Nfkb1 Removal from Proximal Tubule Cells Improves Renal Tubular Outcomes Following Ischemia Reperfusion Injury
Creators
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1.
University of Southern California
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2.
University of Pennsylvania
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3.
California Institute of Technology
Abstract
Key Points
- Persistent NF-κB signaling associates within injured proximal tubule cells (PTCs) that fail to repair on kidney injury.
- Removing activity of Nfkb1, a transcriptional effector of NF-κB signaling, in PTCs enhances PTC repair and decreases injury associated fibrosis.
- Coexpression of Nfkb1 and Relb in injured PTCs suggests additional improvement from comprehensive targeting of NF-κB transcriptional regulators.
Background
CKD is a significant global health burden. AKI is a risk factor of progression to CKD. Recent studies have linked failure in proximal tubule repair as a potential contributing factor to CKD in mouse and human studies. Failed repair proximal tubule cells (FR-PTCs), initially presenting at the site of maximal sensitivity to ischemia reperfusion injury and spreading to more cortical regions over time, adopt a senescence-associated secretory phenotype linked to activation of the NF-kB pathway. Several transcriptional regulatory factors mediate NF-kB pathway action. Of these, Nfkb1 is prominent within FR-PTCs and chromatin studies predict Nfkb1 interactions with pathology-associated gene targets.
Methods
To examine the role of NF-kB in nephron injury outcomes, we removed Nfkb1 activity within the nephron lineage of the mouse kidney and examined the kidney's response to bilateral ischemia reperfusion injury.
Results
Single-cell transcriptional analysis showed a significant reduction of inflammation-associated gene expression, including Ccl2, Birc3, Spp1, Cd47, and Traf1, in Nfkb1-deficient FR-PTCs. A reduced pathological signature correlated with normalized expression of genes associated with healthy proximal tubule function, including Cubn, Kap, and a number of solute carriers. Single-nucleus Assay for Transposase-Accessible Chromatin seq analysis linked transcriptomic changes to enhancer regulation, particularly marked opening of chromatin for targets of hepatocyte nuclear factor family members associated with normal regulation of gene expression in proximal tubule cells.
Conclusions
Examining Assay for Transposase-Accessible Chromatin seq motif predictions and performing direct immunolabeling studies suggested Relb, another transcriptional mediator of NF-κB transcriptional responses with overlapping targeting specificity to Nfkb1, may partially compensate for the loss of Nfkb1. These studies support future efforts to remove ongoing NF-κB signaling within nephrons as a potential therapeutic strategy to target the AKI-to-CKD transition.
Copyright and License
Copyright © 2025 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Nephrology. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CC BY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
Acknowledgement
We thank Janet Romo for instruction on IRI surgery and Dr. Seth Ruffins in the optical imaging facility at University of Southern California for imaging support.
Funding
A.P. McMahon: Division of Diabetes, Endocrinology, and Metabolic Diseases (R01DK126925) and National Institute of Diabetes and Digestive and Kidney Diseases (UC2DK126024).
Contributions
Conceptualization: Shun-Yang Cheng, Andrew P. McMahon.
Formal analysis: Shun-Yang Cheng, Jinjin Guo, Kari Koppitch.
Funding acquisition: Andrew P. McMahon.
Investigation: Shun-Yang Cheng, Jinjin Guo, Kari Koppitch, Nathan Moy, Taylor L. Simonian, Parker C. Wilson.
Project administration: Shun-Yang Cheng.
Software: Shun-Yang Cheng, Parker C. Wilson.
Supervision: Andrew P. McMahon.
Visualization: Shun-Yang Cheng, Jinjin Guo, Parker C. Wilson.
Writing – original draft: Shun-Yang Cheng, Andrew P. McMahon.
Writing – review & editing: Shun-Yang Cheng, Andrew P. McMahon, Jinjin Guo, Kari Koppitch, Nathan Moy, Taylor L. Simonian, Parker C. Wilson.
Data Availability
Data related to transcriptomic, proteomic, or metabolomic data. Original data created for the study are or will be available in a persistent repository upon publication. Raw Data/Source Data. Gene expression omnibus (GEO). All sequencing data have been deposited in the GEO database (accession number is GSE294923). The analysis code is available on GitHub (https://github.com/p4rkerw/mcmahon).
Code Availability
The analysis code is available on GitHub (https://github.com/p4rkerw/mcmahon).
Conflict of Interest
Disclosure forms, as provided by each author, are available with the online version of the article at https://links.lww.com/KN9/B107.
S. Cheng reports the following:
Employer: University of Southern California
J. Guo reports the following:
Employer: University of Southern California
K. Koppitch reports the following:
Employer: University of Southern California (USC)
A. McMahon reports the following:
Employer: California Institute of Technology; Consultancy: GentiBIO; Ownership Interest: Iviva Medical; Trestle Biotherapeutics;; Patents or Royalties: Hedgehog technology licenses through Harvard to Curis. Receive license/royalty payments from Curis;; Advisory or Leadership Role: California Institute of Technology - Jacobs Institute; and Other Interests or Relationships: Board of the University Kidney Research Organization (UKRO).
N. Moy reports the following:
Employer: University of Southern California; Metropolis Dermatology; and Ownership Interest: NVDA; AMGN; AAPL; MSFT; RPM.
T. Simonian has nothing to disclose.
P. Wilson has nothing to disclose.
Supplemental Material
This article contains the following supplemental material online at https://links.lww.com/KN9/B108, https://links.lww.com/KN9/B109, https://links.lww.com/KN9/B110, https://links.lww.com/KN9/B111, https://links.lww.com/KN9/B112, https://links.lww.com/KN9/B113.
Supplemental Figure 1. Nephron lineage–specific ablation of Nfkb1 does not substantially alter injury severity or the subsequent recovery of kidney function after IRI compared with controls. (A) Plasma creatinine concentration on day 2 after IRI. (B) Plasma creatinine concentration on day 28 after IRI. (C) In Nfkb1 knockouts, Nfkb1 GEX is effectively removed from FR-PTCs but remains evident in surrounding CD45+ immune cells.
Supplemental Figure 2. Violin plots showing the expression of marker genes for FR-PTCs and other significant cell clusters.
Supplemental Figure 3. Dot plot shows immune cell type annotation on the basis of CD45+ single-cell transcriptomes. Dots on the dot plot represent cell types with a Fisher's exact test adjusted P value < 0.05; larger dots indicate greater statistical significance.
Supplemental Table 1. Cell types and differentially expressed marker genes for each cluster.
Supplemental Table 2. Differentially expressed gene list for Nfkb1-KO versus wild-type FR-PTCs.
Supplemental Table 3. Pathway enrichment analysis of differentially expressed genes from Nfkb1-KO versus wild-type FR-PTCs.
Supplemental Table 4. Differentially accessible regions identified between Nfkb1-KO and wild-type FR-PTCs.
Supplemental Table 5. Motif enrichment analysis of differentially accessible regions from Nfkb1-KO versus wild-type FR-PTCs.
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Additional details
Identifiers
- PMID
- 40465395
- PMCID
- PMC12407146
Funding
- National Institutes of Health
- R01DK126925
- National Institutes of Health
- UC2DK126024
Dates
- Submitted
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2025-01-10
- Accepted
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2025-05-30
- Available
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2025-08-04Published online