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Abstract: SA-PO290

Whole-Kidney and Single-Tubule RNA Sequencing Reveal Changes of Cell Types and Signaling for Nephrogenic Diabetes Insipidus After Ureteral Obstruction

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Sung, Chih-Chien, Tri-Service General Hospital, National Defense Medical Center, Taipei, TAIWAN, Taiwan
  • Lin, Shih-Hua P., Tri-Service General Hospital, National Defense Medical Center, Neihu, Taiwan
  • Knepper, Mark A., National Heart Lung and Blood Institute, Kensington, Maryland, United States
Background

Unilateral ureteral obstruction (UUO) models in rodents are commonly employed in the study of CKD. Early stages of UUO are marked by polyuria with impaired urinary concentrating ability, associated with loss of aquaporin-2 (AQP2) expression. Most mechanistic work in UUO models has been done at relatively late time points, making it difficult to discriminate ‘first cause’ events from secondary, tertiary, etc. changes in gene expression.

Methods

Preliminary whole-kidney RNA-Seq studies were performed after 0, 3, 6, and 12 hrs of UUO. Based on whole-kidney findings, cortical collecting ducts (CCDs) and cortical thick ascending limbs of Henle (cTALs) were microdissected from rats 3 hrs after UUO. Single-tubule RNA-Seq was carried out independently in 4 UUO rats versus 4 controls.

Results

Whole kidney RNA-Seq time course experiments revealed that Aqp2 and other collecting ducts markers started to decrease between 2 and 6 hrs. Decreases were seen in markers of connecting tubule (Calb1), distal convoluted tubule (Slc12a3) and thick ascending limb (Slc12a1) were also seen within 3 hrs. However, there were no effects on transcripts coding for classical markers of podocytes and proximal tubules within 12 hours. Expression of renal non-epithelial cell markers showed B lymphocytes (Cd19, Cd80) rapidly increased at 3 hrs and decreased at 12 hrs followed by increased abundance of markers of monocytes (Fcgr2b, Sell), macrophages (Gata6), and chemokines (Ccl2, Ccl6, Cxcl14) at 6 to 12 hrs. Several aldosterone-regulated genes showed increases in mRNA including Sgk1, Scnn1a, and Tsc22d3 at 3 hrs. Single-tubuleRNA-Seq data (both CCD and cTAL) showed a large number of transcripts coding for transporters and receptors that were decreased. It also revealedthat immediate early gene transcripts were increased significantly more frequently than expected from random sampling from the full pool of transcripts at 3 hrs.

Conclusion

Whole-kidney RNA-Seq results are consistent with very early effects on the distal nephron but not proximal tubule or glomerulus in terms of gene expression and provided evidence for invasion or activation of inflammatory cell types. Single-tubule RNA-Seq showed cellular signaling changes in CCD and cTAL, consistent with activation of the immediate early response.

Funding

  • Government Support - Non-U.S.