Figure 1.
Muscle and kidney histopathology (hematoxillin-eosin (HE) stain, light microscopy).
(A–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. (C): In the IR group segmental necrosis (circle), disintegrated myofilaments and thicker interstitial spaces (arrow) are detectable. (D): NIM-IR group shows intact muscle fibers (circle) with no expanded interstitial spaces (arrow), similar to the normal structure. (E–F): Sham and NIM-Sham groups show the normal structure of kidney cortical tissue. Tubules have normal appearance. (G): In the IR group massive injury can be detected with loss of cell integrity and intracellular vacuolization (arrows). (H): 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.
Figure 2.
Parameters of muscle injury, muscle fiber viability and muscle wet content.
(A–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). (C): 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). (D): 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).
Table 1.
Laboratory measurements, haemodynamics data and wet/dry ratios.
Figure 3.
Systemic hemodinamycs and microcirculation of skeletal muscle of lower limb.
(A): 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): 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).
Figure 4.
Serum TNF-α and peroxynitrite concentration in the kidney.
(A): 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): 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).
Figure 5.
Renal function and calculated renal parameters.
(A): 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): 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). (C): 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. (D): 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).