Analysis of distal convoluted tubule (DCT) cell de-differentiation due to COP9 signalosome (CSN) dysfunction via enriched DCT single-nucleus RNA-sequencing
Authors: Cornelius Ryan , Su Xiao-Tong , Nelson Jonathan , Reyes Jeremiah , Bradford Tanner , Yang Chao-Ling , Ellison David 
Published: May 2023

Cullin-RING-ligases (CRLs) are a group of E3 ubiquitin ligases that mediate the regulated degradation of proteins. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. To investigate the role of impaired CSN dysfunction in the kidney, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Even though the CSN is expressed all along the nephron, these mice showed remodeling only along the distal nephron, with shortening of the distal convoluted tubule (DCT) and a large reduction in DCT-specific proteins. The DCT is known to remodel due to changes in dietary salt intaked, drug administration, and human disease. Yet, the cellular and molecular basis for this plasticity is unclear. We hypothesized that CSN dysfunction causes de-differentiation of DCT cells leading to DCT remodeling. To test our hypothesis, inducible DCT-specific NCC-Cre-ERT2 mice were bred to INTACT (Isolation of Nuclei TAgged in specific Cell Types, which fluorescently labels nuclei) and Jab1 floxed mice to generate DCT-INTACT- Jab1 -/- mice. One adult male DCT-INTACT- Jab1 -/- mouse and one DCT-INTACT control mouse were treated with tamoxifen to induce nuclear GFP expression and to induce deletion of Jab1 only in DCT cells. Six-weeks post treatment, nuclei from one kidney cortex was isolated and the DCT was enriched using Fluorescence-Activated Nuclei Sorting (FANS). GFP-positive nuclei were used for single-nucleus RNA-sequencing (NovaSeq). Initial results showed that unbiased clustering and UMAP visualization revealed 7 clusters with most cells from the DCT (99.0% for control and 97.6% for Jab1 -/- mice), indicating that the enrichment process was highly successful. Both the control and Jab1 -/- mouse had similar proportions of DCT1 and DCT2 cells; however, we identified two specialized cell types (dDCT1 and dDCT2) which comprised a much larger proportion of the cells in the Jab1 -/- mouse (dDCT1: 2.9% in control vs 41.1% in Jab1 -/-; dDCT2: 2.0% in control vs 13.9% in Jab1 -/-). These cells were highly de-differentiated with lower expression of the canonical DCT identifier genes such as Egf and Umod for DCT1, Calb1 and S100g for DCT2, as well as Slc12a3. Both dDCT1 and dDCT2 cell populations had induced expression of the disease- or carcinoma-related genes Ano3, Nrg1, and Pvt1. In conclusion, Jab1 deletion causes de-differentiation of a population of DCT cells. Further examination of CSN dysfunction via DCT-enriched single-nucleus RNA-sequencing could lead to a better understanding of the molecular mechanisms involved in DCT remodeling. NIH K01DK120790, NIH R01DK51496, Foundation LeDucq: Transatlantic Network of Excellence 17CVD05 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.