Development of a Porcine Slaughterhouse Kidney Perfusion Model
Round 1
Reviewer 1 Report
The authors report perfusion of kidneys retrieved from slaughtered pigs as a potential alternative to laboratory animals. This is an interesting option, although, as correctly pointed out, the limitations are intrinsic to the lack of a proper controlled and reproducible experiment conducted in the lab. Nevertheless, it was possible to assess different warm ischemic times and the levels of ischemic injury using cold storage and hypothermic machine perfusion with or without oxygenation, to be followed by normothermic machine perfusion for 4 hours. The cost-effectiveness of using slaughtered pigs instead of laboratory animals is notable and the information provided is worthy.
I would suggest few comments:
1) Methods: It is unclear what is the rationale behind the two different NKP strategies, and I would recommend to briefly explain the pros and cons of both preservation solutions. The use of aminoacids is later explained in the discussion, but it should be introduced earlier on for a better understanding.
2) Results: Line 226- Please correct "De" with "The".
Reasonably, L-WIT has a detrimental effect on IRI and thus on urine production, but this seems to be happening after 2 hours of NKP, too. Interestingly, kidneys in group HMP0% and HMP100% have a stable clearance over time, as a result of the good urine output. Did the authors think of taking biopsy samples and if not, what was the reason?
3) Discussion: I agree on the fact that HMP is superior in DCD kidneys for DGF rates and eGFR; what is the authors' take in a subsequential usage of HMP and NKP? Ideally HMP100% should prepare the kidneys for the lowest IRI and NKP should not last longer than 2 hours, as per the results presented herein, but I think the readership would benefit of a brief summary (even in the form of a table) of the advantages of the presented strategies.
Author Response
Reviewer 1
The authors report perfusion of kidneys retrieved from slaughtered pigs as a potential alternative to laboratory animals. This is an interesting option, although, as correctly pointed out, the limitations are intrinsic to the lack of a proper controlled and reproducible experiment conducted in the lab. Nevertheless, it was possible to assess different warm ischemic times and the levels of ischemic injury using cold storage and hypothermic machine perfusion with or without oxygenation, to be followed by normothermic machine perfusion for 4 hours. The cost-effectiveness of using slaughtered pigs instead of laboratory animals is notable and the information provided is worthy.
I would suggest few comments:
1) Methods: It is unclear what is the rationale behind the two different NKP strategies, and I would recommend to briefly explain the pros and cons of both preservation solutions. The use of aminoacids is later explained in the discussion, but it should be introduced earlier on for a better understanding.
Response: Thank you for this comment and agree that it indeed need some additional explanation. We added text in the method section on this matter (line 89 – 94).
2) Results: Line 226- Please correct "De" with "The".
Response: Thank you for mentioning. We have adjusted this.
Reasonably, L-WIT has a detrimental effect on IRI and thus on urine production, but this seems to be happening after 2 hours of NKP, too. Interestingly, kidneys in group HMP0% and HMP100% have a stable clearance over time, as a result of the good urine output. Did the authors think of taking biopsy samples and if not, what was the reason?
Response: We see indeed that urine production changes over time and that it differs between treatment groups. The study that we describe in this paper was setup as first pilot experiments in which we did not know what to expect on forehand. We had no experience yet with porcine kidney NMP nor slaughterhouse material. Because of the extensive work in regard to collecting kidneys, preserve and subsequently reperfuse them, we first focused on the practical work in terms of doing the experiment rather than collecting samples. That is why we also only had n=2 or 3 inclusions in every group and this is also why we did not collected biopsies nor sufficient perfusate/urine samples to do extensive analyses. That is why I cannot answer what happens after two hours of NMP in terms of diminished urine production. After finalizing the protocols based on these pilot experiments we now do take a lot of different samples for many purposes. Many experiments have been published in different (transplantation) journals. See reference: 27 -34 in the manuscript. We added an additional paragraph on this matter in the discussion (line 371 – 377).
3) Discussion: I agree on the fact that HMP is superior in DCD kidneys for DGF rates and eGFR; what is the authors' take in a subsequential usage of HMP and NKP? Ideally HMP100% should prepare the kidneys for the lowest IRI and NKP should not last longer than 2 hours, as per the results presented herein, but I think the readership would benefit of a brief summary (even in the form of a table) of the advantages of the presented strategies.
Response: Thank you for this very important comment and agree that such a summary would be valuable. These are already available in several review papers on normothermic machine perfusion of kidneys. We have recently published one ourselves that could shed some light on this matter (doi:10.1097/TP.0000000000003817.). However, in my opinion this subject is outside of the scope of the work presented here. Especially since the proof presented here lacks statistical proof and also the number of analyses are minimal. In this study we are describing the beginning of a NKP model that now has proven its usability in addressing many different research questions regarding normothermic kidney perfusion. But still lacks longterm follow-up and transplantation data that really can be useful in the decision-making regarding the right clinical strategy. We just recently started with the inclusions of human kidneys in our center but still have not transplanted. Normothermic machine perfusion of kidneys is complicated and many questions regarding interpretation of renal function and damage emerge when trying to understand what is happening. In my opinion we are still at the beginning of the era of normothermic kidney perfusion. The reason for addressing the references regarding DGF and eGFR in HMP preserved human DCD kidneys is to show that we were indeed able to see differences in renal function and injury during NKP and that was the first success in using NKP to assess renal function. Which helped in the process of believing that we could indeed see differences in kidney function with the help of normothermic perfusion in slaughterhouse-derived kidneys. But based on the data represented in this manuscript I would not suggest clinical NKP strategies.
Reviewer 2 Report
Dear Authors,
The research work is very well written and justified through suitable evaluation parameters and references. Though it contains sufficient novelty to be accepted for publication, but still minor modifications and suggestions are recommended to improve the quality of the manuscript.
1. I suggest numbering the formulas and improving their readability (line: 166, 167, 168, 175, 176, 177, 187, 188).
2. I suggest describing methods for determining markers of kidney damage.
Conclusion
Overall, the manuscript could be considered as scientific rigor and seems able to add in existing scientific knowledge. Therefore, I recommend the Acceptance of the manuscript with minor modifications on above mentioned suggestions and comments.
Author Response
Reviewer 2
Dear Authors,
The research work is very well written and justified through suitable evaluation parameters and references. Though it contains sufficient novelty to be accepted for publication, but still minor modifications and suggestions are recommended to improve the quality of the manuscript.
- I suggest numbering the formulas and improving their readability (line: 166, 167, 168, 175, 176, 177, 187, 188).
Response: Agree, readability of formulas can be improved. We inserted a table with all formulas used (table 3, line 172).
I suggest describing methods for determining markers of kidney damage.
Response: Added this to the material section (line: 178 – 182).
Conclusion
Overall, the manuscript could be considered as scientific rigor and seems able to add in existing scientific knowledge. Therefore, I recommend the Acceptance of the manuscript with minor modifications on above mentioned suggestions and comments.
Round 2
Reviewer 1 Report
Thank you for revising the manuscript
