Effects Of Contralateral Nephrectomy Timing And Ischemic Conditions On Kidney Fibrosis After Unilateral Kidney Ischemia-Reperfusion Injury Ⅱ
Oct 27, 2023
Results
Protocol 1: effects of the interval between contralateral nephrectomy and uIRI on AKICKD transition Survival rate declined with the extension of the interval between contralateral nephrectomy and IRI
As mentioned in experiment 1, the ischemic duration was fixed at 24 min with a core body temperature of 37 C in uIRI. We observe the effect of contralateral nephrectomy timing after uIRI on the survival of mice. Data showed that in the day 7 group (contralateral intact kidney was resected 7 days after uIRI), all eight mice survived. For the day 10 group, one mouse died the day after contralateral nephrectomy, with a survival rate of 87.5%. For the day 14 group, one mouse died on the first day after the contralateral nephrectomy, and two mice died two days after the contralateral nephrectomy, with a survival rate of 62.5%. These results indicated that the survival rate of mice gradually decreased with the extension of contralateral nephrectomy when the other two influencing factors were fixed (Figure 2(A)).
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Kidney function declined with the extension of the interval between contralateral nephrectomy and uIRI
Compared with the sham control, SCr and BUN were significantly higher in all mice after contralateral nephrectomy (p < 0.05), indicating that they all suffered from kidney damage. Moreover, SCr and BUN levels increased with the extension of the interval between contralateral nephrectomy and uIRI (p < 0.05) (Figure 2(B,C))
Kidney histological injury worsened with the extension of the interval between contralateral nephrectomy and uIRI
HE staining demonstrated that the kidney tissue of the sham group was structurally normal at 8 weeks after uIRI. In all uIRI groups, several post-AKI pathological changes were detected in the kidney (Figure 3(A)), including different degrees of tubular epithelial cell vacuolar degeneration, brush border detachment, focal disintegration necrosis, dilatation of some tubular lumen, and kidney tubular epithelial cell edema with variable morphology. Besides, some glomeruli were wrinkled and sclerotic, with mesangial cells hyperplasia and dilated Bowman’s capsule. Inflammatory cell infiltration was also seen in the kidney interstitial. The tubular and glomerular pathological injury scores of the injured kidney were higher than the sham (p < 0.05), indicating that uIRI with contralateral nephrectomy significantly damaged the kidney. The kidney pathological injury scores increased with the extension of contralateral nephrectomy (p < 0.05) (Figure 3(B,C)). These findings indicate that kidney pathological damage gradually increased with the extension of contralateral nephrectomy after uIRI.
Figure 2. Effects of time intervals between contralateral nephrectomy and unilateral ischemia-reperfusion injury (uIRI) on survival rate and kidney function. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different time points (day 7, day 10, and day 14) with the ischemic duration fixed at 24 min with the core body temperature fixed at 37 C during kidney ischemia. (A): The survival of the mice in each group was scored and presented as a percentage. (B and C): Kidney function was estimated by serum creatinine (SCr), and blood urea nitrogen (BUN). n ¼ 5–8/group. p < 0.05.
Figure 3. Effects of time intervals between contralateral nephrectomy and unilateral ischemia-reperfusion injury (uIRI) on kidney histological injury. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different time points (day 7, day 10, and day14) with the ischemic duration fixed at 24 min with the core body temperature fixed at 37 C during kidney ischemia. Kidney histological injury was determined by hematoxylin-eosin (HE) staining. (A): Representative HE staining of the injured kidney, which showed the typical histopathological features of post-AKI pathological changes, including different degrees of tubular epithelial cell vacuolar degeneration, brush border detachment, focal disintegration necrosis, dilatation of some tubular lumen (#), kidney tubular epithelial cell edema with variable morphology, and inflammatory cell infiltration (%). Some glomeruli were wrinkled and sclerotic, with mesangial cell hyperplasia (&) and dilated Bowman’s capsule (magnification, 400 ). (B): Semi-quantitation of tubular injury. (C): Semi-quantitation of glomerular injury. n ¼ 5–8/group. p < 0.05.
Kidney fibrosis worsened with the extension of the interval between contralateral nephrectomy and uIRI Kidney weight.
The transformation from AKI to CKD is typically characterized by kidney fibrosis [19] and a decrease in kidney weight is a macroscopic indicator of kidney fibrosis [2]. So we measured kidney weight (kidney weight calibrated by body weight) to preliminarily investigate the effect of time intervals between uIRI and delayed contralateral nephrectomy on the AKI-CKD transition. Compared with the sham group, all uIRI groups showed a significant reduction in kidney weight (p < 0.05), indicating that all mice in the uIRI groups had kidney fibrosis. The difference in kidney weight changed significantly when the difference in the time interval between contralateral nephrectomy and uIRI was 3 days, and the kidney weight decreased gradually as the interval time increased (Figure 4(A)).
Masson’s trichrome stain.
Masson’s trichrome staining was performed to further observe the effect of time intervals between contralateral nephrectomy and uIRI on kidney fibrosis. No significant kidney interstitial fibrosis was seen in the sham group while varying degrees of fibrotic changes in the kidney of all uIRI groups were observed at 8 weeks after uIRI (Figure 4(B)). The semi-quantitative analysis of the fibrotic area showed that the fibrotic area of the kidney in the uIRI groups was significantly increased compared with that in the sham group (p < 0.05), indicating that the AKICKD transition model was successfully performed. Moreover, with the delay of the contralateral nephrectomy, the kidney fibrosis became increasingly severe (p < 0.05), indicating that the kidney fibrosis degree changed significantly when the interval between the contralateral nephrectomy and uIRI was 3 days. With the prolonged time interval between contralateral nephrectomy and uIRI, the kidney fibrosis degree became more aggravated (Figure 4(C)).
Figure 4. Effects of time intervals between contralateral nephrectomy and unilateral ischemia-reperfusion injury (uIRI) on kidney fibrosis. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different time points (day 7, day 10, and day 14) with the ischemic duration fixed at 24 min with the core body temperature fixed at 37 C during kidney ischemia. Kidney fibrosis was assessed by kidney weight, Masson’s trichrome staining, and fibronectin (FN), collagen I (COL-I), and a-smooth muscle actin (a-SMA) expressions. (A): Kidney weight to body weight. (B): Representative Masson’s trichrome staining of the injured kidney, which showed the typical of fibrotic changes (%) (magnification, 200 ). (C): Semi-quantitation of the fibrotic area detected by Masson’s trichrome staining. (D): Quantitative RT-PCR analyses for FN expression in the kidney. (E): Quantitative RT-PCR analyses for COL-I expression in the kidney. (F): Representative western blot images for FN, COL-I and a-SMA. (G): Bar chart showing the FN protein expression normalized by b-actin. (H): Bar chart showing the COL-I protein expression normalized by b-actin. I: Bar chart showing the a-SMA protein expression normalized by b-actin. n ¼ 5–8/group. p < 0.05.
Extracellular matrix (ECM) accumulation.
Kidney interstitial fibrosis is characterized by the deposition of ECM [2], such as FN and COL-I, in the interstitial area of the kidney. Western blot and RT-PCR analysis showed that the expressions of FN and COL-I in the kidney were significantly increased in all uIRI groups compared with the sham group (p < 0.05), indicating that all the kidneys underwent fibrotic changes. The expression levels of FN and COL-I in the kidney were higher with the delay of the contralateral nephrectomy (p < 0.05) (Figure 4(D–H)).
Myofibroblasts in the kidney.
Kidney fibrosis is characterized by myofibroblast accumulation, and subsequent ECM production [20]. a-SMA was used to detect myofibroblast quantity at 8 weeks after IRI. Western blot analysis showed that the expression of a-SMA in the kidney tissues significantly increased in all uIRI groups compared with the sham group (p < 0.05), indicating that myofibroblasts were pronouncedly more in the kidney of all injured mice. The expression of a-SMA was higher with the delay of contralateral kidney resection (p < 0.05) (Figure 4(F,I)). These results showed that the myofibroblasts in the uIRI kidney tissue changed significantly in the 3 days between uIRI and the contralateral nephrectomy and that the myofibroblasts gradually increased with longer intervals.
Protocol 2: effects of core body temperatures during ischemia on AKI-CKD transition
Survival rate declined with the increase in core body temperature during ischemia
As mentioned in experiment 2, in order to investigate the effect of core body temperatures during ischemia on AKI-CKD transition, the ischemia duration was fixed at 24 min and the contralateral intact kidney was removed 14 days after uIRI. Data showed that in the 36 C group (the core body temperature of mice during ischemia was controlled at 36 C), one mouse died the first day after the contralateral nephrectomy, and the survival rate was 87.5%. For the 36.5 C group, two mice died in the first 2 days after the contralateral nephrectomy, with 75% of survival rate. Compared with the 36.5 C group, there was one more mouse died on the second day after contralateral nephrectomy with a survival rate of 62.5% in the 37 C group. In the 37.5 C group, except for the three mice that died in the first 2 days after contralateral nephrectomy, one mouse died 3 days after contralateral nephrectomy, with a survival rate of 50%. In addition to what happened in the 37.5 C group, one mouse died 5 days after the contralateral nephrectomy in the 38 C group, and the survival rate was 37.5%. These results indicated that the survival rate of mice gradually decreased with the increase in core body temperature during ischemia (Figure 5(A)).
Kidney function declined with the increase in core body temperature during ischemia
Compared with the sham group, uIRI resulted in a significant increase in SCr and BUN in all uIRI groups (p < 0.05), suggesting that all mice in uIRI groups developed kidney impairment after contralateral nephrectomy. Comparisons between two groups with adjacent temperatures showed that the SCr and BUN of mice increased with an increase of 0.5 C in ischemic body temperature, but there was no statistical difference (p > 0.05), suggesting there were less pronounced changes in the kidney function with a core temperature difference in 0.5 C during ischemia. However, SCr and BUN significantly increased when core body temperature increased by 1 C or above during ischemia (p < 0.05), suggesting that the kidney function significantly changed with a 1 C difference in core body temperature during ischemia. Kidney function declined more significantly with increasing ischemic body temperature (Figure 5(B,C)).
Kidney tissue injury worsened with increased core body temperature during ischemia
According to HE staining, the kidney tissue structure of the mice in the sham group was normal. IRI-induced pathological changes were observed in all user groups, such as different degrees of tubular epithelial cell vacuolar degeneration, brush border detachment, focal disintegration necrosis, dilatation of some tubular lumen, and kidney tubular epithelial cell edema with variable morphology, as well as wrinkled and sclerotic glomeruli, mesangial cells hyperplasia and dilated Bowman’s capsule, inflammatory cell infiltration in kidney interstitial (Figure 6(A)).
The pathological injury scores of kidney tubules and glomeruli showed that compared with the sham group, the pathological injury of the kidney in all uIRI groups was significantly more severe (p < 0.05), suggesting that obvious pathological injury of the kidney appeared in all uIRI groups and that the AKI-CKD model was successfully developed. Comparison between two groups with adjacent temperatures showed that there was an increase in kidney pathological injury scores with a 0.5 C increase in core body temperature during ischemia, but it had no statistical significance (p > 0.05), while kidney pathological injury significantly increased with an increase in core body temperature of 1 C and above during ischemia (p < 0.05) (Figure 6(B,C)). These results indicated that the histopathological damage of the kidney changed significantly with a 1 C difference in core body temperature during ischemia and gradually increased with the increased core body temperature during ischemia.
Kidney fibrosis worsens with the increase in core body temperature during ischemia Kidney weight.
Compared with the sham group, uIRI caused a significant decrease in kidney weight in all uIRI groups, and mice with different ischemic body temperatures developed different degrees of kidney fibrosis. Comparisons between two groups with adjacent temperatures showed that the decrease in kidney weight had no significant difference when the core body temperature increased by 0.5 C during ischemia (p > 0.05), suggesting that the difference of 0.5 C in core body temperature during ischemia had no significant effect on the change of kidney weight. When core body temperature during the ischemic period had a 1 C difference, the kidney weight changed significantly (p < 0.05), and it gradually decreased as the ischemic body temperature increased (Figure 7(A)).
Figure 7. Effects of core body temperatures during ischemia on kidney fibrosis in mice with uIRI and delayed contralateral nephrectomy. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted under different core body temperatures (36 C, 36.5 C, 37 C, 37.5 C, and 38 C) with the ischemic time of the left kidney fixed at 24 min, and the right kidney was removed 14 days after the left kidney IRI. Kidney fibrosis was assessed by kidney weight, Masson’s trichrome staining, and fibronectin (FN), collagen I (COL-I) and a-smooth muscle actin (a-SMA) expressions. (A): Kidney weight to body weight. (B): Representative Masson’s trichrome staining of the injured kidney, which showed the typical of fibrotic changes (%) (magnification, 200 ). (C): Semi-quantitation of the fibrotic area detected by Masson’s trichrome staining. (D): Quantitative RT-PCR analyses for FN expression in the kidney. (E): Quantitative RT-PCR analyses for COL-I expression in the kidney. (F): Representative western blot images for FN, COL-I and a-SMA. (G): Bar chart showing the FN protein expression normalized by b-actin. (H): Bar chart showing the COL-I protein expression normalized by b-actin. (I): Bar chart showing the a-SMA protein expression normalized by b-actin. n ¼ 3–7/group. p < 0.05. NS: non-significant.
Masson’s trichrome stain.
At 8 weeks after uIRI, Masson’s trichrome staining results showed that compared with the sham group, the fibrotic area of kidney tissues in all uIRI groups was significantly increased (p < 0.05), indicating that fibrotic outcome occurred in all uIRI groups. Comparisons between two groups with adjacent temperatures showed that the area of kidney fibrosis increased but with no statistical significance when the core body temperature increased by 0.5 C during ischemia (p > 0.05), suggesting that the difference in 0.5 C in core body temperature during ischemia had no significant effect on kidney fibrosis. For every 1 C increase in core body temperature during ischemia, the kidney interstitial fibrosis area significantly increased (p < 0.05), indicating that the 1 C increase in core body temperature during ischemia had a significant impact on the kidney interstitial fibrosis, and kidney fibrosis gradually worsened with the increase in core body temperature during ischemia (Figure 7(B,C)).
ECM in kidney tissue. The data showed that compared with the sham group, the expressions of FN and COL-I in kidney tissues of all uIRI groups were significantly increased (p < 0.05), which was consistent with the results of Masson’s staining. Comparisons between two groups with adjacent temperatures showed that FN and COL-I expressions in the kidney tissues increased by 0.5 C increase in core body temperature during ischemia, but there was no statistically significant difference (p > 0.05), indicating that a 0.5 C difference in core body temperature during ischemia did not have a significant effect on ECM deposition, while the expression of FN and COL-I increased significantly with a 1 C increase in core body temperature during ischemia (p < 0.05), and they had a more pronounced increase with increasing body temperature. In conclusion, the ECM deposition or the fibrotic outcome in the kidney tissues increased with the higher core body temperature during ischemia (Figure 7(D–H)).
Myofibroblasts in kidney tissue.
Compared with the sham group, the expressions of a-SMA in kidney tissue of all uIRI groups were significantly increased (p < 0.05), suggesting that the myofibroblasts were significantly increased in all uIRI groups. Comparisons between two groups with adjacent temperatures showed that the expression of a-SMA in the kidney increased when core body temperature increased by 0.5 C during ischemia, but there was no significant difference (p > 0.05), suggesting that the difference of 0.5 C in ischemic body temperature had no significant effect on the changes of kidney myofibroblasts. Moreover, the expression of a-SMA in the kidney tissues significantly increased with a 1 C increase in ischemic body temperature, and the a-SMA expression increase was more significant with increasing body temperature (p < 0.05), suggesting that myofibroblasts changed significantly when the difference in core body temperature during ischemia was 1 C or more. Along with the increase in core body temperature during ischemia, myofibroblasts in kidney tissues were gradually increased (Figure 7(F,I)).
Protocol 3: effects of ischemic durations on the AKI-CKD transition
The survival rate decreased as the ischemic duration increased As mentioned in experiment 3, in order to investigate the effect of ischemic durations on the AKI-CKD transition, the ischemic body temperature was fixed at 37 C during uIRI, and the contralateral kidney was removed 14 days after uIRI. The results showed that in the 21 min group (the ischemic duration was 21 min), one mouse died 1d and 2d after removal of the contralateral kidney respectively, with a survival rate of 75%, and one more mouse died 2 days after contralateral nephrectomy in the 24 min group, with a survival rate of 62.5%. In the 27-minute group, four mice, two mice, one mouse, and one mouse died on 1 day, 3 days, 5 days, and 7 days, respectively, after contralateral nephrectomy, and the survival rate was 0%. Similarly, five mice, two mice, and one mouse died on 1 day, 2 days, and 3 days after contralateral nephrectomy in the 30-minute group, and the survival rate was 0%. This indicated that the survival rate decreased as the ischemic duration increased, as all mice in the 27 min group and the 30-min group died 7 days and 3 days, respectively, after contralateral nephrectomy (Figure 8(A)).
Kidney function decreased with longer ischemia duration
Data showed that compared with the sham group, both the 21 min group and 24 min groups showed a significant increase in SCr and BUN (p < 0.05), indicating kidney function declined. Compared with the 21 min group, the 24 min group showed a more pronounced increase in SCr and BUN (p < 0.05), which indicated that the kidney function changed significantly with a 3 min difference in ischemic duration and a more obvious decrease in kidney function could be seen with the longer ischemic duration (Figure 8(B,C)).
Figure 8. Effects of ischemic durations on survival rate and kidney function in mice with uIRI and delayed contralateral nephrectomy. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different ischemic durations (21 min, 24 min, 27 min, and 30 min) with the core body temperature fixed at 37 C during kidney ischemia, and the right kidney was removed 14 days after the left kidney IRI. (A): The survival of the mice in each group was scored and presented as a percentage. (B and C): Kidney function was estimated by serum creatinine (SCr), and blood urea nitrogen (BUN). n ¼ 5–6/ group. p < 0.05.
Kidney histopathological damage worsened with longer ischemia duration
According to HE staining, the kidney tissue structure of the mice in the sham group was normal. Compared with the sham group, the 21 min group and 24 min group had pathological changes in varying degrees, including tubular epithelial cell vacuolar degeneration, brush border detachment, focal disintegration necrosis, dilatation of some tubular lumen, kidney tubular epithelial cell edema with variable morphology, wrinkled and sclerotic glomeruli, mesangial cell hyperplasia and dilated Bowman’s capsule, and inflammatory cell infiltration in the interstitial (Figure 9(A)). Pathological injury scores of kidney tubules and glomeruli were significantly higher in both the 21 min and 24 min groups compared with the sham group (p < 0.05), indicating that significant kidney pathological injury developed in both user groups. Furthermore, the 24 min group had higher kidney pathological injury scores and more severe pathological injury compared with the 21 min group (p < 0.05) (Figure 9(B,C)), suggesting that kidney pathological kidney injury had significant changes with a 3 min difference in ischemic duration. The longer the duration of ischemia, the more severe the pathological damage of the kidney
Kidney fibrosis worsened with longer ischemia duration Kidney weight.
Compared with the sham group, both the 21 min and 24 min groups showed a significant reduction in kidney weight (p < 0.05), and the 24 min group had a more prominent kidney weight decrease compared with the 21 min group (p < 0.05). These indicated that a 3-minute difference in ischemic duration resulted in a significant change in kidney weight, and a more obvious reduction in kidney weight can be seen with longer ischemic duration (Figure 10(A)).
Masson’s trichrome stain. Masson’s trichrome staining and semi-quantification of fibrotic area analysis showed that there was no significant interstitial fibrotic change in the sham group, while different degrees of fibrotic changes were seen in the kidney interstitial in both the 21 min and 24 min groups (p < 0.05). Compared with the 21 min group, the 24 min group had a higher fibrotic degree of kidney tissue (p < 0.05), indicating that kidney fibrosis significantly changed with a 3 min difference in ischemic duration and that more severe kidney fibrosis would become with longer ischemic duration (Figure 10(B,C)).
ECM in kidney tissue. Western blot and RT-PCR analysis showed that compared with the sham group, the expressions of FN and COL-I significantly increased in both the 21 min and 24 min groups (p < 0.05), indicating that IRI-induced ECM increased significantly in uIRI groups, and the kidney underwent significant fibrotic progression. Compared to the 21 min group, the expressions of FN and COL-I were more pronounced in the kidney tissue in the 24 min group (p < 0.05), indicating that the ECM deposition in the kidney tissues changed significantly with a 3 min difference in the ischemic duration, and ECM deposition increased with the prolonged ischemic duration (Figure 10(D–H)).
Myofibroblasts in kidney tissue. Western blot analysis showed that compared with the sham group, the expression of a-SMA of the kidney in the 21 min and 24 min groups significantly increased (p < 0.05), indicating that myofibroblasts had a significant increase in the kidney in the uIRI groups, and significant fibrotic progression occurred. The expression of a-SMA significantly increased with the 3 min increase in ischemic duration (p < 0.05), and more myofibroblasts were found with prolonged ischemic duration (Figure 10(F,I)).
Figure 9. Effects of ischemic durations on kidney histological injury in mice with uIRI and delayed contralateral nephrectomy. The left kidney was subjected to IRI on day 0, and right kidney resection was conducted at different ischemic durations (21 min, 24 min, 27 min, and 30 min) with the core body temperature fixed at 37 C during kidney ischemia, and the right kidney was removed 14 days after the left kidney IRI. Kidney histological injury was determined by hematoxylin-eosin (HE) staining. (A): Representative HE staining of the injured kidney, which showed the typical histopathological features of post-AKI pathological changes, including different degrees of tubular epithelial cell vacuolar degeneration, brush border detachment, focal disintegration necrosis, dilatation of some tubular lumen (#), kidney tubular epithelial cell edema with variable morphology, and inflammatory cell infiltration (%). Some glomeruli were wrinkled and sclerotic, with mesangial cell hyperplasia (&) and dilated Bowman’s capsule (magnification, 400 ). (B): Semi-quantitation of tubulointerstitial injury. (C): Semi-quantitation of glomerular injury. n ¼ 5–6/group. p < 0.05.
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