Schlingmann et al.1 recently reported a novel kidney tubulopathy and cardiomyopathy phenotype that is caused by heterozygous missense variants in Ras-Related GTP-Binding D (RRAGD). Using genetic investigation, they have uncovered new findings which aid in the understanding of renal electrolyte handling and cardiac function.
We were very interested in this study and repeated the RRAGD staining. Immunohistochemistry of C57BL6 mice kidney tissues showed RRAGD colocalized with uromodulin and the sodium-chloride cotransporter. Expression of RRAGD was confirmed in the loop of Henle (thick ascending limb) and in the distal convoluted tubule nephron segments. According to Figure 2, E–G, in their article, we noticed RRAGD mainly expressed in the cytoplasm, consistent with our immunofluorescence staining results in C57BL6 mice kidney tissues (Figure 1, A–C). However, with the same antibody (rabbit anti-RRAGD, NBP2-32106, 1:2000; Novus Biologicals) and suitable conditions for immunofluorescence staining of human kidney tissues, we observed that RRAGD also expressed in the nucleus (Figure 1, D–F), both in the tubular and the glomerular part. In the Human Protein Atlas database,2 RRAGD was expressed in the nucleus, consistent with our result. Is the antibody not suitable for the paraffin sections of the mouse kidney, or is the difference in staining because of the difference between mice and humans?
Immunofluorescence staining showed RRAGD mainly expressed in the mouse kidney cytoplasm while mainly expressed in the human kidney nucleus. Scale bars, 50 μm. (A) RRAGD expression in the mouse kidney cortex, (B and C) at the mouse kidney juxtamedullary and medullar region, (D) at the corpuscle region of the human kidney cortex, and (E and F) in the tubule of the human kidney. DAPI, 4′,6-diamidino-2-phenylindole.
In summary, this is a significant study that has our full attention. Because the expression location of a protein is closely related to its function, we would be glad to hear from the authors.
Methods for Immunofluorescence Staining
The kidney tissues from male C57BL mice and a renal biopsy specimen of a patient with minor glomerular lesion were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 3–4 µm sections. The slides were then deparaffinized, rehydrated, and antigen was retrieved using a citrate buffer. After blocking with goat serum for 30 minutes, the sections were incubated with diluted primary antibodies of RRAGD (rabbit anti-RRAGD, NBP2-32106, 1:2000) overnight at 4°C. Fluorophore-labeled secondary antibody (goat anti-rabbit, ab150077, 1:1000; Abcam) and 4′,6-diamidino-2-phenylindole (1:1000) were used for nuclear staining. The images were captured using a microscope (Eclipse 80i; Nikon, Tokyo, Japan) with a digital camera (DS-U1; Nikon).
Disclosures
L. Chen reports serving as a member of the American Society of Nephrology (ASN), European Dialysis and Transplant Association, International Society for Peritoneal Dialysis, and International Society of Nephrology; receiving research funding from Baxter; and serving as associate editor of JASN. T. Ma reports serving as a member of the ASN and European Renal Association Community. L. Zhang reports serving as a member of the ASN and European Renal Association Community.
Funding
This work was supported by National Natural Scientific Foundation of China grants 82170709 and 81970607 (to L. Chen), and Capital’s Funds for Health Improvement and Research grant CFH 2020-2-4018 (to L. Chen).
Acknowledgments
The funders had no role in study design, data collection, analysis, and manuscript preparation.
Author Contributions
CRediT Taxonomy T. Ma was responsible for formal analysis and methodology, and wrote the original draft; T. Ma and L. Zhang were responsible for investigation; L. Zhang and L. Chen were responsible for resources; L. Chen was responsible for project administration, supervision, and validation; and all authors were responsible for conceptualization and reviewed and edited the manuscript.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
See related reply, “Authors’ Reply: The Subcellular Localization of RRAGD,” on pages 1048–1049, and original article, “mTOR-Activating Mutations in RRAGD are Causative for Kidney Tubulopathy and Cardiomyopathy,” in Vol. 32, Iss. 11, on pages 2885–2899.
- Copyright © 2022 by the American Society of Nephrology
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