An understanding of the role of TRPV4 in mammalian kidney physiology begins in the nematode C. elegans. Worms are repelled by steep osmotic gradients, an adaptive mechanism presumably conserved to minimize the risk of acute changes in cell volume. Worms with mutated copies of a particular gene, while remaining fully motile, are oblivious to potentially harmful osmotic gradients; this gene was dubbed osm-9 [1,2]. The OSM-9 protein bore a striking similarity to the transient receptor potential channel implicated in visual signal transduction in Drosophila [3]—the prototypical member of the now well-recognized TRP superfamily [4]. Several years later, the mammalian orthologue of OSM-9 was identified via homology screening [5,6] and other approaches [7,8]; it eventually became known as TRPV4 [9].

ACTIVATION OF TRPV4

TRPV4 IN CELLULAR OSMOREGULATION

Recent data indicate that TRPV4 regulates water balance at the cellular level. Cells subjected to a hypotonic milieu abruptly swell as a consequence of water entry. Unregulated water entry, facilitated by membrane expression of water channel proteins, constitutes a two-pronged threat: the ensuing increase in hydrostatic pressure jeopardizes cell integrity, while dilution of the cytoplasm adversely affects macro-molecular structure and function. Acutely swollen cells jettison ions and organic solutes to permit water efflux in a process known as regulatory volume decrease. This phenomenon has been studied in great detail in a number of in vitro model systems. In most, hypotonic stress is rapidly followed by calcium entry (reviewed in reference 49); this proximal signaling event then triggers efflux of ions (particularly K⁺ and Cl⁻) and osmotically active organic solutes such as taurine and sorbitol (reviewed in reference 50).

Although many of the volume-regulatory effector mechanisms triggered by swelling-induced calcium transients are conserved across model systems, the mechanism of calcium entry itself is highly cell type–specific. In the model of human airway epithelial cells, as in most models, the regulatory volume decrease following hypotonic stress is calcium dependent [51]. In this model, both the hypotonicity-dependent calcium entry and subsequent regulatory volume decrease require TRPV4 [52]. Downregulation of endogenous TRPV4 expression with a TRPV4-directed antisense prevented activation of a calcium-dependent potassium channel by hypotonicity and completely prevented restoration of normal cell volume. An irrelevant antisense had no effect (Figure 29.1A). These important data were the first to link TRPV4 function to the regulatory volume decrease response itself, and the data imply a pivotal role for TRPV4 in water balance at the cellular level.

FIGURE 29.1

Role of TRPV4 in cellular osmoregulation. (A) LCFSN cells, which natively express TRPV4, were transfected with antisense directed against TRPV4 or an irrelevant control antisense (beta-globin). Increase in cell volume (percent) was then monitored as a (more...)

Becker and colleagues went on to show a similar relationship [53]. Rather than blocking regulatory volume decrease in TRPV4-expressing cells, they investigated the process in the Chinese hamster ovary (CHO) cell line, which lacks TRPV4 expression. Naïve CHO cells failed to undergo regulatory volume decrease following osmotic swelling (Figure 29.1B); heterologous expression of TRPV4, however, was permissive for this restorative change in cell volume [53]. So it appears that, at least in some models, downregulation of TRPV4 expression abolishes regulatory volume decrease, and heterologous expression of the channel is sufficient to confer this adaptive response.

TRPV4 IN SYSTEMIC OSMOREGULATION

Functional Anatomy of TRPV4 in the Mammalian Kidney

Four reports of the cloning of TRPV4 independently noted expression of the channel in the kidney [5–8], particularly in the distal convoluted tubule. In a person of average size, the kidneys convert ~150 liters of daily glomerular filtrate (potential urine or “proto-urine”) into 0.5–1.5 liters of urine; in so doing, they also reabsorb nearly all of the filtered water and inorganic ions, as well as glucose, amino acids, and a wide variety of essential plasma constituents [54]. This 100-fold reduction in urine volume achieved along the kidney tubule is vital; in its absence, lethal volume depletion and hypotension would ensue in minutes. Such robust water conservation is achieved via a combination of apical water permeability of specific kidney tubule segments and a hyperosmotic renal medullary interstitium. Together, these factors establish both a route and a driving force for water reabsorption from the tubule lumen into the systemic circulation. Although no data unequivocally impute a role for TRPV4 in this process, clues have emerged from a detailed investigation of the distribution of TRPV4 expression in the kidney. Glomerular filtrate traverses the hyperosmotic renal medulla via the tight hairpinlike Loop of Henle (Figure 29.2A). The cells lining the “descending” arm of the loop are freely water permeant; the filtrate becomes progressively more concentrated as water traverses the tubule. An abrupt transition occurs at the genu of the loop, where the “descending” limb meets the “ascending” limb: here, apical water permeability abruptly ceases. This architecture “traps” the newly concentrated filtrate in the lumen, preventing its passive dilution by water entry as it exits the hypertonic inner medulla of the kidney. Interestingly, expression of TRPV4 is completely absent along the early parts of the kidney tubule where passive water reabsorption takes place; however, precisely at the transition between tubule water permeability and impermeability (at the hairpin turn at the base of the Loop of Henle), TRPV4 expression abruptly emerges (Figure 29.2A; [30]). TRPV4 expression continues thereafter for the length of the kidney tubule. The solitary exception is the highly specialized tubule segment called the macula densa—the only other tubule segment exhibiting constitutive apical water permeability. Here, as in the more proximal water-permeant tubule segments, TRPV4 expression is absent [30]. Therefore, TRPV4 expression in the kidney is entirely restricted to those tubule segments that lack constitutive apical water permeability and where a transcellular osmotic gradient may be expected to develop.

FIGURE 29.2

TRPV4 in the mammalian kidney. (A) TRPV4 expression along the mouse and rat nephron. Black shading indicates strong TRPV4 expression; gray shading indicates less intense TRPV4 expression. Throughout the course of the collecting duct, expression was stronger (more...)

The Role of TRPV4 in Regulating Water Balance in vivo

Sodium Balance versus Water Balance

As a putative sensor of tonicity, it is likely that TRPV4 responds to perturbations in systemic water balance rather than systemic sodium balance. Serum sodium concentration is largely independent of total body sodium balance; clinically, these two parameters are readily separable. In humans, the differential diagnosis of hypernatremia (a surrogate for plasma hypertonicity) requires an assessment of total body sodium content and water content. In nearly all circumstances, hypernatremia reflects water loss with or without sodium loss, although in rare instances it may follow ingestion or administration of concentrated sodium-containing salts (reviewed in references 58 and 59). Total body sodium balance cannot be assessed quantitatively in humans in clinical practice; rather, it is inferred from a physical examination. Findings consistent with hypovolemia (a deficit in total body sodium content) include increased heart rate and decreased blood pressure. In addition, urinary sodium excretion is quantified as a crude estimate. That is, abnormally low urinary sodium excretion is a marker for decreased total body sodium content (hypovolemia), whereas a “normal” degree of urinary sodium excretion is consistent with intravascular volume repletion and a physiological level of total body sodium content.

Classification of Hypernatremia

Despite the total body sodium content of the individual, hypernatremia nearly always reflects water loss—water loss in the absence of sodium loss in the “euvolemic” hypernatremias and water loss in excess of ongoing sodium loss in the “hypovolemic” hypernatremias. The presence or absence of associated sodium loss, while not impacting the hypernatremia per se, assists in identifying the pathological abnormality [58,59]. Euvolemic hypernatremia is caused primarily by excess urinary water loss (diabetes insipidus) or hypodipsia (decreased thirst). Nonurinary sites of water loss are also possible (e.g., water loss through the gastrointestinal tract or through perspiration). Increased water loss alone, unless overwhelming, rarely produces significant hypernatremia because it is limited by AVP action on the kidney-collecting duct (when urinary concentrating ability is preserved), and because it produces a powerful urge to drink. In the absence of an appropriate thirst response or when water is unavailable, hypernatremia can quickly ensue despite maximal urinary water conservation. Correction of euvolemic hypernatremia requires water alone. Hypovolemic hypernatremia, in contrast, results from loss of more than just pure water; “wasting” of sodium in the urine or from other sites (e.g., gut, sweat glands, etc.) is also required. If the sodium loss occurs from a nonrenal site, urinary sodium conservation is seen. Aldosterone, upregulated by the hypovolemia, minimizes urinary sodium excretion. In contrast, if the kidneys are the culprit in the sodium loss, this adaptive response is conspicuously absent. Correction of hypovolemic hypernatremia requires repletion of both water and sodium (albeit more of the former).

Water Balance in TRPV4^−/− Mouse Models

It is helpful to interpret the available data for each of the two independently reported TRPV4 knockout models in light of this clinical paradigm. It is also important to emphasize, as had previously been noted, that some discrepancies between these authors’ findings might have arisen because of the different ways in which the models were generated [14].

Mizuno et al. [60] compared plasma sodium concentration in TRPV4^−/− and wild-type mice. In their relatively small sample (n = 10 per group), they were unable to demonstrate a difference between the mice under basal conditions, although there was a nonsignificant trend toward higher plasma sodium concentration in the TRPV4^−/− mice (140.7 + 0.8 vs. 137.9 + 1.0 mEq/l). This group also reported lower urinary sodium levels for the TRPV4^−/− mice (123 + 15 vs. 165 + 21 mEq/l). In addition, they noted an insignificant but seemingly substantial decrease in systolic blood pressure among the knockout mice (105 + 4 vs. 116 + 5 mmHg). These two latter parameters are perhaps consistent with a modest relative decrease in total body sodium content and effective intravascular volume (hypovolemia); however, because these were not balance studies, the discrepancy in urinary sodium concentration might reflect in part the greater dilution of the −/− urine (see below). Therefore, no firm conclusion can be drawn about the total body sodium (“volume”) status in these mice. With respect to water balance in this study, there was a trend toward higher water loss (urine volumes of 1.8 + 0.5 vs. 1.4 + 0.4 ml/d) and greater water intake (4.5 + 1.0 vs. 4.0 + 0.8 ml/d) among the TRPV4^−/− mice, although this did not reach the threshold for statistical significance. Urine volume is not a surrogate for urinary water excretion, of course, as the urine may be solute rich or solute poor, and the latter will contribute more to free water clearance. Nonetheless, consistent with these findings was the relative (and again insignificant) decrease in urinary creatinine, an indirect index of the degree of urinary concentration. With elevated plasma osmolarity, this constellation of findings could represent either (1) excess water diuresis driving thirst or (2) osmotic diuresis (e.g., from glycosuria) driving thirst. Discrimination between these scenarios requires an assessment of urine osmolarity; a relatively diluted urine is consistent with the former, whereas an “isosthenuric” urine (i.e., urine with a relatively fixed osmolarity approximating that of plasma) suggests the latter. However, the “basal” urine osmolarities were identical in the +/+ and −/−mice in this model.

Unregulated renal water loss (diabetes insipidus), which was likely a factor in the Mizuno model (Figure 29.3), generally reflects either a defect in hypothalamic production of the water-retentive peptide arginine vasopressin (AVP) or a defect in the renal response to this hormone. Perhaps consistent with the former, basal levels of serum AVP were slightly (albeit insignificantly) lower in the −/− mice. Consistent with a nephrogenic picture, however, AVP levels in response to an acute osmotic load were dramatically higher in the −/− mice relative to wild-type mice. The osmotic stimulus in this exercise was supraphysiological; the serum osmolarity was acutely increased from 310 to 410 using propylene glycol infusion. But in vitro exposure of brain slices to much more subtle hyperosmotic stimuli resulted in a similarly enhanced release of AVP from TRPV4^−/− slices relative to slices from the +/+ brains. In addition, independent of AVP, excessive water diuresis can also be seen with gross abnormalities in plasma calcium or potassium concentrations, but neither were present in this model. In aggregate, the data seem most consistent with diabetes insipidus in the TRPV4^−/− mice in the model of Mizuno et al. (Figure 29.3). Of note, this interpretation is based primarily on nonsignificant data trends; nonetheless, these “trends” are internally consistent and hence credible.

FIGURE 29.3

Genesis of a water deficit in mammals. In diabetes insipidus (A), excess urinary water loss (filled arrow) relative to normal (B) is the primary disorder; in primary hypodipsia (C), decreased water intake is primary. The thickness of the horizontal arrows (more...)

In the model of Liedtke and Friedman, plasma osmolarity was measured rather than plasma sodium concentration [61]. In contrast to the study of Mizuno et al., Liedtke found a clear-cut increase in plasma osmolarity among the TRPV4^−/− mice (300 vs. 295 mosmol/kgH₂O). To “unmask” this difference, however, mice were single-housed and deprived of food for 24 hours prior to assessment; in the absence of these refinements, no difference was observed. Whereas assessment of plasma osmolarity rather than plasma sodium appears to be a more direct measurement of this physiological parameter, it is subject to confounding in practice. Specifically, osmotically “ineffective” substances in the plasma such as urea (i.e., blood urea nitrogen) can contribute to measured osmolarity. However, because urea readily crosses most cell membranes, it is not considered an “effective” osmole in vivo. In contrast, other nonsodium solutes are osmotically effective in vivo (e.g., glucose) and do contribute to plasma osmolarity. Therefore, from a clinical perspective, plasma sodium concentration is frequently a more reliable index of a relative water deficit or excess than is plasma osmolarity; when the latter is used in the evaluation of human disease, simultaneous determination of blood urea nitrogen and glucose is generally obtained. Also of note in this study, mice were deprived of food but access to water was not restricted. Dietary intake is the major source of urinary urea, which contributes osmoles to the urine and can act as an osmotic diuretic. Protein intake also dictates blood urea nitrogen, which, although osmotically ineffective, influences measured osmolarity of the plasma. Therefore, protein starvation can significantly impact plasma and urine osmolarity and urine volume independently of water balance. It is unclear if these may have been factors in their study.

With respect to an assessment of volume status (total body sodium content) in the study of Liedtke and Friedman, blood pressure and urinary sodium content were not reported. It is in the realm of water balance where the striking findings emerge. In concert with an elevated plasma osmolarity, and in marked contrast to the observations of Mizuno et al., Liedtke noted dramatically reduced (~30 percent less) water intake among the TRPV4^−/− mice [61]. When water was restricted for 24 hours, the discrepancy in plasma osmolarity was magnified, increasing by a further 6 percent in the −/− mice compared to only 3 percent in wild-type mice. This suggested accelerated water loss in the TRPV4 knockout mice, although data comparing urine volume or osmolarity were not available. In aggregate, these data seem most consistent with hypodipsia (i.e., a primary decrease in thirst or osmosensation; Figure 29.3) and, potentially, an inappropriate renal response to this disorder.

The data of Mizuno and colleagues [60] and of Liedtke and Friedman [61] suggest dissimilar disorders of water balance. In general, when evaluating a state of plasma hypertonicity, maneuvers designed to impact water access or water loss are paramount. Water restriction, with serial monitoring of an increment in plasma sodium (and osmolarity); detailed analysis of urine volume, osmolarity, sodium, and potassium content (i.e., in metabolic cages); and assessment of water intake would be optimal. The impact of supplemental AVP, if any, on these parameters could then be assessed. It is possible that TRPV4^−/− mice will exhibit partial abnormalities at multiple levels. Tissue-specific targeting of a TRPV4 knockout to either the distal nephron or the lamina terminalis might permit dissection of these events. In the early and ground-breaking studies of these two groups of investigators, it could not be envisioned beforehand that hypertonicity would ensue in the TRPV4^−/− mice; in vitro data with TRPV4 as a sensor of hypotonic stress suggested that genetic absence of this protein might predispose to water excess rather than water loss. But now the foundation has been laid for a detailed investigation into the role of TRPV4 in the regulation of systemic water balance under basal and pathological conditions.

SUMMARY

TRPV4 is activated by a variety of fatty acid metabolites and by a number of physical stimuli including thermal and osmotic stresses. It is likely that tonicity-dependent activation of TRPV4 requires SRC family kinase–dependent tyrosine phosphorylation. Other data support a role for phospholipase A₂ and cytochrome P450 epoxygenase activity and involvement of epoxyeicosatrienoic acids. The architecture of subcellular localization of TRPV4 in the mammalian kidney suggests a role for this channel in responding to interstitial signals related to systemic water balance. The TRPV4-expressing tubule segments may then integrate and respond to these signals by altering lumenal solute reabsorption in a “feed forward” regulatory mechanism. The two TRPV4^−/− mouse models generated to date have given rise to conflicting data: genetic absence of this sensor of hypotonicity induces a state akin to diabetes insipidus in one model and primary hypodipsia in the other. It is likely that more detailed water (and solute) balance studies will establish whether these models are as dissimilar as they initially appear, and will clarify the role of TRPV4 in regulating systemic water balance.

Acknowledgments

ACKNOWLEDGMENT

This work was supported by the National Institutes of Health, the Department of Veterans Affairs, and the American Heart Association.

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