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Abstract: TH-PO482

Cilia Disruption May Regulate the Renal Inner Medulla NOS Pathway

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Sedaka, Randee S., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Yoder, Bradley K., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Inscho, Edward W., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Jin, Chunhua, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Pollock, David M., University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Pollock, Jennifer S., University of Alabama at Birmingham, Birmingham, Alabama, United States
Background

Primary cilia act as flow-mediated mechanosensors, signaling via intraflagellar transport (IFT). We previously reported that NO synthase 1 (NOS1) is the major isoform contributing to flow-induced nitric oxide (NO) production in the inner-medullary collecting duct (IMCD) and contributes to natriuretic responses. In this study, we hypothesized that functional ciliary IFT contributes to IMCD NOS1 abundance and activation.

Methods

Mouse IMCD cells were studied under static or flow (10 dynes/cm2) conditions for 1-hr to assess NO production (HPLC) and ciliary marker expression (western blot). Inner medullary (IM) NOS isoform abundance and urinary NO excretion were measured in 2 models of cilia disruption, PCK rats and conditional IFT88 knockout mice.

Results

Exposure to flow significantly increased IMCD NO production (static: 168±18, flow: 312±26 pmol/mg pr/hr; n=6/group, p<0.05) and ciliary marker expression, IFT88 (static: 1.0±0.3, flow: 1.7±0.1 RDU/b-actin; n=6/group, p=0.02) and acetylated-α-tubulin (static: 1.0±0.2, flow: 1.9±0.2 RDU/b-actin; n=6/group, p=0.01) compared to static controls. IM NOS1α abundance was not elevated in PCK rats (SD: 1.00±0.1, PCK: 1.22±0.1 RDU/b-actin; n=4/group, p>0.05), but NOS1β abundance was increased compared Sprague Dawley (SD) control rats (SD: 1.0±0.2, PCK: 3.9±0.2 RDU/b-actin; n=4/group, p<0.01). NOS3 abundance was similar between SD and PCK rats (1.0±0.1 and 0.9±0.1 RDU/b-actin, respectively; n=4/group, p>0.05). However, pNOS3 Thr495 expression, a NOS3 inhibitory phosphorylation site, was significantly higher in PCK rats (SD: 1.0±0.3, PCK 3.1±0.5 RDU/total NOS3; n=4/group, p<0.01). Similarly, NOS1β expression in IFT88KO mice trended to be higher compared to controls (Cre-: 1.0±0.4, Cre+ 3.0±0.6 RDU/b-actin; n=3/group, p=0.06). No significant changes in NOS3 abundance were observed (Cre-: 1.0±0.1, Cre+ 1.2±0.2 RDU/b-actin; n=3/group, p>0.05). Urinary NO excretion was similar in SD and PCK rats (SD: 6.1±2.2, PCK 10.0±0.4 umol/day; n=4-5/group, p>0.05) as well as Cre- and Cre+ IFT88KO mice (Cre-: 211±61, Cre+ 220±13 nmol/day; n=3/group, p>0.05) on normal salt diet. These data suggest that ciliary disruption influences NOS isoform abundance in the renal IM.

Conclusion

In conclusion, functional cilia may contribute to acute flow-induced NO production and NOS isoform abundance.

Funding

  • NIDDK Support