Why is osmolarity important to water movement and fluid balance in the body?

Control of Urine Dilution

For the kidney to generate a large volume of urine, a sufficient volume must reach the diluting segment. Oligoanuric acute renal failure (Chapter 112) and severe chronic renal failure (Chapter 121) are clinical situations in which a low glomerular filtration rate impairs delivery. In hypotension (Chapter 98), extracellular volume depletion, or edematous states associated with arterial underfilling, increased proximal tubular reabsorption results in decreased delivery to the diluting segment. However, the reduced delivery itself is a relatively minor effect compared with the accompanying elevation in ADH.

To form dilute urine, salt must be reabsorbed and water left behind by healthy cells with vigorous active transport processes. To excrete large volumes of dilute urine, the tubule must remain impermeable to water after a dilute urine has been formed. To achieve this goal, ADH secretion must be inhibited.

ADH secretion can be in response to osmotic stimuli or volume stimuli. Hypotension, extracellular volume depletion, and the edematous states (heart failure, cirrhosis, nephrosis) associated with arterial underfilling can cause an increase in ADH levels (Table 108-2). ADH also is elevated in hypothyroidism (Chapter 213) and in hypocortisolism (Chapter 214), especially when it is caused by hypopituitarism (Chapter 211). In these patients, depressed cardiac output provides a volume stimulus for ADH secretion, even though patients can be euvolemic on physical examination. Some diuretics (e.g., the thiazides) exert their effects by poisoning salt absorption in the cortical diluting segment. Severe hypokalemia decreases sodium transport out of the renal tubule cells and thus decreases the amount of salt that can be reabsorbed. Furthermore, drugs such as chlorpropamide increase ADH release, thereby impairing water excretion, and also increase the sensitivity of the renal tubule to ADH. When the tubule becomes more sensitive, hyponatremia might develop even with small intakes of water. Finally, a collection of disorders associated with elevated ADH in the absence of volume stimuli or drugs is grouped under the heading syndrome of inappropriate ADH (SIADH;Table 108-3).

Hypo-osmolality Due to Volume Disorders

In conditions such as extracellular volume depletion or depletion of the effective plasma volume, such as occurs in heart failure or cirrhosis, the ability of the kidney to make large quantities of dilute urine is severely impaired. This impairment involves two defects: ADH is secreted despite hypo-osmolarity and, to a lesser extent, inadequate delivery of tubular fluid to the diluting segment.

The afferent mechanisms in each of these clinical situations stimulate release of renin and formation of angiotensin II. The volume of fluid that leaves the proximal tubule to go to the loop of Henle will, therefore, be less than the amount delivered to the loop in states of volume expansion. Hence, the amount of free water that can be formed and later excreted will decrease, even though the function of the loop of Henle is normal. Because the amount or salt and water reabsorbed in the proximal tubule is greater than normal, the tubular concentration of other substances not as freely permeable as water will also increase. Because urea and uric acid are diffusible across the tubular membrane, they will be reabsorbed to a much larger extent than normal because the concentration difference across the membrane is greater. Hence, in these situations the plasma concentration of urea and uric acid tend to be higher than normal.

In all of these conditions—extracellular volume depletion, heart failure, and cirrhosis—ADH secretion is increased owing to the activation of volume receptors.These patients require a lower osmolality to suppress ADH secretion and have higher ADH levels at osmolarities above the set point. This high ADH level, which is the dominant factor for the development of hypo-osmolality in these conditions, will further decrease the volume of free water that is excreted and accentuate the hyponatremia. The decrease in the volume of dilute urine that can be excreted will not lead to hypo-osmolality unless the patient’s water intake is larger than the amount of water excreted. For a given defect in water excretion, the severity of the hypo-osmolality is proportional to the water intake. In extracellular volume depletion and heart failure, the thirst mechanism is activated, with consequent increases in water intake. Not only is water excretion decreased, but also water intake is increased.

13.11.4 Osmolarity (Ashara and Shah, 2017)

Mean osmolarity of human tears is about 310 mOsm/kg and a tonicity is equivalent to 0.9% sodium chloride solution. The osmotic pressure controls the blood–aqueous barrier permeability, which is maintained by the presence of sodium, chlorine, and bicarbonate transport as well as by the physiochemical properties of drugs. The pH of the administered formulations must be ideally about 7.4, which is the pH of the tears, and osmolarity must be around 310 mOsm/kg. The various ocular formulations show several advantages (for e.g., hydrophilic ointment is easy to wash) but their use restricted due to discomfort caused by the osmotic effect (Dubald et al., 2018). Thus, the osmolarity of the tested ophthalmic preparations is to be calculated by the following equation.

mOsm/L=Concentration(g/L)Molecular weightx100

An adjusting substance, usually sodium chloride, is to be added to the exact amount, which is calculated by using the following equation to make an isotonic solution from hypotonic solution.

Weight of sodium chloride required=0.52−freezing point of unadjusted solutionfreezing point depression of1%solution of drug

Toxins: The Osmolar Gap in Toxin-Induced Acidosis

The so-called “osmolar gap” can indicate the presence of certain toxins. Under most physiologic conditions, Na+ (and its accompanying anions), urea, and glucose generate much of the osmotic pressure of blood. Serum osmolality is calculated according to the following expression:

(16.16)Osmolality=2 [Na+]+BUN2.8+Glucose (mg/dL)18

The calculated and determined osmolalities usually agree within 10 mOsm/kg. When the measured osmolality exceeds the calculated osmolality by more than 10 mOsm/kg, one of two possibilities usually exists. First, the serum Na+ may be spuriously low, as occurs with hyperlipidemia or hyperproteinemia (pseudohyponatremia). Second, osmolytes other than sodium salts, glucose, or urea may have accumulated in plasma. Examples are infused mannitol, radiocontrast media, or other solutes, including the alcohols, ethylene glycol, and acetone, which can increase the osmolality in plasma. For these examples, the difference between the osmolality and the measured osmolality is proportional to the concentration of the unmeasured solute. Such differences in these clinical circumstances have been referred to as theosmolar gap. In the presence of an appropriate clinical history and index of suspicion, the osmolar gap becomes a very reliable and helpful screening tool in assessing for toxin-associated high AG acidosis. An increase in the osmolar gap can be associated with AKA and lactic acidosis as discussed in the previous section, but in general, ethanol intoxication per se does not cause a high AG acidosis, although it can cause an elevated osmolar gap. Isopropyl alcohol (rubbing alcohol) poisoning similarly is not metabolized to a strong acid and does not elevate the AG, although the osmolar gap can be elevated and there can be a positive nitroprusside reaction from the metabolism to acetone.

Osmolarity

The osmolarity laboratory provides a clear, easy technique for identifying and quantifying cell response to changes in osmotic conditions. Contractile vacuoles in Tetrahymena are large and visible even with the fairly low-quality microscopes generally available in high school biology labs, and changes are readily captured for detailed analysis using the single frame, time lapse, and movie options available with low-cost digital cameras. Tetrahymena contractile vacuoles gather and expel water in periodic fashion, with the rhythm and rate of contraction dependent on environmental factors. The laboratory addresses membrane permeability and osmoregulation in a free-swimming cell under a variety of conditions. The exercise can be varied according to class level, from simply observing changes to collecting and graphing data on rates of contraction under different conditions to student-designed experiments altering the cell environment in specific ways and recording and analyzing the results.

Students learn concepts related to water and chemistry of life; physiological regulation; relationship of structure to function; homeostasis; feedback mechanisms; and structural similarity between single cell and multicellular organisms.

The specific protective mechanism in response to sorbitol osmotic stress signal

At high osmolarity, two branches of the HOG pathway, the SHO1 branch and the SLN1 branch, are observed to sense osmotic changes and rapidly make internal adjustments. In Fig. 6.10, sorbitol osmotic stress is shown to have many more mutual interactions and feedforward loops in the HOG pathway than hypo-osmotic stress (Fig. 6.9). The connections may make pathways more rapid and more robust (acting against external noise) in response to sorbitol osmotic stress.

According to Table 6.2B, the new found proteins are the 12 proteins (highlighted by gray color) which interact with a significant number (>5) of preselected proteins. The 12 proteins are grouped and will be discussed in the following paragraphs based on the research shown in Table 6.2B.

Two proteins, namely WSC3 and SPA2, are the new found proteins which are known as members of the cell-wall integrity pathway. WSC3 is involved in the maintenance of cell wall integrity [82], while SPA2 acts as a scaffold protein for MKK1 and MPK1 [64]. In addition, BEM4 is probably involved in the RHO1-mediating signaling pathway [78]. BEM4 is functionally relevant to RHO1 and should play a novel role in the signaling pathway mediated by RHO1. One possible role of BEM4 is to act like chaperone in the stabilizing or folding of RHO1. According to the cell-wall integrity pathway shown in Fig. 6.10, we suggest that RHO1, PKC1, and SLT2 may play important roles in the inactive cell-wall integrity pathway under sorbitol osmotic stress.

In the pheromone response pathway, four proteins, including GPA1, SST2, FAR1, and GIC2, are the new found proteins as shown in Table 6.2B. GIC2, whose function is still unknown, can interact with CDC42, and therefore GIC2 is grouped with the pheromone response pathway and the SHO1 branch of the HOG pathway [79]. GPA1, a Gα subunit, has been involved in mediating pheromone response pathway [64,80]. SST2 is required to prevent receptor-independent signaling of the pheromone response pathway [81]. Additionally, Far1 is a cell cycle arrest mediator [76].

In the SLN1 branch of the HOG pathway, SKN7 and NBP2 are new found proteins participating in this important pathway under sorbitol osmotic stress. SLN1-YPD1-SKN7 has been proven to act as a phosphorelay system that turns on the HOG pathway until the yeast suffers from cell shrinking (Fig. 6.10) [37,64]. In addition, SKN7 appears to have different functions, such as acting as a transcription factor or a protein in signaling systems, not only mediating different stresses but also linking the cell-wall integrity pathway to the HOG pathway mediated by interacting directly with RHO1 (Fig. 6.7). During yeast adaptation, NBP2 is predicted to act as an adapter, recruiting PTC1 to the PBS2-HOG1 complex in the PTC1 inactivation of HOG1. We suggest that the activated HOG pathway under sorbitol osmotic stress is due to the unbound NBP2-PBS2 complex which results in HOG1 which cannot be inactivated by PTC1 [75].

In the SHO1 branch of the HOG pathway, the new found proteins OCH1, SKM1, and RGA1 are probably important in response to high osmolarity. The promoter of OCH1, which encodes a mannosyltransferase, responds to the presence of SLN1, and KSS1 is activated by the mutation of OCH1 [64]. Therefore, we suggest that OCH1 participates in the SHO1 branch of the HOG pathway under sorbitol osmotic stress. SKM1, which is similar to STE20 and CLA4, is probably a downstream effector of CDC42, but the function of SKM1 is still unclear [37]. According to [92], CDC42 may promote the phosphorylation of GIC2 by recruiting STE20 and SKM1. Therefore, we suggest that SKM1 is a member of the SHO1 branch of the HOG pathway under sorbitol osmotic stress. RGA1 is suggested as a link between CDC42 and pheromone pathway components [83] (Fig. 6.7). Although the 12 new found proteins are probably important in response to sorbitol osmotic stress, most of them, such as BEM4, GIC2, FAR1, OCH1, SKM1, and RGA1, are functionally unclear, and likely even participate in multiple pathways with complicated roles. We can only infer some possible protective mechanisms according to previous studies and these results.

3 The HOG Signaling System

The HOG pathway (Fig. 2.1) is one of the best understood and most intensively studied MAPK systems. First, components (Hog1 and Pbs2) were identified in a genetic screen for osmosensitive mutants deficient in glycerol accumulation (Brewster et al., 1993). In parallel, inactivation of SLN1, encoding the single yeast sensor histidine kinase, was found to be lethal (Ota and Varshavsky, 1993). This lethality, which was later shown to be because of inappropriate overactivation of the Hog1 kinase, was suppressed by knockout of any of the genes SSK1, SSK2, PBS2, and HOG1, thereby defining a linear pathway from Sln1 to Hog1. In addition, overexpression of PTC1, PTP2, or PTP3 suppressed lethality of the sln1Δ mutant, defining those as negative elements of the pathway (Maeda et al., 1994; Posas and Saito, 1997; Posas et al., 1996). Finally, the observation that ssk1Δ as well as ssk2Δ ssk22Δ mutants were osmotolerant while deletion of PBS2 and HOG1 caused osmosensitivity prompted genetic screens employing synthetic enhancement that identified SHO1, STE20, and STE11 as encoding components of the Sho1 branch (Maeda et al., 1995). Identification of the pathway components and characterization of their order of function represent textbook examples of the power of both targeted and global yeast genetics approaches. In fact, forward genetics, suppressor mutation, multicopy suppression, synthetic enhancement, epistasis analysis, and yeast two hybrid screens were all employed in this context. Particular powerful genetic tools are mutations that activate signaling constitutively. Significant knowledge has emerged on the flow of information through the pathway and hence the mechanisms of signal transduction by combining the genetic tools with in vitro and in vivo protein interaction assays, as well as in vitro protein kinase assays (de Nadal et al., 2002; Hohmann, 2002; O'Rourke et al., 2002; Saito and Tatebayashi, 2004; Tatebayashi et al., 2006).

The HOG signaling system consists of two branches that converge on the MAPKK Pbs2, the Sln1, and the Sho1. Components of the Sho1 branch also take part in pseudohyphal development and mating in S. cerevisiae (O'Rourke and Herskowitz, 1998). In many fungi, it appears that the Sho1 module is not connected to Pbs2 and hence is not involved in osmotic responses (Furukawa et al., 2005; Krantz et al., 2006). This indicates that the Sho1 module might not primarily have a role in osmosensing but rather perceives signals related to cell shape and/or cell surface conditions, in accordance with the role in activation played by the cell polarity machinery. Sho1 is specifically located at sites of cell growth and does not appear to sense turgor changes (Reiser et al., 2000, 2003).

The Sho1 branch consists almost exclusively of proteins shared with the pseudohyphal development pathway and the pheromone response pathway. Signaling specificity seems to be assured by recruitment to scaffold proteins (Sho1, Opy2, Pbs2) and requires the Hog1 kinase. In hog1Δ mutants, exposure to osmotic stress causes activation of the pseudohyphal and pheromone response pathways and morphological aberrations (Davenport et al., 1999; O'Rourke and Herskowitz, 1998; Rep et al., 2000). The mechanism by which Hog1 prevents such cross talk has not yet been elucidated. Mechanisms involved in activation of the Sho1 branch following osmotic shock have been described in detail using constitutively active Stell and Sho1 mutants as well as protein interaction studies (Tatebayashi et al., 2006). As indicated earlier, the sensing mechanism of osmotic changes in the Sho1 branch is not understood at this point but must be closely related to Sho1 (Tatebayashi et al., 2006). The observation that Sho1 can be replaced by engineered proteins that recruit Pbs2 to the plasma membrane suggests that Sho1 does not function as a sensor itself (Raitt et al., 2000). Sho1 shows much less variation in size than in primary sequence (Krantz and Hohmann, 2006), indicating a structural rather than an enzymatic function.

Sln1 is a sensor histidine kinase related to bacterial two‐component systems. Such proteins are widespread in fungi and plants (Catlett et al., 2003). Sln1 has a similar domain organization as the bacterial osmosensing histidine kinase EnvZ. Both proteins have two transmembrane domains at their N terminus, which are connected by a large extracellular loop, about 300 amino acids in yeasts. It is believed that the extracellular loop and the transmembrane domains sense turgor changes (Reiser et al., 2003), perhaps by responding to movements of the plasma membrane relative to the cell wall. The homodimer is likely regulated by a structural change, which is propagated from the extracellular sensing domain to the intracellular histidine kinase domain of Sln1 (Posas et al., 1996; Reiser et al., 2003). In S. cerevisiae the Sln1 histidine kinase is a negative regulator of the downstream MAPK cascade; deletion of SLN1 or inactivation of the kinase results in lethal Hog1 overactivation (Maeda et al., 1994). When active (i.e., under ambient conditions), the Sln1 histidine kinase cross‐phosphorylates within a dimer (Posas et al., 1996), and the phosphate group is transferred via the Sln1 receiver and response regulator domains as well as the Ypd1 phosphotransfer protein to the Ssk1 response regulator protein. Hyperosmotic shock causes inactivation of Sln1 kinase activity and dephosphorylation of Ssk1. This scenario is well supported by mutational analysis of all steps in the phosphorelay system (Posas et al., 1996). Unphosphorylated Ssk1 mediates activation of the redundant MAPKKKs Ssk2 and Ssk22, which in turn activate Pbs2.

The activity and the relative contribution of the two pathway branches to Hog1 kinase activity are usually measured in mutants that are blocked in either branch (Maeda et al., 1995; O'Rourke and Herskowitz, 2004). Whether such experiments reflect activity of the two branches in wild‐type cells is presently unknown. It appears that the Sho1 branch has a higher stress threshold for activation (Maeda et al., 1995; O'Rourke and Herskowitz, 2004) and that it is insufficient to mediate maximal pathway activation alone (unpublished data).

Osmolarity

The increase in osmolarity (in milliosmoles per liter [mOsm/L]) that occurs in DKA must be differentiated from the increase in osmolarity seen in hyperosmolar-hyperglycemic nonketotic (diabetic) coma (HHNC). The osmolarity can be measured by freezing point depression or estimated with the use of the following formula:

Osmolarity=(2×sodium)+(glucose/18) + (BUN/2.8)+(ethanol/4.6)

Patients with DKA not uncommonly present with hyperosmolarity and coma. In HHNC, the osmolarity is usually greater than 350 mOsm/L, and it can exceed 400 mOsm/L. The serum sodium and potassium levels can be high, normal, or low and do not reflect total-body levels, which are uniformly depleted. The glucose concentration is usually greater than 600 mg/dL, and levels higher than 1000 mg/dL are common. In pure HHNC, there is no significant metabolic acidosis or anion gap.

Patients often present with combinations of the preceding findings. HHNC can involve mild to moderate ketonemia and acidosis. Alcoholic ketoacidosis can contribute to either DKA or HHNC. Lactic acidosis is common in severe DKA and HHNC. Any patient with hyperglycemia greater than 250 mg/dL and an anion gap metabolic acidosis should be treated by the general principles outlined in the following section, with special consideration for other possible contributing metabolic acidoses.

Why is osmolarity of body fluids important?

How does osmolality maintain water balance?

How osmolality affects the movement of fluid between the fluid compartments?

What is osmolarity of body fluids?