Evaluation Of Renal Haemodynamics In Rat Models Of Cold Ischemia Reperfusion Injury And Acute Renal Allograft Rejection With Contrast-enhanced Ultrasound

Part one Evaluation of renal haemodynamics in rat models of cold ischemia-reperfusion injury with CEUSObjective: We quantified the regional renal blood flow by real-time contrast-enhanced ultrasound (CEUS) and analyzed the correlations of the parameters between renal haemodynamics and serum renal function or the degree of acute tubular necrosis. The aim of this study was to investigate the value of CEUS in the diagnosis of acute tubular necrosis (ATN) after cold ischemia-reperfusion injury.Methods: There were total 15 male Sprague-Dawley rats recruited into CEUS examinations in each group, namely sham-surgery group(SSG) , which were just removed their right kidneys, and renal cold ischemia - reperfusion injury group (CIRI). Every five rats were performed CEUS examinations on days one, three and seven, respectively, after surgery in each group. After adequate anesthesia by intraperitoneal injection of 40 mg.kg-1 sodium pentobarbital, contrast agent SonoVue was infused continuously via jugular vein at a rate of 0.8ml.kg-1.min-1 and the left kidney CEUS was performed with the commercial ultrasound system IU22 (Philips Medical Systems, Amsterdam,Netherlands) with pulse inversion harmonic imaging technology in this study. All data were digitally recorded and analyzed off-line with QLAB advanced quantification software (Philips Medical Systems, Amsterdam,Netherlands) by two independent radiologists. ROI (region of interest) was positioned on the renal cortex, out medulla and inner medulla opposite to the renal hilum, respectively. The plot of renal signal intensity versus time after FLASH was fitted to an one minus exponential function: y=A (1-e-βt) automaticly, where A is the peak signal intensity,βindicating signal velocity, and the product of A andβrepresenting as the regional renal blood flow of the ROI. After CEUS examination, blood samples were obtained from the inferior vena cava for renal function examination before removing the left kidney. Rats were sacrificed by abdominal aorta dissection. Renal tissue fixed by formalin and sectioned for HE staining later. Tubular necrosis score (TNS) was calculated according to the pathological findings under light microscope. Correlations between renal hemodynamics parameters and serum renal function parameters or TNS of CIRI rats were analyzed statistically.Results: The value of parameters A was no significant difference compared at different time points within the same group of SSG or CIRI and compared between SSG and CIRI groups at the same time points after the surgeries (P>0.05). Renal blood flow in all regions were lower in SSG group on day one compared with those on days three and seven after the surgeries, but there was no significant difference (P>0.05) except the value ofβwith significant difference (P<0.05). Just as the same, no significant differences were found while comparing the serum parameters of renal function and tubular necrosis score of pathological evaluation on days one, three and seven after the surgeries (P>0.05). The renal blood flow in the cortex, the outer medulla and the inner medulla were significant lower on day one compared with those on days three and seven after the surgeries in CIRI group and renal cortical blood flow was significant lower compared with those of SSG group on day one after surgeries (P<0.01). The reduction of renal blood flow was mainly due to the decreaced value ofβ. The regional blood flow in cortex, outer medulla and inner medulla of CIRI group were still significant lower compared with those of SSG group(P<0.01) on days three after the surgeries, but the renal cortical blood flow rose progressively while renal outer and inner medullary blood flow remained relatively unchanged. The regional blood flow in cortex, outer medulla and inner medulla of CIRI group further increased and returned to normal compared with those of SSG group on days seven after the surgeries (no significant difference, P>0.05). Accordingly, the values of the parameters of serum renal function and pathological score of tubular necrosis were significantly higher than those of SSG group on day one (P<0.01), and was gradually decreased on days three but still significantly higher than those of SSG group on day one (P<0.01) and returned to normal on days seven with no significant difference between SSG group and CIRI group (P>0.05). The values of parameters of serum renal function and tubular necrosis score were no significant correlation with the parameters of A in all renal regions (P>0.05), while they have negative correlation with the values of parametersβand A×βin all renal regions, especially those in cortical region. Conclusion: CEUS can display the different perfusion pattern and quantify the blood flow in renal cortex, outer medulla and inner medulla in a real-time status, and confirmed that renal blood flow in cortex and medulla were significantly decreased in rats after cold ischemia - reperfusion injury. At the same time, renal cortical blood flow was preferentially increased than those in renal medullary regions during the recovery phase and renal blood flow varied with the values of signal velocity (β) after renal cold ischemia-reperfusion injury. Bothβand A×βwere sensitive parameters in detecting the microcirculation disturbances after renal cold ischemia-reperfusion injury.Part two Evaluation of renal haemodynamics of acute renal allograft rejection in rat models with CEUSObjective: We investigated the changes of regional renal haemodynamics of acute renal allograft rejection (AR) consecutively and comprehensively in rat models in the early stages by CEUS with the pathological evaluation. The aim of this study was to investigate the regularities of intrarenal hemodynamics in different renal regions during AR occurence and the value of CEUS in the diagnosis of AR in the early stages. Methods: Eighteen male rats of orthotopic kidney transplantation were collected in both isograft group (Lewis→Lewis, n=9) and allogrft group (F344→Lewis, n=9) into CEUS examinations. Every three rats were performed CEUS examinations on days one, three and seven, respectively. The methods of CEUS examination and quantitative test of serum renal function were just as the same as those mentioned in part one. Renal tissue fixed by formalin and sectioned later for conventional HE staining, special staining (masson trichrome, periodic acid Schiff and methenamine silver staining) and ultrastructural observation under transmission electron microscope. The diagnoses of acute renal allograft rejection were made according to Banff 2007 criteria. Correlations between renal hemodynamics parameters and serum renal function parameters or Banff classification of acute rejection were analyzed statistically. Results: Prominent reduction of renal blood flow in cortex, outer medulla and inner medulla were seen on day one after transplantation in both isograft and allograft groups. Comparisons of parameter A,βas well as A×βshowed no significant differences between isograft and allograft groups (P>0.05). Renal cortical blood flow began to improve on days three and increased significantly on days seven after transplantation in isograft group. Significant differences were found while comparing the value of parametersβand A×βin days seven with those in days one and three (P<0.01), but no significant differences while comparing the value of parameter A among days one, three and seven in isograft group (P>0.05). Renal blood flow in cortex, outer medulla and inner medulla further decreased from days three by presenting as the reduction of the value of parameters A,βand A×βand showed significant difference comparing the values on days seven with those on days one and three (P<0.01) in allograft group. Comparison with those in isograft group, significant reduction was found with the value of parameters A,βas well as A×βin cortical and outer medulla and parameter A×βin inner medulla on days three (P<0.05) which exacerbated on days seven (P<0.01) in allograft group. No significant differences were seen of the values of SCr,BUN in both isograft and allograft groups on day one after transplantations (P>0.05). Statistical significant differences were seen of data SCr and BUN from days three to seven with the gradually decreased in isograft group but obvious increased in allograft group (P<0.01). Histological evaluation with HE and special staining showed different degree of tubular necrosis on days one and three in isograft group and the renal tissue returned to normal on days seven with no obvious inflammatory cell infiltration and vascularities. But inflammatory cell infiltrations were found in renal interstitial and were graded as acute cellular rejectionⅠA -ⅠB according to Banff 2007 criteria on days seven in allograft group. On days seven, more lymphocytic infiltrations were seen in renal interstitial, renal tubules and small arteries with the hypertrophy of Bowman capsule and vascular wall. Their pathological diagnoses were acute cellular rejection ofⅡ-Ⅲclassification with no evidence of humoral rejection in allograft group. The ultrastructure of renal tissue under the electron microscope showed no obvious abnormalities in isograft group. But the ultrastructure of renal tissue in allograft group showed larger clearance among tubular epithelial cells, mild hyperplasia of the capillary endothelial cells, inflammatory cell infiltration in renal interstitial and the hypertrophy of vascular smooth muscle on days three and exacerbated on days seven with markedly thickening of Bowman capsules and arterial wall after transplantations. All the values of parameters A,β,A×βin cortex, outer medulla and inner medulla showed negative correlations with those of the parameters of serum renal function and Banff classification (P<0.05), especially the value of parameter A. Conclusion: Renal blood flow in cortex and medulla regions decreased significantly in acute renal allograft rejection in rat models after transplantations. Parameters A,βand A×βwere sensitive in detecting hemodynamic abnormalities in acute renal rejection. Consecutive and dynamic monitoring the changes of renal hemodynamics can provide early diagnosis of acute rejection after transplantation.Part three Comparative study of renal haemodynamic abnormalities of acute tubular necrosis and acute renal allograft rejection in rat modelsObjective: The aim of this study was to investigate the different renal haemodynamic abnormalities of acute tubular necrosis after ischemia-reperfusion injuries and acute renal allograft rejection in the early stages and provide possible basic theory for differential diagnosis of ATN and AR after renal transplantion.Methods: Every five rats in both SSG and CIRI groups with every three rats in both isograft and allograft groups were performed CEUS examinations on days one, three and seven, respectively. The methods of CEUS examination and quantitative test of serum renal function were just as the same as those used in experimental one and two. Analysis of variance was used in comparison among each groups.Results: Renal cortical blood flow decreased in CIRI, Isograft and Allograft groups compared with that in SSG group on day one after sugeries, which presented as the reduction of the value of parametersβ,A×β(P<0.05) with no significant difference of parameter A (P>0.05). All the value of every parameters of renal haemodynamics in cortex showed no significant difference among CIRI, Isograft and Allograft groups on days one after the surgery (P>0.05). Renal outer medullary blood flow also decreased in Isograft and Allograft groups compared with those in CIRI and SSG group on day one after sugeries by presenting as the reduction of the value of parameters A×β(P<0.05). Renal cortical blood flow began to improve in CIRI and Isograft group on days three and increased significantly on days seven with no significant difference compared with those in SSG group (P>0.05). Renal blood flow in cortex, outer medulla and inner medulla were exacerbated and further decreased by presenting as the reduction of the value of parameters A,βand A×βand showed significant difference compared with those in the other three groups on days three (P<0.05) and on days seven (P<0.01). Accordingly, the values of the parameters of serum renal function in CIRI, Isograft and Allograft groups were significantly higher than those of SSG group on day one (P<0.01) with no significant difference among CIRI, Isograft and Allograft groups (P>0.05). The values of of the parameters of serum renal function in CIRI and Isograft groups decreased from days three and returned to normal on days seven with no signifiacant difference compared to those in SSG group (P>0.05). But The values of of the parameters of serum renal function in allogrfat group exacerbated and further increased significantly compared to those in the other three groups on days three and seven (P<0.01).Conclusion: ATN occurred in the early phase after the surgeries and renal blood flow in cortex and medulla were significantly reduced which were due to the changes of blood flow velocity. AR occurred later than that of ATN after renal transplantation with more severe reduction of renal blood flow in cortex and medulla which was due to both reduction of blood flow velocity and vascular volume. Our studies identified that CEUS examination may be the useful methods in differential diagnosis between AR and ATN in the early stage.