Role of HAO2 in rats with chronic kidney disease by regulating fatty acid metabolic processes in renal tissue

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1. 

   PREreview

   Mar 1, 2024

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10732785.

   In this study, the authors generated a single-cell RNA-seq atlas of blood and kidney in Sprague-Dawley rats with chronic kidney disease. They show a dysregulation of fatty acid metabolism genes that points to a dysfunction in this pathway as a feature of CKD in rats. This supports recent publications that have found similar findings with human and mice samples. In this context, authors performed further experiments based on the identification of the gene HAO2, encoding one of the 2-hydroxyacid oxidase enzymes, as downregulated in CKD samples. They show some experiments that suggest that this gene is involved in the pathogenesis of CKD and its modulation could be a therapeutic target. …

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10732785.

   In this study, the authors generated a single-cell RNA-seq atlas of blood and kidney in Sprague-Dawley rats with chronic kidney disease. They show a dysregulation of fatty acid metabolism genes that points to a dysfunction in this pathway as a feature of CKD in rats. This supports recent publications that have found similar findings with human and mice samples. In this context, authors performed further experiments based on the identification of the gene HAO2, encoding one of the 2-hydroxyacid oxidase enzymes, as downregulated in CKD samples. They show some experiments that suggest that this gene is involved in the pathogenesis of CKD and its modulation could be a therapeutic target. However, there is no detail about how this experiment was done, preventing its evaluation. The authors mention the word "mouse knock out" in the introduction, but the figure suggest knockdown was used. Methods do not reveal at all this information and lack key information about the rats used, and no statement about ethical approval. Overall, I find this paper interesting but without these important details missing in the study, and no validation of the CKD model, the conclusions are not sustained by the data

   Major issues

   • After reading the manuscript, it is not clear to me the relevance of the RNA-seq of blood cells for this study? Why include it together with the kidney RNA-seq to later focus only on kidney cells?

   • A fundamental aspect missing in this study is the validation of the CKD model with the adenine diet. Authors should add robust evidence that these rats developed CKD, otherwise it is difficult to interpret the single-cell RNAseq data.

   • Tone down the HAO2 findings. In my opinion, considering the conclusion on the role of HAO2 is only based on overexpression experiments in vitro, this should be toned down, and reflected in the title. The way I understand the paper, it could be more of a "resource" type of study, providing a single-cell RNA-seq atlas of the rat kidney in CKD

   • When talking about HAO2, Wu et al (PMID 31537650) have shown a pronounced sexual dimorphism of Hao2 expression in the mouse kidney, with a pronounced decrease upon kidney injury that is female-specific. This should be at least discussed in this manuscript, considering it is focusing on this gene. There is no mention of the sex of the rats in the methods section, and this is an important aspect to add, together with the age of these rats.

   • There is no mention of the ethical approval for the animal experimentation performed in this manuscript. This must be added.

   • Another important aspect missing in the methods section is the statistical analysis. For example in comparisons between groups in WB and qPCR data. Authors could add individual data points over the bars to better understand the variability in the data.

   • The authors say that HAO2 is expressed in the tubules based on the IHC data (change the immunofluorescence mentioned in the legends to immunohistochemistry, by the way), and I agree with them. But without showing other renal areas, it cannot be discarded that is expressed in other cells. This nuance should be reflected in the way of describing the results, if no other images are shown. If the authors have data on which proximal tubule segment (S1, S2, S3) HAO2 is expressed, it would be interesting to add in the discussion for example.

   • Oil Red O images in Figure 6B and 7A are not convincing. It is difficult to see the difference with that magnification. I suggest to include an inset with a higher magnification, and include a quantification.

   • Full gels should be provided to analyze the western blot images. If the same loading control was used to normalize the other proteins, this should be clearly stated as well as the reasons to do that.

   • It is crucial to explain in which model the experiments shown in Figure 7 and 8 were performed. The authors mention the word "mouse knock out" in the introduction, but the figure and the discussion text suggest knockdown was used, even they talk about in vitro data? 

   Minor issues

   • I would suggest to replace "model" with "CKD" across the figures, the message would be more direct

   • The overall understanding of the paper would improve if the abbreviations were explained across the manuscript. For someone that is not in the field, it could be difficult to understand some of them. In particular this should be carefully revised in the abstract.

   • I am not sure about the use of "antigen repair" in an IHC protocol. Maybe the authors meant "antigen retrieval"?

   • Please refer to specific equipment across the methods (machine for qPCR, gel imaging instrument…). This is an important aspect for reproducibility.

   • What is the housekeeping gene used in qPCRs and the primer sequences? Please add that information.

   • This could be a personal thing, but the color schema and legend in Fig 1H is quite difficult to follow, as it requires going back and forth to the explanation of the legend to understand the difference between groups. I suggest that the authors find a different way of visualizing the groups to make the figure easier to understand.

   • Graph headings in Figure 5 could be improved so it is easily grasped without having to check the figure legend. For example, 5B and 5C have exactly the same heading, addition of "Control" in 5B, and "Model" to 5C would improve the understanding of the figure.

   • Also in Figure 5, it is confusing for me to read that downregulated pathways in the "Control vs Model" graph correspond to upregulated enriched pathways in the model when mentioned in the text. Although I understand they are the same, it can easily lead to confusion. Please harmonize the way to refer to them.

   • Minor writing thing but also could lead to confusion: In the results section in this sentence: "In the functional enrichment analysis of PT cells, fatty acid metabolic process-related genes were found to be downregulated in the model group, where HAO2 is the gene involved in regulating fatty acid metabolic process." The use of "is the gene" sounds like it is the most important one, or the only one. As the authors know, this is not true, so they should change the writing here to improve the understanding.

   • Scale bar info missing in figure legends of Figure 6.

   • The possible role peroxisomal fatty acid metabolism in this context should be mentioned somewhere, especially when Acox1 expression is measured in this study.

   • If ATP and FFA data are measured in kidney tissue, it should be reported per tissue weight

   Competing interests

   The author declares that they have no competing interests.

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2. 

   Version published to 10.1101/2022.12.13.520357v1 on bioRxiv

   Dec 15, 2022