The effect of water deprivation on the expression of atrial natriuretic pepide and its receptors in the spinifex hopping mouse, Notomys alexis

This study investigated the mRNA expression of the atrial natriuretic peptide (ANP) system (peptide and receptors) during water deprivation in the spinifex hopping mouse, Notomys alexis, a native of central and western Australia that is well adapted to survive in arid environments. Initially, ANP, NPR-A and NPR-C cDNAs (partial for receptors) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. Using a semi-quantitative multiplex PCR technique, the expression of cardiac ANP mRNA and renal ANP, NPR-A, and NPR-C mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control mice (access to water). The levels of ANP mRNA expression in the heart remained unchanged, but in the kidney ANP mRNA levels were increased in the 7-day water-deprived mice, and were significantly decreased in the 14-day water-deprived mice. NPR-A mRNA levels were significantly higher in 7-day water-deprived mice while no change for NPR-A mRNA expression was observed in 14-day water-deprived mice. No variation in NPR-C mRNA levels was observed. This study shows that water deprivation differentially affects the expression of the ANP system, and that renal ANP expression is more important than cardiac ANP in the physiological adjustment to water deprivation.

Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis

Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation.

The effect of water deprivation on signalling molecules that utilise cGMP in the Spinifex Hopping Mouse Notomys alexis

In mammals the natriuretic and guanylin peptides influence renal and intestinal fluid content and electrolyte transport by binding to and activating guanylyl cyclase (GC) receptors that in turn stimulate production of the intracellular second messenger guanosine 3':5'-cyclic monophospate
(cGMP). However, the role of natriuretic and guanylin peptides in desert mammals is not understood. The spinifex hopping-mouse (Notomys alexis), has a suite of behavioural and physiological mechanisms that permits survival for extended periods without access to free water. Because signalling molecules that generate cGMP are known to promote water excretion, it was predicted that natriuretic and guanylin peptide synthesis would be down regulated in water-deprived N. alexis, and thus reduce the amount of water lost in the urine and faeces. However, in the kidney ANP and GC-A mRNA levels were increased in water-deprived mice, but CNP and GC-B mRNA levels were decreased. Water deprivation increased guanylin and uroguanylin mRNA expression in the distal colon, but it remained unchanged in the kidney and proximal colon. The expression of GC-C mRNA increased in the proximal colon but not in the distal colon. This study shows that water deprivation differentially affects the expression of regulatory molecules that stimulate cGMP producti

The response of the natriuretic peptide system to water deprivation in the desert rodent, Notomys alexis

Natriuretic peptides (NPs) are regulatory molecules that cause cGMP-mediated diuresis and natriuresis in mammals. Accordingly, it is interesting to consider their role in desert-adapted animals in which water is often limited. This study investigated the response of the natriuretic peptide (NP) system to varying periods of water deprivation (WD) in the Australian desert rodent species, Notomys alexis. It was hypothesised that the expression of the NP system will be down-regulated in water-deprived N. alexis compared to water-replete animals. The plasma levels of ANP were significantly reduced after 3 days of WD, but were unaffected by 7, 14 and 28 days of WD. Water deprivation for 3, 7, 14 days had a variable effect on the mRNA expression of ANP, CNP, NPR-A, NPR-B, and NPR-C, and a uniform down-regulation was not observed. However, after 28 days of WD, mRNA expression was similar to water-replete animals, except for NPR-A. Surprisingly, 7 and 14 days of WD caused an up-regulation in the ability of ANP to stimulate cGMP; this also occurred at 14 days for CNP. Taken together, the mRNA expression and peptide mediated guanylyl cyclase activity data after WD were in the opposite direction to what was predicted. Interestingly, after 28 days of WD, most parameters were similar to those of water-replete animals, which indicates that a down-regulation of the NP system is not part of the physiological response to an absence of free water in N. alexis.

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex Hopping Mouse, notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.

The effect of water deprivation on the tonicity responsive enhancer binding protein (TonEBP) and TonEBP-regulated genes in the kidney of the spinifex hopping mouse, Notomys alexis

In desert rodents, the production of concentrated urine is essential for survival in xeric environments in order to conserve water. Reabsorption of water in the kidney is dependent on large osmotic gradients in the renal medulla. This causes the renal cells to be bathed in a hypertonic extracellular fluid that can compromise cellular function. In response to hypertonicity, kidney cells accumulate compatible, non-ionic osmolytes that lower the ionic strength within the cells to isotonic levels by replacing intracellular ionic electrolytes. The tonicity-responsive enhancer binding protein (TonEBP) is a transcription factor that regulates the expression of genes that encode proteins that catalyse the accumulation of compatible osmolytes. We investigated the expression of TonEBP mRNA and protein and compatible osmolyte genes in the Spinifex hopping mouse, Notomys alexis, an Australian desert rodent that produces a highly concentrated urine. TonEBP mRNA expression was unchanged after 3 days of water deprivation but was significantly increased after 7 and 14 days of water deprivation. Immunohistochemistry showed that during water deprivation TonEBP had translocated from the cytoplasm into the nucleus of cells in the renal medulla and papilla. In addition, 3, 7 and 14 days of water deprivation caused a significant increase in aldose reductase (AR), myo-inositol (SMIT), betaine/GABA (BGT-1) and taurine (TauT) transporter mRNA expression, which is indicative of an increase in TonEBP activity. In desert rodents, TonEBP regulation of gene transcription is probably an important mechanism to protect renal cells in the face of the large corticomedullary gradient that is required to concentrate urine and conserve water.

Water deprivation induces appetite and alters metabolic strategy in Notomys alexis : unique mechanisms for water production in the desert

Like many desert animals, the spinifex hopping mouse, Notomys alexis, can maintain water balance without drinking water. The role of the kidney in producing a small volume of highly concentrated urine has been well-documented, but little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. In Notomys, we found that water deprivation (WD) induced a sustained high food intake that exceeded the pre-deprivation level, which was driven by parallel changes in plasma leptin and ghrelin and the expression of orexigenic and anorectic neuropeptide genes in the hypothalamus; these changed in a direction that would stimulate appetite. As the period of WD was prolonged, body fat disappeared but body mass increased gradually, which was attributed to hepatic glycogen storage. Switching metabolic strategy from lipids to carbohydrates would enhance metabolic water production per oxygen molecule, thus providing a mechanism to minimize respiratory water loss. The changes observed in appetite control and metabolic strategy in Notomys were absent or less prominent in laboratory mice. This study reveals novel mechanisms for appetite regulation and energy metabolism that could be essential for desert rodents to survive in xeric environments.

The role of leptin and ghrelin in appetite regulation in the Australian Spinifex hopping mouse, Notomys alexis, during long-term water deprivation

Water deprivation of the Spinifex hopping mouse, Notomys alexis, induced a biphasic pattern of food intake with an initial hypophagia that was followed by an increased, and then sustained food intake. The mice lost approximately 20% of their body mass and there was a loss of white adipose tissue. Stomach ghrelin mRNA was significantly higher at day 2 of water deprivation but then returned to the same levels as water-replete (day 0) mice for the duration of the experiment. Plasma ghrelin was unaffected by water deprivation except at day 10 where it was significantly increased. Plasma leptin levels decreased at day 2 and day 5 of water deprivation, and then increased significantly by the end of the water deprivation period. Water deprivation caused a significant decrease in skeletal muscle leptin mRNA expression at days 2 and 5, but then it returned to day 0 levels by day 29. In the hypothalamus, water deprivation caused a significant up-regulation in both ghrelin and neuropeptide Y mRNA expression, respectively. In contrast, hypothalamic GHSR1a mRNA expression was significantly down-regulated. A significant increase in LepRb mRNA expression was observed at days 17 and 29 of water deprivation. This study demonstrated that the sustained food intake in N. alexis during water deprivation was uncoupled from peripheral appetite-regulating signals, and that the hypothalamus appears to play an important role in regulating food intake; this may contribute to the maintenance of fluid balance in the absence of free water.

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

Molecular physiology of water balance in the Spinifex hopping mouse

Many animals live in the deserts of Australia and survive without drinking water. This research examined desert adaptations in the Spinifex hopping mouse (Notomys alexis), and is the first study to demonstrate how many genes and exzymes in the kidney are controlled to maintain water balance in the absence of drinking water.