Abstract: TH-OR21
Primary Cilia and the Glycocalyx Are Flow Sensors for Nitric Oxide Production by Thick Ascending Limbs
Session Information
- Molecular Insights in Ion, Acid, and Water Handling
October 22, 2020 | Location: Simulive
Abstract Time: 05:00 PM - 07:00 PM
Category: Fluid, Electrolyte, and Acid-Base Disorders
- 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Garvin, Jeffrey L., Case Western Reserve University, Cleveland, Ohio, United States
- Hong, Nancy J., Case Western Reserve University, Cleveland, Ohio, United States
Background
The primary cilium is an organelle found on essentially all epithelial cells. Similarly, the glycocalyx is a matrix-like layer of proteoglycans, glycosaminoglycans (GAGs) and plasma proteins covering the surface of all cells. In vascular endothelial cells, primary cilia and GAGs such as heparan sulfate and chondroitin sulfate mediate responses to the mechanical forces exerted by blood flow. In thick ascending limbs, increases in luminal flow enhance nitric oxide (NO) production, an important regulator of kidney function including sodium reabsorption; however, the role of primary cilia and the glycocalyx in NO production by thick ascending limbs is unknown. We hypothesized that primary cilia and the glycocalyx act as flow sensors and thus mediate flow-induced NO production by thick ascending limbs.
Methods
We measured flow-induced NO in isolated rat thick ascending limbs using DAF-FM. Intracelllular NO was first measured during the control period without and with luminal flow. NO was measured again during the experimental period after treating tubules for 15 min to deciliate cells or to degrade major glycocalyx GAGs. Dibucaine (0.1 mM) was used to remove cilia from cells. Heparinase III (0.2 or 0.4 U/ml) and chondroitinase ABC (0.2 U/ml) were used to degrade heparin sulfate and chondroitin sulfate, respectively.
Results
In untreated control tubules, flow-induced NO did not differ between the two periods, 4.33 ± 1.03 vs 4.68 ± 0.84 arbitrary units (AU)/min. Dibucaine decreased flow-induced NO from 4.25 ± 0.62 to 1.19 ± 0.65 AU/min (p < 0.002). Heparinase (0.2 U/ml) attenuated flow-induced NO from 4.02 ± 0.84 to 1.80 ± 0.74 AU/min (p < 0.04); a higher concentration (0.4 U/ml) caused a greater decrease from 4.24 ± 0.86 to 1.56 ± 0.41 AU/min (p < 0.006). Heat inactivation of heparinase (0.2 U/ml) abolished its effect (3.01 ± 0.34 to 2.83 ± 0.22 AU/min). Chondroitinase (0.2 U/ml) decreased flow-induced NO from 4.17 ± 0.96 to 2.45 ± 0.49 AU/min (p < 0.038).
Conclusion
We conclude that both primary cilia and the glycocalyx act as flow sensors in thick ascending limbs and transduce mechanical stimuli into chemical signals that ultimately result in NO production by this segment.
Funding
- Other NIH Support