Effects Of Contralateral Nephrectomy Timing And Ischemic Conditions On Kidney Fibrosis After Unilateral Kidney Ischemia-reperfusion Injury Ⅲ

Discussion 

Animal models of kidney IRI, particularly the uIRI model with delayed contralateral nephrectomy, are commonly used to investigate the AKI-CKD transition, but the modeling conditions vary widely among different research [8,10,12,17]. In our study, we assessed the effects of timing of contralateral nephrectomy after uIRI, core body temperatures during ischemia, and kidney ischemic durations on the post-AKI fibrotic outcome. The results showed that a longer interval between contralateral nephrectomy and uIRI, higher ischemic body temperature, or longer ischemic duration, caused more severe kidney injury and fibrosis when the other two variables were fixed.

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We first observed the effect of time intervals between contralateral nephrectomy and uIRI on the kidney injury. The time intervals between contralateral nephrectomy and uIRI in previous reports varied widely. In some cases, kidney fibrosis of the ischemic kidney was not obvious at 42 days after uIRI when contralateral nephrectomy was conducted 3 days after uIRI [21], and the fibrotic outcome could be seen at 56 days after uIRI when contralateral nephrectomy was conducted 7 days after uIRI [10]. Significant fibrotic changes of the ischemic kidney happened at 28 days after uIRI when contralateral nephrectomy was conducted 8 days after uIRI [12], while some researchers found this result only 11 days after uIRI when contralateral kidney was removed 10 days after uIRI [8]. Moreover, some researchers even observed kidney fibrosis at 98 days after uIRI when contralateral kidney was resected at 14 days after uIRI [15]. However, few researchers have investigated the effect of different time intervals between contralateral nephrectomy and uIRI on renal fibrosis. Based on the results reported in the literature and our pre-experiment, we chose 7 days, 10 days and 14 days after uIRI to remove contralateral kidney respectively, with fixed ischemic duration and core body temperature of ischemia during uIRI. The results showed that the survival rate decreased, and the degree of kidney injury, kidney histopathological damage, and kidney fibrosis worsened with the extension of the interval between the two operations. This suggested that the time interval between contralateral nephrectomy and uIRI significantly affected the outcome of the kidney. As far as our current results are concerned, the 3 days’ difference in the time intervals resulted in a significant difference in the severity of AKI progression to CKD. Kidney fibrosis after AKI became more severe with later contralateral nephrectomy. The possible reason for this is that kidney function after IRI is mainly retained by the compensatory work of the contralateral intact kidney, and the blood supply of the injured kidney decreases over time and leads to fibrosis [11,12]. If we remove the contralateral kidney at this point, the function of the injured kidney would not be compensated. The earlier the contralateral intact kidney is removed, the better the compensation effect for the injury will be. On the contrary, when the contralateral nephrectomy is conducted later, the more severe kidney fibrosis will be. Consistent with the previous study [7], uIRI is more likely to induce fibrosis in the injured kidney if the contralateral kidney is preserved, whereas the removal of the intact kidney is conducive to the long-term repair of the IRI kidney [21].

We also found core body temperature during kidney ischemia had an important impact on kidney injury. There are cold and warm kidney IRI. Conducting kidney IRI at 32  C or below is called the cold one, which is commonly used in kidney transplantation studies [2]. We used the warm ischemia model to investigate AKICKD transition, and our pre-experiment showed no obvious fibrotic changes happened when we controlled core body temperature during ischemia at 33  C, 34  C or 35  C. However, most mice could not tolerate high ischemic temperature and died during surgery when it was higher than 39  C. As a result, we chose 36-38  C for the ischemic temperature in our study. We observed the effect of core body temperature at 36  C, 36.5  C, 37  C, 37.5  C and 38  C on kidney injury with a fixed ischemic duration of 24 min and removal of the contralateral kidney 14 days after uIRI. The results showed that higher core body temperature during kidney ischemia resulted in a lower survival rate, more pronounced deterioration in kidney function and more severe kidney fibrosis. Similar to previous reports [2], the 1  C difference in core body temperature led to statistically significant changes in kidney function and the fibrotic outcome mentioned above. We found that a 0.5  C difference in core body temperature during ischemia did not result in a significant difference in fibrosis severity. Consistent with the need to control core body temperature fluctuations within 0.5  C during ischemia among the groups, which was highlighted by only a few studies [22]. These results provided strong evidence for the temperature control range for IRI modeling. Our group previously also found that if the core temperature was too low during ischemia, the kidneys did not have significant fibrosis manifestations even if the ischemia duration was long enough (results not shown), which also suggested the importance of strict control of ischemic temperature. However, few papers published to date have mentioned core temperature during kidney ischemia, Skrypnyk et al. [12] fixed mice on a water bath system at 38  C without mentioning the core body temperature during ischemia. Colombaro et al. [14] and Li et al. [16] did not mention the ischemic temperature in their studies, indicating that the effect of ischemic temperature on modeling has not been adequately emphasized.

Finally, we investigated the effect of the ischemic duration on CKD progression after AKI. The ischemic duration described in earlier studies ranges from 15 min [15] to 45 min [10]. When the ischemic duration was less than 21 min in our pre-experiment, no significant fibrotic changes were found. When the ischemic duration was longer than 30 min, all mice died in 24 h after contralateral nephrectomy. Therefore, in our study, the ischemic duration was set at 21–30 min. Ischemic durations were set at 21 min, 24 min, 27 min and 30 min respectively, with the core body temperature at 37  C and the time interval between contralateral nephrotomy and uIRI of 14 days. As described in the results, all mice in the 27-minute and 30 min groups died within 7 days after contralateral nephrectomy, indicating that the injury was severe and exceeded the survival threshold of mice. Fortunately, the effect of ischemic time on kidney injury has now been noted by other research and our results were consistent with the study by Wei et al. [15]. Kidney ischemic duration above 27 min with strict control of the core body temperature at 37  C during ischemia resulted in fatal injury, and 21 min and 24 min were appropriate ischemic times. Regarding the ischemia times and their corresponding degrees of kidney injury, our results clearly differ from parts of previous studies. In the study of Guan et al., the longest ischemia time was 45 min [10]. According to Li et al., the ischemia time of 35 min induced moderate to severe injury [16]. However, the ischemia time of 30 min in most research caused moderate to severe injury [2,8,12,17,23]. The reason for the above differences may be due to the lack of strict control of core body temperature during ischemia. As far as our current results are concerned, 3 min’ difference in ischemic duration made a significant difference in the severity of AKI 

In summary, our results showed that although the uIRI model with delayed contralateral nephrectomy resulted in kidney function impairment, histopathological changes, and fibrotic outcome in most cases, which meant AKI-CKD transition, the interval between contralateral nephrectomy and uIRI, core body temperature during ischemia, and ischemic duration significantly affected the survival rate, kidney function, and kidney fibrosis, i.e., the severity of CKD. Therefore, controlling experimental conditions strictly is very important for the stability and consistency of the animal model, which is the basis for completing later studies and should be of great importance to researchers. Our study also provided a reference for other researchers to successfully establish AKI-CKD models.

The survival rate in our uIRI model with delayed contralateral nephrectomy was somewhat lower than other studies under the same conditions [15]. It’s probably because the difference in laboratory conditions, operators’ techniques and surgical proficiency, experimental environments with different temperatures, temperature control equipment, and micro-arterial clampers, which might affect the results. So a constant environment temperature, and trained and skilled operators are also important to establish a consistent and reliable ischemic AKI model.

Our study also had limitations. Limited by the huge workload, we only studied the effects of contralateral nephrectomy performed on day 7, day 10 and day 14 after uIRI and did not observe the effect of more subtle time interval differences (e.g., day 8 and day 9 after uIRI) when investigating the effect of the time points of contralateral nephrectomy after uIRI on kidney injury. The ischemic durations were only set at 21 min, 24 min, 27 min and 30 min when studying the effect of different ischemic durations on kidney fibrosis, as described in our pre-experimental results and previous reports. The effect of smaller differences in ischemic time (e.g., 22 min, 23 min) on fibrotic outcome was not observed. Therefore, based on the results currently available, we can only draw the conclusion that 3 days’ difference in time interval or 3 min’ difference in time length has a meaningful impact on kidney injury, while further research is still needed to determine the impact of shorter time intervals. A higher core body temperature during ischemia or longer ischemic duration caused higher mortalities when other conditions were fixed. The number of surviving mice was relatively small when the core temperature was above 37  C or when the ischemic time was longer than 24 min, which may have an impact on the later data analysis. But the results obtained were still statistically significant due to the small differences within the groups, so no additional mice were added.

In conclusion, in the uIRI model with delayed contralateral nephrectomy, kidney fibrosis after AKI becomes more severe as the interval between contralateral nephrectomy and uIRI is prolonged, the core body temperature rises during ischemia and the ischemic duration increases. The experimental conditions should be strictly controlled in future studies to set a universal standard for the AKI-CKD animal model.

References 

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