Centhaquine Restores Renal Blood Flow and Protects Tissue Damage After Hemorrhagic Shock and Renal Ischemia

Abstract

Background: Centhaquine (CQ) (Lyfaquin^®) is in late stage clinical development as a safe and effective first-in-class resuscitative agent for hemorrhagic shock patients (NCT02408731, NCT04056065, and NCT04045327). Acute kidney injury (AKI) is known to be associated with hemorrhagic shock. Hence, effect of CQ on protection of kidneys from damage due to hemorrhagic shock was investigated. Methods: To assess effect of CQ on AKI in shock, we created a rat model with hemorrhagic shock and AKI. Renal arteries were clamped and de-clamped to induce AKI like ischemia/reperfusion model and hemorrhage was carried out by withdrawing blood for 30 min. Rats were resuscitated with CQ (0.02 mg/kg) for 10 min. MAP, heart rate (HR), and renal blood flow (RBF) were monitored for 120 min. Results: CQ produced a significant improvement in RBF compared to vehicle (p< 0.003) even though MAP and HR was similar in CQ and vehicle groups. Blood lactate level was lower (p = 0.0064) in CQ than vehicle at 120 min post-resuscitation. Histopathological analysis of tissues indicated greater renal damage in vehicle than CQ. Western blots showed higher HIF-1α (p = 0.0152) and lower NGAL (p = 0.01626) levels in CQ vs vehicle. Immunofluorescence in the kidney cortex and medulla showed significantly higher (p< 0.045) expression of HIF-1α and lower expression of Bax (p< 0.044) in CQ. Expression of PHD 3 (p< 0.0001) was higher, while the expression of Cytochrome C (p = 0.01429) was lower in the cortex of CQ than vehicle. Conclusion: Results show CQ (Lyfaquin^®) increased renal blood flow, augmented hypoxia response, decreased tissue damage and apoptosis following hemorrhagic shock induced AKI, and may be explored to prevent/treat AKI. Translational Statement: Centhaquine (CQ) is safe for human use and currently in late stage clinical development as a first-in-class resuscitative agent to treat hemorrhagic shock. In the current study, we have explored a novel role of CQ in protection from hemorrhagic shock induced AKI, indicating its potential to treat/prevent AKI.

Keywords: acute kidney injury; centhaquine; hemorrhage; hypoxia inducible factors; resuscitation; shock.

FIGURE 1

Schematic representation of in vivo experiment in rats involving hypovolemic shock, acute kidney injury with kidney ischemia/reperfusion (I/R) and resuscitation with CQ. Procedure from anesthesia to the end of the in vivo experiment is shown with brief description of each step and time intervals of some important steps. B.G.–indicates a small volume blood collection for analyzing blood gas components.

FIGURE 2

Effect of CQ on kidney blood flow in a rat model of kidney ischemia/reperfusion with hypovolemic shock. (A), 1 and 2, representative images of blood flow in kidneys of vehicle 1) and CQ 2). Upper panel in 1 and 2 shows kidney images obtained from PeriCam PSI blood flow imaging system. Red represents highest blood flow and blue represents the lowest. The lower panels in 1 and 2 show the blood flow graphs–Blue line indicates blood flow in complete imaged area, Black line indicates blood flow in the kidney. N = 7 for vehicle and N = 8 for CQ. (B), a graphical representation of kidney blood flow at different time points (1–baseline, 2–after hemorrhage, 3–after de-clamping renal arteries, 4–0 min post-perfusion, 5–30 min post-perfusion, 6–90 min post-perfusion and 7–120 min post-perfusion). Data represents mean ± SEM values. (C), the graph represents percent change in blood lactate levels in CQ and vehicle rats at different time points–after hemorrhage (0 min of resuscitation), at 30 and 120 min of resuscitation. Baseline is indicated with dotted lines. Data represents mean ± SEM values. N = 7 for vehicle and N = 8 for CQ. Statistical analysis–unpaired t-tests.

FIGURE 3

Effect of CQ on cardiovascular activities, HR and MAP. (A, B), a graphical representation of mean HR (A) and MAP (B) at different time points (1–baseline, 2–after hemorrhage, 3–after de-clamping renal arteries, 4–0 min post-perfusion, 5–30 min post perfusion, 6–90 min post-perfusion and 7–120 min post-perfusion). Data represents mean ± SEM values. N = 7 for vehicle and N = 8 for CQ. Statistical analysis–unpaired t-tests.

FIGURE 4

Effect of CQ on expression of hypoxia responsive factors in kidneys. A and B, representative western blots of HIF-1α (A) and HIF-1β (B) and their respective GAPDH (loading control). (C, D), graphical representation of HIF-1α/GAPDH (C) and HIF-1β/GAPDH (D) normalized data. The error bar represents mean ± SEM. N = 4. Black vertical lines in A and B show that the bands were cropped from the same blot (full blots are provided as Supplementary Figures S1, S2). Statistical analysis Ordinary One Way ANOVA and Fisher’s test. E and F, representative images of immunofluorescence of HIF-1α (red) and HIF-1β (green) in cortex (E) and medulla (F) in sham, vehicle (control) and CQ. (G–J), graphs of mean fluorescence intensity of HIF-1α in cortex (G) and medulla (H), and of HIF-1β in cortex (I) and medulla (J). N = 4. Nuclei in all immunofluorescence microscopy images were stained with DAPI (blue). Statistical analysis- Ordinary One Way ANOVA and Fisher’s test.

FIGURE 5

Expression of hypoxia responsive factor, PHD 3 in kidney cortex. (A), representative images of immunofluorescence of PHD 3 (red) in cortex (A) in sham, vehicle (control) and CQ. (B), a graph of mean fluorescence intensity of PHD 3 in cortex. N = 4. Nuclei in all immunofluorescence microscopy images were stained with DAPI (blue). Statistical analysis Ordinary One Way ANOVA and Fisher’s test.

FIGURE 6

Effect of CQ on kidney tissue damage. (A), representative microscopy images of H and E stained kidney tissue sections from sham, saline and CQ treated rats. Some of the dilated cortical tubules are marked with “black arrows”, vacuoles (clear spaces) in tubules are shown by “blue arrow heads”, a highly vacuolated tubule is “encircled with blue line”, necrotic cells are indicated by “thin black arrows” and tubules with hyaline casts are shown with “white arrows”. Bar scale = 50 µm. (B), a graph showing pathological damage score of the kidney tissues in sham, vehicle and CQ treated rats, Calculated on the basis of scores of tubular dilatation (C), hyaline casts (D) and tubular necrosis or degeneration (E). Error bars represent mean ± SEM. N = 4. Statistical analysis- Ordinary One Way ANOVA and Fisher’s test.

FIGURE 7

Effect of CQ on expression of kidney damage markers. (A), representative western blots of acute kidney damage marker, NGAL and GAPDH (loading control). Black vertical lines in A show that the bands were cropped from the same blot (full blots are provided as Supplementary Figure S3). (B), graphical representation of NGAL/GAPDH normalized data. The error bar represents mean ± SEM. N = 4. (C, D), representative images of immunofluorescence of apoptotic proteins, Bax (red) and Cytochrome C (green) in cortex (C) and medulla (D) in sham, vehicle (control) and CQ. (E–H), graphs of mean fluorescence intensity of Bax in cortex (E) and medulla (F), and of Cytochrome C in cortex (G) and medulla (H). N = 4. Nuclei in all immunofluorescence microscopy images were stained with DAPI (blue). Statistical analysis- Ordinary One Way ANOVA and Fisher’s test.

FIGURE 8

Effect of CQ on mitochondrial DNA in kidneys. (A), representative in situ PCR images vehicle and CQ treated rat kidney tissues after 120 min of resuscitation. Probe represents the negative control (background fluorescence) of the in situ PCR reaction. Red fluorescence indicates amplified MT-ATP8 DNA in mitochondria. Image magnification 200x. (B), fluorescence intensity graph of MT-ATP8 DNA. N = 4. Values are expressed as mean ± SEM. Statistical analysis- Ordinary One Way ANOVA and Fisher’s test.