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Abstract: FR-PO515

RNAseq of Microdissected Collecting Ducts Revealing the Early Signaling Mediating the Loss of Aquaporin 2 After K+ Deprivation

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Sung, Chih-Chien, Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
  • Chen, Min-Hsiu, Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
  • Hsu, Yu-Juei, Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
  • Lin, Shih-Hua P., Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
Background

Potassium (K+) deficiency could cause a reduction in urinary concentrating ability, resulting in nephrogenic diabetes insipidus (NDI), but the detailed mechanism remains unclear. Recently, transcriptomic and proteomic data from acquired NDI models reveals that oxidative stress, apoptosis, and inflammatory signaling are associated with AQP2 loss. We aim to explore the early signaling after K+ deprivation in cortical collecting ducts (CCDs).

Methods

Immunoblotting and bulk kidney RNAseq were performed at 0, 12, 24, and 48 hours after K+ deprivation in rats. Serum and urine biochemistry were also recorded. Based on immunoblotting and bulk kidney RNAseq, CCDs were microdissected from rats at 6 hrs after K+ deprivation versus time controls. Single-tubule RNA-Seq was carried out independently in K+ deprivation rats versus controls (n=4).

Results

Immunoblotting of bulk kidney showed a decrease in AQP2 protein abundance at 12 hours of K+ deprivation diet, and urine osmolality was significantly decreased at 24 hours, confirming the animal model of K+ deprivation-induced NDI. Preliminary bulk kidney RNA-Seq time course experiments also revealed that Aqp2 and other collecting ducts markers such as Aqp3, Aqp4, and Fxyd4 mRNA started to decrease at 12 hrs. Single-tubule RNA-Seq data of CCDs at 6 hrs after K+ deprivation showed Aqp2, Aqp3, and Atp1a1 were significantly downregulated. It also revealed that chemokine transcripts (Ccl20 and Ccl28) were increased significantly. We also carried out analysis of Gene Ontology Biological Process terms that are statistically over-represented in the list of 88 “Increased Transcripts”at 6 hrs of K+ deprivation in CCDs, and many of the terms are related to glutathione metabolic process (Gstm1, Gsta1, Gstt3, Txnrd3), positive regulation of ERK1 and ERK2 cascade (Nrp1, Ccl20, Ripk2, Fgfr4, Fgfr3), cell chemotaxis (Ccl20, Ccl28, Hbegf), cellular response to lipopolysaccharide (Ccl20, Ripk2, Cd14, Tfpi), consistent with an inflammatory response.

Conclusion

Our small samples RNA-Seq from microdissected CCDs in rats showed early cellular signaling changes in activation of oxidative stress and inflammatory signaling causing loss of aquaporin-2 in K+ deficiency induced NDI.