared to the Sham and NIM-Sham groups. In the NIM-IR group, urine output is significantly higher compared with the IR group (# p<0.01 vs. Sham; † p<0.05 vs. NIM-Sham; § p<0.05 vs. IR; ‡ p<0.01 vs. Sham; & p>0.05 vs. NIM-Sham). (<b>B</b>)<b>:</b> Serum creatinine level of the IR group is significantly elevated compared with the Sham and NIM-Sham groups. The serum creatinine level of the NIM-IR group is significantly lower compared with the IR group (# p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.05 vs. IR). (<b>C</b>)<b>:</b> Serum BUN/creatinine ratio showed a lower value compared with the Sham and NIM-Sham groups. In the NIM-IR group, a significantly increased value was detectable than in the IR group, however the difference was not significant. (<b>D</b>)<b>:</b> The fractional Na⁺-excretion is significantly increased in the IR group compared with the Sham and NIM-Sham groups. There is a significantly lower value in the NIM-IR group than in the IR group (# p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.01 vs. IR).</p

Parameters of muscle injury, muscle fiber viability and muscle wet content.

<p>(<b>A–B</b>)<b>:</b> Serum creatine-kinase (CK) and lactate-dehydrogenase (LDH) concentrations were significantly elevated in the IR group compared with the Sham and NIM-Sham groups. Significantly lower value was detectable in the NIM-IR group compared with the IR group, leading to the conclusion that there may be a lower extent of muscle necrosis (CK: # p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.01 vs. IR; ‡ p<0.01 vs. Sham; & p<0.01 vs. NIM-Sham; LDH: # p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.01 vs. IR; ‡ p<0.01 vs. Sham; & p<0.01 vs. NIM-Sham; U/l – Unit/liter). (<b>C</b>)<b>:</b> Muscle fiber viability was assessed by NADH-tetrazolium staining and was expressed as percentage of viability measured in untreated controls (%). In the IR group there was a significant decline in viability compared with the Sham and NIM-Sham groups. Significantly higher value was found in the NIM-IR group, compared with the IR group (# p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.01 vs. IR; ‡ p<0.01 vs. Sham; & p<0.05 vs. NIM-Sham). (<b>D</b>)<b>:</b> Wet/Dry ratio is suitable to determine the amount of interstitial edema. The wet content of the skeletal muscle tissue was significantly lower in the NIM-IR group compared with the IR group (# p<0.01 vs. Sham; † p<0.01 vs. NIM-Sham; § p<0.05 vs. IR; ‡ p<0.05 vs. Sham; & p<0.05 vs. NIM-Sham).</p

Muscle and kidney histopathology (hematoxillin-eosin (HE) stain, light microscopy).

<p>(<b>A–B</b>)<b>:</b> An almost physiologically histological picture is observable in the Sham (A) and NIM-Sham (B) groups, with intact muscle fibers and normal wide interstitial spaces. (<b>C</b>)<b>:</b> In the IR group segmental necrosis (circle), disintegrated myofilaments and thicker interstitial spaces (arrow) are detectable. (<b>D</b>)<b>:</b> NIM-IR group shows intact muscle fibers (circle) with no expanded interstitial spaces (arrow), similar to the normal structure. (<b>E–F</b>)<b>:</b> Sham and NIM-Sham groups show the normal structure of kidney cortical tissue. Tubules have normal appearance. (<b>G</b>)<b>:</b> In the IR group massive injury can be detected with loss of cell integrity and intracellular vacuolization (arrows). (<b>H</b>)<b>:</b> In the NIM-IR group cell necrosis is less severe (arrow) and the tubular integrity remained intact (circle), an almost normal picture can be seen.</p

Systemic hemodinamycs and microcirculation of skeletal muscle of lower limb.

<p>(<b>A</b>)<b>:</b> Microcirculation of the lower limb skeletal muscle was monitored by laser Doppler flowmeter (LDF). Data are shown as percentage of baseline flow before ischemia (%). In the IR group a significant decline can be observed compared with the Sham and NIM-Sham groups after the onset of reperfusion. Microcirculation became stabilized at a significantly higher level in the NIM-IR group than in the IR group (# p<0.05 vs. Sham; † p<0.05 vs. NIM-Sham; § p<0.05 vs. IR). (<b>B</b>)<b>:</b> Mean arterial pressure (MAP) was registered during blood pressure monitoring. MAP of the Sham and NIM-Sham groups remained constant during the entire experimental period, whereas values of both the IR and NIM-IR groups decreased at the beginning of reperfusion. MAP of the NIM-IR group was significantly higher after the onset of reperfusion as compared with the IR group (# p<0.05 vs. Sham; † p<0.05 vs. NIM-Sham; § p<0.05 vs. IR).</p

Laboratory measurements, haemodynamics data and wet/dry ratios.

<p>LDH: lactate dehydrogenase; CK: creatine kinase; W/D: wet/dry ratio; LDF RA: Laser Doppler flowmeter, reperfusion area; TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; MAP: mean arterial pressure; BUN: blood urea nitrogen; FENa: fractional Na⁺ excretion.</p

Representative H&E-stained liver sections.

<p>In the control groups (A: “early” control; D: “late” control) mild tissue injury and sinusoidal dilatation were observed. In the IR groups (B: “early” IR; E: “late” IR) confluent necrotic areas were detected accompanied by significant leukocyte infiltration and tissue hemorrhage. The levosimendan pretreated groups (C: “early” levosimendan pretreatment; F: “late” levosimendan pretreatment) were characterized by focal necrosis associated with milder tissue hemorrhage and less severe leukocyte infiltration.</p

Immunohistochemical analysis.

<p>S: sham-operated; C_E: “early” control; IR_E: “early” ischemia-reperfusion; L_E: “early” levosimendan pretreated; C_L: “late” control; IR_L: “late” ischemia-reperfusion; L_L: “late” levosimendan pretreated.</p