Vasopressin, protein metabolism and water conservation

Abstract

Purpose of Review

Highlight the mechanisms through which vasopressin and hypertonic stress regulate protein metabolism

Recent Findings

• Mammals have an “aestivation-like” response in which hypertonic stress increases muscle catabolism and urea production

• Vasopressin can directly regulate ureagenesis in the liver and the kidney

• In humans chronic hypertonic stress is associated with premature aging, diabetes, cardiovascular disease, and premature mortality

Summary

There is an evolutionarily conserved “aestivation-like” response in humans in which hypertonic stress results in activation of the vasopressin system, muscle catabolism, and ureagenesis in order to promote water conservation.

Introduction

Water is key to life; thus, water restriction is one of the most extreme stresses on biological systems. As such, all living organisms have developed mechanisms to defend against dehydration stress. With its evolutionary origins some 650 million years ago in Urbilateria, the vasopressin system is the preeminent regulator of water homeostasis in mammals [1]. In humans, vasopressin is a nine amino-acid peptide hormone that is mainly produced in the hypothalamus and released into the blood by the posterior pituitary in response to low blood pressure or hypertonic stress. Circulating vasopressin then binds to V1a, V1b, and V2 receptors that are primarily expressed in the vasculature and kidney, leading to increased blood pressure and increased water reabsorption. In addition to its roles regulating blood pressure and water balance, vasopressin has been identified as a key regulator of metabolism [2] and poor hydration status is associated with a myriad of poor health outcomes [3]. More specifically, vasopressin has been shown to directly regulate the metabolism of carbohydrates, lipids, and proteins. We and others have reviewed the role of vasopressin in lipid and carbohydrate metabolism [4–7]. The focus of this review will be the relationship between vasopressin and protein metabolism as it relates to urea and water conservation.

Urea metabolism in the kidney and the liver

When the body is faced with hypertonic stress, the first task is to conserve water. In most cases, water is lost through sweat, stool, and ventilation (termed insensible losses) and urine. While vasopressin can regulate insensible fluid losses [8–11], urine accounts for most of the water lost from the body. Thus, vasopressin’s major role in limiting water losses is to utilize urea to concentrate the urine and maximize water reabsorption.

Urea is an organic osmolyte produced in the liver through the urea cycle [12] (Figure 1). The urea cycle is required for the disposal of ammonia, which is one of the byproducts of protein catabolism. Urea production rate is known to be proportional to protein intake and urea production is impaired in liver disease, while urea excretion is impaired in kidney disease [13].

Figure 1 – “Aestivation like” response to hypertonic stress –

Hypertonic stress leads to higher vasopressin levels, increases in glucocorticoid mediated muscle catabolism, increased ureagenesis in the liver, and accumulation of urea the kidney. Together this “aestivation-like” response promotes water conservation. Lightning bolts denote steps that are regulated by vasopressin in vitro and/or in vivo. NKCC2 – Na:K:2Cl cotransporter 2, AQP2 – Aquaporin 2, UT-A1-3 – Urea Transporter A1-A3 respectively. Figure created with BioRender.

Urinary concentration is the process through which the kidney reabsorbs water from the tubular fluid to minimize water excretion. A detailed description of the mechanisms of urinary concentration and dilution is beyond the scope of this manuscript and have been reviewed in-depth previously [14] [15]. Briefly, the kidney concentrates urine through the “counter current multiplier” system in which, the recirculation of NaCl and urea between the collecting ducts, the vasa recta, and the ascending loop of Henle of juxtamedullary nephrons creates an osmotic gradient from the outer medulla to the tip of the tip of the inner medulla. This osmotic gradient favors water reabsorption from the ultrafiltrate (Figure 1). Vasopressin is a key regulator of both the urea transporters and water channels involved in urinary concentration. When vasopressin concentration increases, V2R stimulation in the kidney leads to increased expression and membrane abundance of the water channel aquaporin-2 (AQP2), and urea transporters (UT-A1, UT-A2, and UT-A3) [14–18] (Figure 1). Mice lacking AQP2, UT-A1, UT-A3 develop severe polyuria, highlighting that these transporters are necessary to maximize urinary concentration [19–22]. Specifically, the role of urea and protein metabolism in urinary concentration was highlighted in UT-A1/3 knockout mice. When fed a high protein diet (which increases urea production) UT-A1/3 knockout mice were unable to appropriately concentrate urine after water restriction; this effect was lost when placed on a low protein diet [20, 22]. These observations confirmed the original hypothesis by Wirz and Berliner who proposed that urea would act as an osmotic diuretic unless the kidney collecting duct were able to concentrate urea in the medulla of the kidney [23–25] . Therefore, urea and urea recycling are critical elements without which urinary concentration is impaired. Much of the relationship between vasopressin and urea has focused on the mechanisms through which vasopressin regulates both urea and water transport in the kidney. Less is known about the relationship between vasopressin and the synthesis of urea.

Given the unique ability of the liver to synthesize urea, together with the absolute requirement of urea and vasopressin signaling for maximal urinary concentration, it would be potentially advantageous for vasopressin to regulate both processes, thus favoring more efficient urinary concentration. In fact, multiple groups have shown that vasopressin can increase ureagenesis in liver cells in vitro [26–28]. Moreover, Staddon and McGivan showed that vasopressin and glucagon can have additive effects increasing ureagenesis in isolated hepatocytes [27]. The regulation of ureagenesis and ammonia clearance in the liver by vasopressin likely requires functional V1aR, as the V2R is not expressed in the liver [29, 30]. Exactly how vasopressin regulates ureagenesis by V1aR is unclear. Hiroyama et al. found that mice deficient in V1aR had hyperammonaemia, increased rates of proteolysis, reduced intrahepatic blood volume, and varying levels of circulating blood amino acids. However, there were no differences in blood urea nitrogen levels between V1aR deficient mice and wild-type controls [31, 32]. These results suggest that V1aR regulates ammonia entry into the urea cycle, but V1aR mice did not display the typical characteristics of a urea cycle defect. Thus, it is unclear why V1aR deficient mice have impaired ammonia clearance. An alternative activation pathway for urea synthesis triggered via renin-angiotensin-aldosterone system might be playing a role [33]. However, in aggregate these observations suggest that V1aR stimulation in the liver can modulate nitrogen waste handling and ureagenesis [34].

Arginase is the last enzyme in the urea cycle and converts arginine to ornithine and produces urea (Figure 1). There are two isoforms of arginase, Arg1 which is mainly expressed in the liver and Arg2 which is highly expressed along the nephron and in particular in the S3 segment of the proximal tubule in the kidney (Figure 1). Since the kidney does not express all the enzymes required to complete the urea cycle, arginase-2 does not produce urea to eliminate nitrogenous waste, rather urea production by arginase-2 regulates L-arginine and L-ornithine and can influence cell proliferation [35] [36]. The specific function of arginase-2 in the kidney, has been difficult to elucidate. Stimulation of the V2R with desmopressin (dDAVP) leads to an increase in arginase-2 protein expression by 60% [37, 38] yet total-body arginase-2 deficient mice had increased levels of AQP-2 and more efficient urine concentration [39]. These results would suggest that arginase-2 expression blunts urinary concentrating ability. In contrast, Ansermet et al. showed kidney tubular epithelial cell-specific arginase-2 knockout impaired the corticomedullary urea gradient and impaired urinary concentration [36]. Although the specific function of arginase-2 remains unclear, these data suggest that in addition to regulation of membrane abundance and permeability of urea transporters, vasopressin can regulate renal specific ureagenesis [40].

Aestivation, water conservation, and vasopressin

What, if any, is the physiological and clinical relevance of vasopressin, protein metabolism and ureagenesis? Enhorning and colleagues reviewed the long-term health outcomes with poor hydration status and showed that measures attributed to underhydration including high plasma vasopressin, urinary concentration, and increased plasma sodium, are associated to chronic diseases and premature mortality [3]. Thus, it is of paramount importance to have a mechanistic understanding of the role that vasopressin, protein metabolism, and ureagenesis play in the development of chronic disease.

It is known that water restriction increases the rate of protein catabolism in both birds and mammals [41]. During hibernation, which primarily occurs in winter, protein and fat are catabolized to provide energy, heat, and conserve water. During aestivation, a state of lower metabolic rate and inactivity in animals in response to heat and/or arid conditions, both invertebrate and vertebrate species utilize protein and fat catabolism as a source of metabolic water [11, 42]. In aestivation, organic osmolyte production increases as a result of protein catabolism to promote net water conservation, and as discussed above, the main protein derived organic osmolyte is urea [5, 43].

Like aestivation, vasopressin signaling is conserved across multiple invertebrate and vertebrate species and is known to play a role in the osmoregulation of animals living in arid climates [5]. Thus, both aestivation and vasopressin signaling favor net osmolyte and urea production. In fact, the primacy of urea synthesis to maintain water balance is conserved across nature. For example, Australian green-striped burrowing frogs prioritize breakdown of non-essential jumping muscles for urea production during aestivation [44]. However, one of the best examples of reliance on protein metabolism and urea production to conserve water is the lungfish. Lungfish are obligate air-breathers that live in climates that alternate between wet and arid conditions. During the dry seasons, African lungfish will produce mucus from the skin, burrow in the mud and encase themselves in a cocoon for up to three years [45]. During this period of aestivation, lungfish shift their nitrogen balance from excreting ammonia through the gills to producing urea which accumulates in the body and is excreted on arousal by wet conditions [46]. Moreover, lungfish have a functional vasopressin signaling system which express V1aR and V2R orthologs and vasotocin, a biologically active evolutionary precursor of vasopressin that is highly upregulated during aestivation [45–48]. Importantly, it has now been shown that there is an “aestivation-like” response in mammals which aids in preventing dehydration and re-prioritizes muscle catabolism for urea generation and maintenance of water homeostasis [49–52]. In particular, Titze et al. have shown that high salt feeding induces muscle catabolism and ureagenesis in both mice and humans [52]. Moreover, Hultström et al. showed that dehydration activates an “aestivation-like” responses in critically ill patients with high osmolality [53, 54]. Hultström’s group showed that in critically ill patients with and without COVID-19, a high osmolality was associated with a shift in metabolism towards amino acid production from muscle breakdown and urea production [53, 54]. Whether vasopressin has direct effects on muscle catabolism is unresolved; both synthesis and breakdown have been reported in vitro and in vivo [34]. Of note, one provocative study found that underhydration was associated to sarcopenia in elderly adults [55]. Moreover, the “aestivation-like” response reported by various groups has shown both elevated vasopressin and a glucocorticoid dependent increase in muscle catabolism. Interestingly, glucocorticoid secretion by the adrenal medulla can be augmented by vasopressin [34, 52, 56]. Therefore, aestivation represents a conserved response in which organisms increase vasopressin production and prioritize protein catabolism for osmolyte generation and net water conservation [43, 49–52].

The mechanistic molecular pathways linking muscle catabolism, increased ureagenesis, and vasopressin remain incompletely understood. However, we will focus on the role of the nuclear factor of activated T cells 5 (NFAT5) also known as the tonicity-responsive enhancer-binding protein (TonEBP), which is the master regulator of osmolyte accumulation in cells and is required for the maintenance of the renal medullary hypertonicity [57–60]. NFAT5 is a transcription factor that is induced by hypertonic stress, leading to its translocation from the cytoplasm to the nucleus where it can engage its target genes [61, 62]. NFAT5 regulates the expression of transporters that increase organic osmolytes such as the betaine (SLC6A12) and myo-inositol (SLC5A3) transporters, and well as the enzyme aldose reductase (AKR1B1) which converts glucose to sorbitol[63–66]. Increase abundance of these osmolytes is necessary for cells to the defend against intracellular water loss under the hypertonic conditions in the medulla and to contribute to the gradient necessary for urine concentration. For example, loss of aldose reductase results a diabetes insipidus phenotype[67, 68].

While it has not been directly studied if NFAT5 is stimulated by vasopressin, NFAT5 is a critical regulator of the vasopressin response. The increased tonicity in the renal medulla following stimulation of the V2R is sufficient for NFAT5 phosphorylation and activation [69]. In addition to organic osmolyte regulatory genes, NFAT5 target genes include AQP2 and the urea transporters UT-A1 and UT-A2 [70], and it has been implicated in AVP-independent regulation of these genes. Using mice expressing a dominant-negative NFAT5 in collecting duct cells, Lam et al showed that mice had submaximal urine concentration following water restriction or dDAVP administration [71]. While AQP2 and UT-A1 were upregulated, UT-A2 was not induced under these conditions, suggesting loss of NFAT5 activity results in impaired urea recycling within the kidney [71]. Whole body NFAT5 deletion in mice leads to early lethality and atrophy of the kidney medulla [72], while tissue-specific deletion of NFAT5 from principal cells results in nephrogenic diabetes insipidus that does not respond to dDAVP [59]. This suggests that NFAT5 is required for appropriate vasopressin signaling and maximal water conservation. NFAT5 expression and dysregulation has been linked to a multitude of metabolic diseases that closely mimic those associated with chronic underhydration [3, 60, 73–75], NFAT5 expression is upregulated under “aestivation-like” conditions [49], and NFAT5 in muscle has been linked to skeletal muscle formation [76]. Thus, understanding in what tissue and the specific molecular mechanisms through which NFAT5 carries out its biologic effects is critical to understanding the “aestivation-like” response to hypertonic stress.

Conclusion

Ancient, evolutionarily conserved systems in which vasopressin, protein metabolism and water conservation are regulated in parallel is present in humans. These systems are relevant to human disease, and more work is needed to understand the underlying biology to be able to develop new therapeutics for diseases like diabetes, coronary artery disease, hypertension and metabolic syndrome.

Key Points.

1. Aestivation is an evolutionarily conserved response to promote water conservation which promotes urea synthesis and muscle catabolism

2. Hypertonicity promotes muscle catabolism and increase urea synthesis

3. Vasopressin can directly regulate protein metabolism in the liver and the kidney