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Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney
M Echevarria1, G Frindt, G M Preston
1Department of Physiology and Biophysics, Cornell University Medical College, New York 10021.
Abstract:
To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.
Insights
Researchers found distinct water channels in rat kidney tissue. Different kidney sections express specific water channels, some influenced by cyclic AMP (cAMP), suggesting varied roles in renal water transport.
Area of Science:
- Molecular Biology
- Renal Physiology
- Xenopus Oocyte Expression Systems
Background:
- Renal water reabsorption is crucial for maintaining body fluid homeostasis.
- The presence and diversity of water channels in different kidney segments remain incompletely understood.
- Aquaporins, a family of water channel proteins, are key to regulating water transport.
Purpose of the Study:
- To investigate the hypothesis that rat renal tissue expresses multiple distinct water channels.
- To identify and characterize water channels present in the renal cortex, medulla, and papilla.
- To explore the regulation of these water channels by cyclic AMP (cAMP).
Main Methods:
- Messenger RNA (mRNA) was extracted from rat kidney cortex, medulla, and papilla.
- Extracted mRNA was injected into Xenopus oocytes to express putative water channels.
- Osmotic water permeability (Pf) was measured using a standard light microscopy technique.
- Inhibition studies with p-chloromercuriphenylsulfonate and antisense oligonucleotides for CHIP28 were performed.
- Oocyte exposure to cAMP was used to assess channel regulation.
Main Results:
- Injection of renal mRNA significantly increased oocyte osmotic water permeability (Pf).
- Expressed water permeability was sensitive to p-chloromercuriphenylsulfonate and had a low activation energy, characteristic of water channels.
- CHIP28 antisense oligonucleotide reduced water permeability expression by 70% (cortex), 100% (medulla), and 30% (papilla).
- cAMP exposure further increased Pf in medulla mRNA-injected oocytes, an effect not blocked by the CHIP28 antisense oligonucleotide.
Conclusions:
- Rat kidney expresses multiple distinct water channels, including CHIP28 water channels in the cortex and medulla.
- The renal medulla contains cAMP-activated water channels distinct from CHIP28.
- The renal papilla expresses cAMP-insensitive water channels.