Abnormal osmotic regulation in trpv4-/- mice

Abstract

Osmotic homeostasis is one of the most aggressively defended physiological parameters in vertebrates. However, the molecular mechanisms underlying osmotic regulation are poorly understood. The transient receptor potential channel, vanilloid subfamily (TRPV4), is an osmotically activated ion channel that is expressed in circumventricular organs in the mammalian CNS, which is an important site of osmotic sensing. We have generated trpv4-null mice and observed abnormalities of their osmotic regulation. trpv4-/- mice drank less water and became more hyperosmolar than did wild-type littermates, a finding that was seen with and without administration of hypertonic saline. In addition, plasma levels of antidiuretic hormone were significantly lower in trpv4-/- mice than in wild-type littermates after a hyperosmotic challenge. Continuous s.c. infusion of the antidiuretic hormone analogue, dDAVP, resulted in systemic hypotonicity in trpv4-/- mice, despite the fact that their renal water reabsorption capacity was normal. Thus, the response to both hyper- and hypoosmolar stimuli is impaired in trpv4-/- mice. After a hyperosmolar challenge, there was markedly reduced expression of c-FOS in the circumventricular organ, the organum vasculosum of the lamina terminalis, of trpv4-/- mice compared with wild-type mice. This finding suggests that there is an impairment of osmotic sensing in the CNS of trpv4-/- mice. These data indicate that TRPV4 is necessary for the normal response to changes in osmotic pressure and functions as an osmotic sensor in the CNS.

Fig. 1.

Generation of trpv4-null mice. (A) Targeting of exon 12 of the trpv4 gene to obtain a null allele. The figure depicts the targeted trpv4 locus on mouse chromosome 5. The trpv4 gene is comprised of at least 15 exons. Exon 12 codes for the supposed pore-loop domain and the adjacent transmembrane domains 5 and 6. This exon was flanked by loxP genetic elements, and an adjacent neomycin selection cassette was inserted, followed by another loxP site. All loxP sites were in the same orientation. A BamHI site occurring in wild type after exon 12 was eliminated in the targeting construct. The neo cassette contained an upstream BamHI site. P, PacI site; EP, external probe used in genomic Southern blotting. (B) Southern blot of mouse tail genomic DNA after mating to EIIa-cre⁺ mice. Depicted here are homozygous null mice, a heterozygous mouse, and a homozygous wild-type mouse. The wild-type allele measures 6.5 kb, and the targeted allele measures 8.4 kb after excision of exon 12 and the neo selection marker, as in A. DNA was digested with BamHI, and the same external probe (see A) that was used for screening of embryonic stem cells was used. Genotyping was also performed with PCR by using primers p2 and p4. Wild-type mice had a 3-kb band, null mice a 1.6-kb band, and heterozygotes had both (results not shown). (C) Absence of TRPV4 protein by Western blotting. Kidney protein preparations were examined by Western blotting for TRPV4 and for β-actin. Note the absence of TRPV4 protein in trpv4-null mice. In the wild-type mice, there is the expected 107-kDa protein, as well as a shorter 75-kDa isoform, perhaps indicative of a yet undescribed splice variant.

Fig. 2.

Impaired response to hypertonicity in trpv4^-/- mice. (A) Impaired osmotic regulation in trpv4^-/- mice when they are single-housed. Normal systemic osmotic pressure was apparent in both genotypes when mice were group-housed with freely available food and water (data not shown). When mice were single-housed without food and free access to water, a slight, yet significant, rise in systemic tonicity in trpv4^-/- mice was seen. (B) Impaired drinking of trpv4^-/- mice without challenge. Single-housing, no access to food as described in A. Drinking volumes were adjusted per 20 g of body weight. A significantly reduced water intake was measured in trpv4^-/- mice, which was at its most pronounced in the first 6 h of the experiment. (C) Greater hyperosmolality in trpv4^-/- mice in response to dehydration. Mice were single-housed without food and water, and systemic tonicity was determined after 48 h. A significant rise of blood osmolality in trpv4^-/- mice was observed. (D) Impaired defense against hypertonicity in trpv4^-/- mice after systemic osmotic challenge with 0.5 M NaCl (i.p. administration). Mice were single-housed without food and water during the experiment. Blood osmolality was determined 90 min after administration of 0.5 M NaCl. A significant increase in systemic osmotic tonicity was observed in trpv4^-/- mice. (E) Impaired drinking of trpv4^-/- mice after systemic osmotic challenge with 0.5 M NaCl (i.p. administration). Water intake is shown here. The setting was the same as in D. A significantly reduced amount of water intake in trpv4^-/- mice is apparent. (F) ADH response after systemic hypertonic challenge. Depicted is the amount of plasma ADH after i.p. administration of 0.15 and 0.5M NaCl. A significantly lower amount of ADH could be noticed in trpv4^-/- mice in response to hypertonic saline administration.

Fig. 3.

Response to dDAVP in trpv4^-/- mice. (A) Response to chronic systemic administration of the ADH analogue, dDAVP. Blood osmolality is shown here. trpv4^-/- mice and wild-type littermate controls were implanted with osmotic minipumps loaded first with saline, then pumps were switched to chronic infusion of dDAVP (1 μg per 20 g of body weight in 100 μl, chronic infusion over 3 days). Systemic osmotic pressure was slightly increased in trpv4^-/- mice while receiving saline. Chronic infusion of dDAVP led to systemic hypotonicity in trpv4^-/- mice, whereas wild-type littermates remained normotonic. During the entire experiment, mice were single-housed, water was freely available, and food was withheld for periods of 24 h, after which blood osmolality and drinking amounts were recorded. (B) Response to chronic systemic administration of the ADH analogue, dDAVP. Shown here is fluid intake (percent of saline amount). The average percentage of fluid intake while receiving dDAVP is shown. One hundred percent is the amount drunk by each individual mouse while receiving saline. After dDAVP treatment, each animal drank only a percentage of this baseline amount. The average percentage was calculated. The difference between genotypes is statistically significant (P < 0.05, t test). Whereas the kidneys of trpv4^-/- mice responded to dDAVP treatment (as in C), their increased drinking in comparison to wild-type littermates (77% as compared with 51% reduction of drinking) led to systemic hypotonicity (as in A).

Fig. 4.

Specific impairment of CNS sensing of systemic osmotic pressure in trpv4^-/- mice. (A) Immunohistochemistry for TRPV4 in the lamina terminalis. (Upper) Depicted is the circumventricular organ, SFO, and adjacent to it is the choroid plexus. The SFO is prominent in the rostral part of the third ventricle and is located caudally to the ventral hippocampal commissure. Note the absence of specific staining in the nulls. (Lower) Depicted is the circumventricular organ, the OVLT. The rostral portion of the anterior end of the third ventricle is sectioned; note specific staining in the OVLT in wild-type littermate mice, but not in trpv4-null mice. (Bar, 50 μm.) (B) Colabeling of TRPV4⁺ cells in the OVLT (Left, green fluorescence) with neuronal marker NeuN (Center, red fluorescence). Merged images are on the right. (Bar, 10 μm.) (C) Micrograph of the circumventricular organ, the OVLT, in the lamina terminalis. Shown here is immunohistochemistry for the c-FOS immediate early response gene. Representative micrographs of the lamina terminalis in a trpv4^-/- mouse and in a wild-type littermate control mouse are shown. Note activation of the OVLT (inverted-U shape) and the rostrally positioned median preoptic area, MnPO. Mice were injected i.p. with 0.5 M NaCl, and their brains were fixated in formalin 90 min later. (Bar, 50 μm.) (D) Morphometry of c-FOS⁺ cells in circumventricular organs, the OVLT and the SFO. A significantly lower count of cells was noted in the OVLT of trpv4^-/- mice, whereas the lower count in the SFO did not reach statistical significance. (E) Response to cold stress. Core body temperature was recorded by a rectal probe at 0 and 150 min. Mice were single-housed without food and water and exposed to 4°C for 150 min. Core body temperature dropped <36°C in both genotypes. Mean body temperature after the cold stress test was slightly lower in trpv4^-/- mice, but the difference did not reach statistical significance.