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My concerns are addressed. I think this revised version can be considered for publication in this journal, except for this point:
In the main text of Fig. 6, these results are labeled [a-c], however in the legend of Fig. 6, [a-d] are demonstrated. They only revised the main text, but seem to have forgotten to modify the legend of Fig. 6. This legend issue needs to be corrected at the Proof stage.
[# PeerJ Staff Note - this decision was reviewed and approved by Gwyn Gould, a PeerJ Section Editor covering this Section #]
**PeerJ Staff Note:** Although the Academic and Section Editors are happy to accept your article as being scientifically sound, a final check of the manuscript shows that it would benefit from further editing. Therefore, please identify necessary edits and address these while in proof stage.
Issues that need to be revised or improved:
1. Line 96 [minced into approximately 1 mm3 s granules]: [s] was shown before [granules]. Is this a spelling typo?
2. Line 188 [IGFBP3 or IGFBP-3]: Please use a consistent writing style throughout the main text and figures.
3. Line 192 [identified as] or [identified in]?
4. Lines 192-204: Results involving TMEM176A, CTSB and PBLD cannot be confirmed in the Figure 3 or other parts of the Results section. Please carefully check and clearly highlight in which figure (a, b, c, d, etc.) or table these results are displayed.
5. Lines 205-212: As with the above issue, these descriptions must be able to be found by the readers in the corresponding results section.
6. Line 294 [Supplementary Material 8] should be revised to [Supplementary Table 8].
7. Line 309 [Supplementary Material 9] should be revised to [Supplementary Table 9]? Also, please revise [Supplementary Material 10].
8. Line 296 [CXCR2] was incorrectly written out as [CXXR2] in Figure 5d. Please be careful and fix this error.
9. Lines 321-375: The writing in Figure 6 is completely unsatisfactory. This part of the description, in which figure (a, b, c, d, etc.) or table can it be found? This section must be thoroughly re-written to allow the precise meaning of a, b, c and d of Figure 6 to be fully revealed. And finally, the authors need to give an overall summary that encapsulates the entire Figure 6. Without these modifications, the authors cannot understand why the results of Fig. 6a-6d are necessary to be displayed in their paper.
10. Abstract section [These cells showed a pronounced chemotactic response and had an activating effect on neutrophils. This was substantiated by the widespread overexpression of pro-inflammatory genes in these cells]: The authors should soften their tone because their results only uncover possibilities rather than certainties.
11. Abstract section [Notably, the gene expression profiles of inflammatory PTCs in PBMCs, urine, and kidney tissues were closely correlated]: The meaning of [correlated] is not quite so understandable. Please carefully clarify this by explaining which results support this claim. If the authors just want to emphasize that they found a certain degree of similarity in the gene expression profiles of PTCs in PBMCs, urine, and kidney tissues, re-organizing their language is what is needed. Otherwise, they could consider deleting this sentence.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
1. Lin et al.’s study utilized scRNA-seq to investigate the heterogeneity of kidney diseases at the single-cell level. An important finding is the detection of PTCs in both PBMCs and urine samples of nephritis patients while not detected in healthy blood samples. Further analysis of the gene expression profiles indicated that multiple populations of fibroblast cells, mesangial cells, and PTCs exhibited pro-inflammatory or pro-apoptotic responses. In general, this study has great clinical significance and scientific research value.
2. The structure of the article is consistent with the standard chapters, and the diagrams are clear and understandable.
1. By using single-cell RNA sequencing, the study was able to identify the pathological changes in renal cells of nephritis patients at the single-cell level and uncover the immune mechanisms involved in the progression of the disease. The chemotactic and activating effects of inflammatory PTCs and fibroblasts on neutrophils were found to be crucial for the development and advancement of nephritis. Furthermore, the study suggests that non-invasive urine PTCs testing could potentially improve the early diagnosis and intervention of nephritis, providing a less invasive alternative to kidney biopsies. However, it’s important to note that further research is needed to validate these findings, particularly in a larger sample size and including additional types of nephritis. Continued exploration of the roles of different cell populations and immune mechanisms in nephritis will contribute to a better understanding of the disease and aid in the development of more effective diagnostic and treatment strategies.
2. The unraveling of transcriptional profiles of kidney at the single-cell resolution improved the better understanding and managing of kidney disease.
Overall, the findings from this study provide novel insights into the cellular and molecular aspects of nephritis, including the identification of subtype-specific signatures, the presence of disseminated PTCs, the developmental trajectory of PTCs, and the coexistence of functionally normal and abnormal cells. These discoveries have the potential to advance our understanding of nephritis pathogenesis and may lead to the development of new diagnostic and therapeutic strategies for this complex renal condition.
Authors responded to all my previous comments and suggestions sufficiently and correctly, the article now reads much better and persuasive.
Overall, the author has made a relatively adequate revision, but there is still a small problem that needs to be revised. I notice that Figure 6 is marked abcd, but I only find figure 6 in the manuscript. Is it Figure 6a,Figure 6b, or Figure 6a-d?
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Please respond and make appropriate revisions based on the Reviewers' suggestions and my comments (below). This will greatly improve the quality of the manuscript.
My comments:
1. Clarification or definition of terms like "multipapillary kidney" and "glomerulosclerosis" may be beneficial for readers not familiar with these terms.
2. The term "bulk transcriptome profiles" may need to be explained for a broader audience.
3. The introduction could better delineate between current diagnostic methods and the promising approaches that single-cell RNA sequencing offers, thereby highlighting the novel contribution of the study more clearly.
4. The introduction might benefit from a more explicit statement of the research question or hypothesis, to guide the reader to what specifically is being investigated in this study.
5. [Biopsy collected from consented subjects during a clinically indicated biopsy procedure] should be [Biopsy samples were collected from subjects who had provided informed consent, and the samples were obtained during a clinically indicated biopsy procedure]?
6. Some abbreviations are introduced but not used consistently throughout (e.g., "single-cell RNA-Seq" vs. "scRNA-Seq").
7. "The multipapillary kidney of contains roughly one million glomerulus."? Please revise.
"It not only provides disease classification, prognostic information, but also elucidates of the mechanisms of pathogenesis..." – The sentence structure here is awkward and needs to be revised.
8. Multiple instances of missing articles such as "a" or "the.
9. The citation format is inconsistent (some are in brackets and some are parenthetical). The style should be uniform throughout the text.
10. "The raw data is available" should be "The raw data are available."
11. The usage of Proximal tubule cells (PTC) and Proximal tubule cells (Inflammation, I-PTC) can be confusing. Clear differentiation or explanation may be needed.
12. "Decending" should be spelled "Descending," "neutrophil-mediated kidney inflammation" should be "neutrophil-mediated," and "anesis" appears to be a typo?
13. "accounting 1.58% of the total cells" should be "accounting for 1.58% of the total cells."
14. "5 healthy control" should be "5 healthy controls."
15. "Top 20 DEGs are showed" should be "Top 20 DEGs are shown."
16. "It is worthy to noting" should be "It is worthy of noting."
17. "neutropil" may be a typo and should be "neutrophil.""neutropil" appears to be a consistent misspelling; this should likely be corrected to "neutrophil" throughout.
18. "controvsal resoult about it role" should be "controversial results about its role."
19. "capillary density[55]. It also involved" should be "capillary density [55]. It is also involved."
20. "information diagnosis" might be better as "informative diagnosis."
21. "none-invasive" should be "non-invasive."
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
**Language Note:** The Academic Editor has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at copyediting@peerj.com for pricing (be sure to provide your manuscript number and title). Alternatively, you should make your own arrangements to improve the language quality and provide details in your response letter. – PeerJ Staff
This manuscript is a well-written and researched study. The manuscript is thorough, the literature cited in the introduction is sufficient to attract readers with a broader field of knowledge. Data of the manuscript is solid and well supported the conclusion.
Membranous nephropathy, interstitial nephritis, proliferative and sclerosing glomerulonephritis and diabetic nephropathy are the representative renal diseases. The analysis of pathological change of these renal in single cell level could provide mechanistic insights into their common pathobiology. The experimental design is reasonable. The methods are generally appropriate, and data were processed correctly.
The early and precise diagnosis of renal disease is one of the increasingly serious public health concerns worldwide. The current diagnostic indicators, such as proteinuria and serum creatinine, are greatly interfered by the physiological conditions of patients, and the changes in the indicator level are always not synchronized with renal damage. More importantly, the golden standard diagnosis of suspected chronic kidney disease is biopsy, which is traumatic and not suitable for contraindication patients. The main drawback of biopsy is its insensitive to early progression. The manuscript analyzed gene expression profiles of cells of renal tissue, PBMCs and urine from four nephritis patients in single cell level, and found that chemotactic and activating of inflammatory PTCs and fibroblasts on neutropils play an important rule in development and progress of nephritisthis process. It indicates that the inhibition of inflammation of targeting cells could be the therapeutic targets of kidney disease. The existence of inflammatory PTCs in blood and urine sample is really a rather remarkable finding. The none-invasive urine PTCs test may be developed based on the results to improve the early and none-invasive diagnosis of nephritis. The However, more samples with different renal diseases are needed in the future.
1. Lin et al.’s study utilized scRNAseq to investigate the heterogeneity of kidney diseases at the single-cell level. An interesting find is the detection of PTCs in both PBMCs and urine samples of nephritis patients, while not detected in healthy blood samples. In general, this study has great clinical significance and scientific research value.
2. Wordings need native speakers’ checking, grammar and spacing in sentences etc..
E.g. line 43, Line 50 (et al. is for persons, etc. are for things), Line 161, 166, 176, 293. Statue in main text might be state? Also, it is necessary to check the format uniformity of the references, such as (1) and [2]...
3. The diagrams are clear and understandable.
1. By using single-cell RNA sequencing, the study was able to identify the pathological changes in renal cells of nephritis patients at single-cell level and uncover the immune mechanisms involved in the progression of the disease. The study also suggests that non-invasive urine PTCs testing could potentially improve the early diagnosis and intervention of nephritis, providing a less invasive alternative to kidney biopsies. However, it’s important to note that further research is needed to validate these findings, particularly in a larger sample size and including additional types of nephritis.
2. The unraveling of transcriptional profiles of kidney at the single-cell resolution improved mechanism understanding of kidney disease. The author should discuss the advantages of the transcriptional profiles of kidney at the single-cell resolution.
3. The materials and methods need to be further refined, such as the number of patient samples, the detailed parameters of the analysis method screening. In addition, scRNASeq data of healthy blood was obtained from Gene Expression Omnibus at NCBI (GSE157278), which was further analyzed with the same procedure. The author should state why GSE157278 was selected here.
4. Figure 2 is missing sub figure g. For all heatmaps, authors should put clear statement, e.g. heatmap for top 20 DEGs etc. in figure legend. When saying pathways, authors should tell whether it is gene ontology or kegg pathways etc. in figure legend.
1. A large number of references appear in the description of the results, which can be reflected in. discussion section instead of results section. Please simplify the description of the results.
2. There were 558 DEGs with >0.5 fold change, including 556 up-regulated genes and two down-regulated genes (Supplemental Table 4). The author needs to clarify the basis for the selection of >0.5 fold change.
3. Overall, the findings from this study provide novel insights into the cellular and molecular aspects of nephritis, including the identification of subtype-specific signatures, the presence of disseminated PTCs, the developmental trajectory of PTCs, and the coexistence of functionally normal and abnormal cells.
These discoveries have the potential to advance our understanding of nephritis pathogenesis and may lead to development of new diagnostic and therapeutic strategies for complex renal condition. It is better for the authors to have an in-depth discussion on future clinical application of these findings.
This study found a high correlation in the gene expression profiles of inflammatory PTCs between PBMCs, urine, and kidney samples. The findings shed light on the cellular subsets involved in nephritis and highlight the potential of urine and PBMC PTCs as non-invasive diagnostic markers for kidney disease. Further research in this area has the potential to improve early diagnosis and treatment strategies for kidney diseases. However, there remains some issues need to be addressed.
1.There are some writing style need to be revised, such as“Cellular gene expression profiles of cells of renal tissue,....” in abstract should be “Gene expression profiles of cells of renal tissue,....”.
2.Studies in 2022 and 2023 are not included in the references. Please confirm whether there has been no research progress in this field in the past two years or whether the references need to be updated.
3.It is necessary to clearly describe the results obtained by the detection method in Figure 1a. Is it immunohistochemistry or something else? In addition, the magnification needs to be specified in the legend, and the scale needs to be marked in each diagram.
1.Is the detection of PTCs in PBMCs or urine a diagnostic indices of renal disease? It is a common marker or specific to one or some nephritis? The exfoliation of PTCs is an early or late stage of nephritis. It determines the possibility of urine PTCs detection in the early diagnosis of nephritis.
2. It’s worth noting that the identification of specific marker genes in these clusters provides insights into the functional state and potential roles of different cell types within the kidney tissue of the patients. The overexpression of LTA4H and LTB4 in fibroblast cells suggests a potential role in promoting inflammation and fibrosis. High levels of mitochondrial genes and IGFBP-3 in mesangial cells indicate a preapoptotic state and potential involvement in kidney disease progression. The upregulation of TMEM176A in proximal tubule cells suggests ongoing tubular injury and inflammation, while the high expression of SLC22A8 in another subset of proximal tubule cells indicates active reabsorption function. These findings contribute to our understanding of the cellular and molecular mechanisms underlying different kidney diseases, such as membranous nephropathy, interstitial nephritis, proliferative and sclerosing glomerulonephritis, and diabetic nephropathy. Further investigation and validation of these markers may lead to the development of diagnostic and therapeutic strategies targeting these specific cell types and their associated pathways.
1. Among these 12 cell clusters, 10 clusters contained cells from samples taken from the four different patients (Figure 1c), indicating common cell types across the different kidney diseases. However, Cluster 8 (FC) exclusively included cells from one patient, while Cluster 11 (Neutrophils) comprised cells from two patients. The distribution of cells from the four samples within the 12 clusters can be found in supplemental table 2. The study provides valuable insights into the cellular composition of renal tissue and highlights the heterogeneity of cell types across different kidney diseases. The scRNA-seq data obtained from PBMCs, blood samples, and urine samples may offer further insights into the systemic cellular changes associated with these kidney diseases. Researchers can utilize this data to investigate disease-specific gene expression profiles, identify potential biomarkers, and gain a better understanding of the underlying cellular mechanisms driving these conditions.
2. There were 448 DEGs with fold-change>2 or <0.5. Heatmap of top 20 DEGs in the two clusters were shown in Figure 3a (Supplemental Table 6). 58 GO terms were enriched, most of which were involved in biological process (BP, 45 terms), followed by 11 terms of cellular component (CC) and 2 terms of molecular function (MF). The author needs to elaborate on the actual content of the obtained results rather than simply describe them.
3. To understand the state transitions during the development of nephritis, the potential developmental trajectories of PTCs were construct by using unsupervised inference method Monocle (Figure 4, supplementary material 10)[42]. The author should describe the state transitions during the development of nephritis rather than a lengthy description of a single gene.
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