James Cooper, D2, 00509474

Comparing the effects of isotonic saline and water on urinary output to investigate how different water balance control systems differ
Cooper, James A, Imperial College School of Medicine. Completed May 23rd 2010, submitted May 24th 2010 Introduction The kidneys are vital as one of the bodys major sources of osmolarity and blood volume control. They control excretion of water and electrolytes by Abstract: Isotonic saline and water theoretically activate different systems affecting renal output. Saline should elicit an effect through the reninangiotensin system and baroceptors whilst water should influence osmoreceptors and vasopressin. As these systems both affect the kidney but through different if overlapping mechanisms, we tested the extent of the differences between mechanisms by giving subjects a litre of 0.9% saline or a litre of water and measuring subsequent urine output and specific gravity against subject baseline values. We also gave subjects other stimuli such as caffeine to see how they interacted with these two systems. We found that water caused a greater increase in flow rate than saline, which makes sense as it would partially activate both systems, and that water also caused a greater decrease in specific gravity, due to saline blocking the salt retention of the angiotensin

filtering them out from the blood and reabsorbing the amounts of each it needs with specific channels and transporters (1). If you give a patient an excess of water, there is a drop in osmolarity detected by osmoreceptors that causes the hypothalamus to produce less vasopressin to be released in the neurohypophysis(2). Vasopressin acts on the collecting duct to increase Aquaporin production, and aquaporin is the specific channel for water to be reabsorbed(3,4) so there are fewer aquaporins expressed in the collecting duct so less water is absorbed, thus more water is excreted with fewer electrolytes, amending changes to both osmolarity and volume.

in osmolarity to be detected but there will still be an increase in volume, so the body must detect this change in another manner to deal with the change. Increase in volume will cause an increase in blood pressure. Baroceptors sense an increase in pressure and fire more frequently. Via the hypothalamus the sympathetic nervous system will be inhibited and the parasympathetic system will be stimulated, causing vasodilation and decreased cardiac output(5). This will reduce blood pressure in its own right but also increase renal perfusion which increases urinary output. An increase in blood pressure is also detected by the juxtaglomerular cells at the distal convoluted tubule of the kidney, which then produce less renin (6). The increase also leads to atrial natriuretic peptide production in the heart, which inhibits production of renin and aldosterone (7). Renin is an enzyme that converts angiotensinogen to Angiotensin which eventually causes (see diagram 1): vasoconstriction eventually causing more water reabsorption down the water potential gradient; increased aldosterone production, which increases water and sodium reabsorption through increased expression of sodium-potassium antiporters and sodium channels in the distal convoluted tubule; increased vasopressin production; increased expression of sodiumhydrogen antiporters on the apical membranes of cells in the proximal tubule and thick ascending limb of the loop of Henle leading to sodium retention; and finally increased osmotic pressure

However if we add normal saline (0.9%) then we are adding something with the same osmolarity as blood. Thus there will be no change

Diagram 1: The Renin Angiotensin

James Cooper, D2, 00509474

relative to hydrostatic pressure in the renal arterioles leading to more reabsorption(8). If renin is downregulated, all these effects are downregulated and less water and salt are reabsorbed.(8)(8)(7) So these pathways are theoretically affected differently by water or saline consumption, but to what degree do they differ in activation and effect? I aim to compare the effects on urinary output of water and saline and a few other variables, in order

saline and water and then comparing their trends to those of subjects receiving caffeine, caffeine with glucose, glucose, exercise/dehydration and water with DDAVP, as these variables might add to our understanding of how each pathway interacts. We chose specific gravity because it can be measured easily and is an indicator of the urines osmolarity and thus the excretion of solutes relative to that of water. We chose flow rate because it is an indicator of water excretion and takes different timings of results into account. Method Population the study group for this trial was taken from 2nd year medical students, all of Imperial College, London. Thus the age of participants was predominantly 19-21 year olds, and if we assume that there are an equal number of omissions in boys and girls and an equal distribution amongst randomised groups, a male: female ratio of 4:3. No study was undertaken to look at any underlying health problems or differences or ethnic variation in groups. Due to medical school procedure, the population had already been broken up into 28 randomised groups so we used these as our randomisation process, then paired 4 groups together into 7 supergroups (as it were) undergoing a specific stimulus. The total numbers were 47 (+1), 43, 37 (+1), 36, 37, 37, and 40 in each group respectively as listed below (those who dropped out of the trial before its completion in brackets) giving a total of 277 participants. We did not take a power calculation to see what sample Ingredients of bolt energy drink : diagram 2

size we needed, we just used the sample available to us. Interventions-The selected stimuli were (in order): drinking a litre of water, a litre of isotonic saline at 0.9%, a litre of diet bolt (a drink containing 300 ml of caffeine per litre with negligible sugar content), a litre of bolt (a drink containing 300ml of caffeine and 100g of glucose per litre, see diagram 2), a litre of water followed by an injection of DDAVP (containing 1 microgram of DDAVP, in 2.5ml of Sodium chloride solution) an hour later, a litre of water followed by 20 minutes of exercise (of their choice) beginning an hour later, and finally a litre of 5% dextrose solution. If anyone could not/really did not want to receive their allocated stimulus, they could request to switch to another group. Procedure Each volunteer was to arrive, note the time of their last urination, and pass water into a collecting jug. Then they would measure the specific gravity and the volume of their urine using standard dipsticks and a measuring cylinder. Then they consume their fluid (e.g. 1l of water or saline), note the time they finished and continuing to take urine samples as in the first instance. Each time they would note the time since the last reading, the volume passed and its specific gravity. If they had a further intervention they would take it at the times indicated above. The samples were meant to be at 20 intervals but this was not always possible, even though a pilot study indicated it should be. This only had 2 people in it, and did not take

to investigate the differences in the results and stimulation of each of these pathways. I hypothesis that adding water should increase excretion of water and decrease excretion of solutes through osmoregulation to maintain normal osmolarity and adding saline will also increase water excretion but without a decrease in solute excretion through effects on the renin-angiotensin system. Objectives I intend to compare the flow rate and specific gravity of urine over time in subjects receiving

James Cooper, D2, 00509474

interventions into account, accounts for this discrepancy.

so

this

probably

drinking saline, it does prevent different peaks interfering where in fact the effect of the saline would have been the same.. Exclusion criteria- the exclusion criteria for my results were anyone who hadnt finished their saline/water. Other groups also excluded on this basis. I also excluded participants that hadnt posted at least 6 results. I felt that we did not need more results than this to establish a trend of some sort which would make the data useful, and omitted results in general would not skew the trends of results there was no reason to believe that urine production is not constant between readings. I refuse to extrapolate results as it has no benefit over extended interval rates other than to create the illusion of having more samples at a given time, falsely increasing the significance of results. Results The first thing we did was compare saline and water group mean baseline values. Both groups had quite high baseline SGs (kg/l) of 1.0189 and 1.0196 respectively, and as both groups saw their SG values lower, this implies mild dehydration. The baseline flow rates (ml/min) were 0.94 and 3.10 respectively, so it would seem that the saline group started off significantly more dehydrated than the water group. (see figure 1) Therefore as well as other results, I calculated changes in both flow and SG from the baseline to try and allow for this difference, though Ill discuss why that might not

Post experimental analysis As samples were at variable intervals, we took volumes and divided by the time between samples to find a flow rate. Whilst technically we cannot know that this flow rate was constant within time-spans longer than the standard 20 minutes, this is irrelevant as we also could not know if the rate was constant within 20 minute intervals either and we will just have to make an assumption that unless there is a specific extrinsic stimulus, urine production is consistent. I then slotted each flow rate into a 20 minute time interval to make the data comparable. We then would collate subgroups on each treatment into supergroups and calculate means, standard deviation, standard error of the mean and confidence intervals. Then using t-tests we would see if there was a significant difference between saline and water groups, and plot the trends of all groups on a graph to see if there were any obvious effects with these other stimuli. In order to work from a baseline we used the volume of the baseline sample and divided by the time since they last passed urine.

be enough later. Looking at the trend of changes from the baseline, there is a greater change in the water groups flow rate, but relative to the baseline, the change is much less (see figure 2). Also, by the end of the trial at 240 minutes, the water groups flow rate had dropped below the baseline whilst the saline groups was still significantly above it. The peak change of flow rate was at 60 minutes in both groups, and in both groups it decreased back towards the norm at a similar rate. The SG in the water group hit a trough at 80 minutes then slowly rose again, whilst the saline group had a sharp trough at 60 minutes and a wavering line that did not deviate as much from the baseline (see figure 3). As differences and relative changes were calculated (foolishly, by me) from the mean values at each reading and not the raw results, there are only standard deviations, errors and confidence intervals for SG and absolute flow rate which is unfortunate. The saline group data on the whole was calculated with fewer results at each timing, but still had a comparable standard error to that of the water group throughout the experiment. Some statistics - Using an independent t-test, we found that trough SG difference had a p-value of <0.01, which was significant. The p-value for the flow rate apparently does not exist, so something strange happened there.

Diagram 2

For my saline group in particular, it was impossible to maintain a standard time for finishing the saline, so in my results I took values from the baseline as zero, then (assuming minimal effects on output with no input, and that all stored urine was passed initially) all further samples at times after finishing the saline. Whilst this doesnt account for rates of

James Cooper, D2, 00509474

Personal and general trend data- My own personal data shows that my SG did not change very much at all, with no clear trend, and that my flow rate also remained very constant at approximately 2.5 ml/min throughout (I believe this is related to a dehydrated status as my SG was relatively high) (table 1 and 2). If we look at the data comparing SG (figure 5) and flow rate (figure 6) for all interventions, we can see that in all cases there was a sharp increase in flow rate around 60 minutes after consuming water, and SG plummeted in all groups at 60 minutes then rose back to baseline again. The effect was greater in every cohort other than saline. In the dextrose group, the trend followed waters, except with a marginally later peak flow rate. In the caffeine group, the peak flow rate and trough SG values lasted for longer than waters. In the caffeine and dextrose group, the differences were less than in just caffeine or water, but the same elongated peak and trough were present. In the exercise group, both trends followed water, but the flow rate decreased at a higher initial rate after peak. In the DDAVP, the trend followed water up to 60 minutes then the flow rate fell sharply to very low levels by 100 minutes and the SG rose at a faster rate than in any other variable. Adverse effects Some saline recipients suffered from vomiting and diarrhoea. These participants invariably failed to post at least 6 results, so they were excluded from the data. Discussion

Limitations- Our experiment had quite a large number of limitations. Firstly, there were guidelines for caffeine intake, alcohol consumption and hydration before the experiment but these were not enforced. Hence initial hydration states were not standardised, leading to very different baselines between the water and saline groups. The rate and timings in drinking the saline in the saline group were not uniform, which presented difficulties in determining the effect of saline that have only partially been compensated for. Some people could not take samples at set 20 minute intervals, which led to mathematically fiddling with times which confused results. The use of specific gravity dipsticks was prone to human error (some participants read the incorrect marker on the dipstick) and relied on subjective judgement against a colour code. People finished the experiment at different times, as there was no concrete endpoint, people were told they could leave when they wanted towards the end and some people felt they could not continue due to hunger, dehydration and fatigue. People were of different sizes, ethnicities and may have had other different factors demographically and without subset analysis of these, there could be many confounding factors. People could switch groups if they wished to (though not that many did this) which could lead to selection bias, as fitter people would be more likely to adhere to exercise or injections and that could introduce a confounding factor. The was also no blinding (it

would be difficult to single or double blind a needle or exercise or make all the solutions taste the same without adding more confounders) which means that there could easily be a placebo effect in many of the interventions. Scientific discussion of results- the absolute higher flow rate in flow rate in the water compared to saline is probably because the saline group were initially very dehydrated. This means that any added volume in the saline was predominantly used to bring the volume and blood pressure back to normal rather than causing an excess that the body wished to dispose of. However, the much higher peak relative flow rate in the saline could also be attributed to the dehydration, as the dehydration caused an initially low flow rate so if it were overcome, any changes would be magnified by this small starting value. It is difficult to test this theory without either subgroup analysis taking hydration states into account or standardised initial hydration. If the differences in relative flow rate are due to actual difference between the groups and not confounding due to dehydration, then this experiment could possibly tell us that activation of baroceptors has a stronger influence on urine output than vasopressin control alone, though dehydration would mean that the saline was actually relatively hypotonic and thus would have an effect on vasopressin regulation through osmoreceptors as well. Water would activate both the osmoreceptors due to osmotic change and the baroceptors due to

James Cooper, D2, 00509474

increased blood volume, whilst the saline should only increase blood volume and should not affect the osmoreceptors. This would also be an explanation for why the absolute change from baseline flow rate was greater in the water group, as the kidney is receiving orders to lose water through two systems not one. The fact that the peak in flow rate was at the same time in each case implies that each system takes approximately the same time (60 minutes) to have an effect on the kidneys output. The greater decrease in SG seen in the water group would be explained by the fact that it is predominantly acting on osmoreceptors, whilst the saline acts (mostly) on baroceptors, and downregulating the renin-angiotensin system would reduce sodium reabsorption(9) and thus not decrease the SG of the urine (except maybe partially through potassium excretion). The noticeable decrease in saline SG might be due to K+ loss or prior dehydration leading to some osmoregulation. The oscillating SG values in the saline group are probably due to measurement subjectivity and problems in collated data, as are not very great and do not make scientific sense. In the trends of other variables data, the peak was generally around 60 minutes, which agrees with the pilot study. In dextrose and diet bolt, the peak output was slightly later than in saline or water, but with the timing difficulties it is impossible to say whether that is a real difference or not. The greatest flow rate peak was seen in the diet bolt

group, but with differences between participants and different initial hydration states, we would need to undergo an ANOVA test together with subset analysis for this to be useful. The dextrose group trend makes sense, as oral dextrose should pass through the hepatic portal vein before the main circulation, storing most glucose as glycogen and using the rest, so within minutes it is effectively water. The extended peak in the caffeine group could be due to caffeines diuretic properties, which are presumed to be through A1 receptor antagonism, which is paradoxical and may or may not involve phosphodiesterase inhibition (10)(11). The same would apply for Bolt, but the lower values are probably due to demographic differences within the groups. The exercises quicker fall of flow rate after peak would be due to vasoconstriction reducing renal perfusion and water and salt loss through sweating. DDAVPs peak effect should be 1-1.5 hours after i.v. infusion, but it probably caused a maximal reduction in output before it could reach its own potential maximal effect on Aquaporin synthesis. It should work 2 minutes after administration, explaining the drop after 60 minutes. Looking at trends in specific gravity is very difficult due to the subjectivity and mistakes in the use of dipsticks. My own datas lack of change after saline introduction implies I was dehydrated and the addition of fluid did not cause a change in blood pressure to trigger an increase in urinary output.

This makes sense, as though (low pressure) baroceptors can be more potent than osmoreceptors (especially in dealing with hypovolaemia), they are less sensitive to changes, so a large volume change is needed to cause a response. In order to further investigate my aim in future, it could be possible to repeat the above experiment (with some more standardisation and information on individual subjects before hand to subgroup analyse) but using hypertonic saline (and a water control) and splitting the participants into those receiving just their fluid, those receiving fluid and a vasopressin inhibitor and those receiving the fluid and an ACE-inhibitor or aldosterone antagonist, those receiving fluid and i.v. desmopressin and those receiving fluid and (if possible) i.v. angiotensin . Whilst there is some overlap between vasopressin and renin-angiotensin, this would try to isolate the effects of each system. Conclusion- we could not find any significant difference between water and 0.9% saline on urine output, but there was some evidence of less urine production from the baseline in saline users, and saline gave a significantly different urine specific gravity. The limitations in experiment design and initial dehydration states meant the experiment could not test its target hypothesis as it could not isolate baroception and osmoregulation. The issue needs further research with less overlap between these two systems.

James Cooper, D2, 00509474

Figure 1 (error bars calculated from 95% confidence intervals) Figure 2

Figure 3 (error bars calculated from 95% confidence intervals) Figure 4

James Cooper, D2, 00509474 Figure 5

Table 1
Time (mins) 0 20 40 60 80 100 120 140 Personal specific gravity (kg/l) 1.02 1.02 1.02 1.015 1.02 1.02 1.015 1.02

Time (mins) 0 20 40 60 80 100 120 140

Personal Urine Flow Rate (ml/min) 2.16 2.55 2.15 2.1 2.44 2.4 2.7 2.7

Figure 6

Table 2

Table 3

James Cooper, D2, 00509474

Physiological Reviews 1980 Oct;60(4): pp. 961-1048. (3) Knepper MA, Inoue T. Regulation of aquaporin-2 water channel trafficking by vasopressin. Current opinion in cell biology 1997 Aug;9(4): pp. 560-564. (4) Dibas AI, Mia AJ, Yorio T. Aquaporins (water channels): role in vasopressinactivated water transport. Proceedings of the Society for Experimental Biology & Medicine 1998 Dec;219(3): pp. 183-199. (5) Reid IA. Interactions between ANG II, sympathetic nervous system, and baroreceptor reflexes in regulation of blood pressure. American Journal of Physiology 1992 Jun;262(6 Pt 1): pp. E763-78. References (1) Greger R. The mechanisms of renal tubule electrolyte and water absorption, 100 years after Carl Ludwig. Pflugers Archiv - European Journal of Physiology 1996;432(3 Suppl): pp. R82-6. (2) Bie P. Osmoreceptors, vasopressin, and control of renal water excretion. (6) Thrasher TN. Baroreceptor regulation of vasopressin and renin secretion: lowpressure versus high-pressure receptors. Frontiers in neuroendocrinology 1994 Jun;15(2): pp. 157-196. (7) Cuneo RC, Espiner EA, Nicholls MG, Yandle TG, Livesey JH. Effect of physiological levels of atrial natriuretic peptide on hormone secretion: inhibition of angiotensin-induced aldosterone secretion and renin release in

normal man. Journal of Clinical Endocrinology & Metabolism 1987 Oct;65(4): pp. 765-772. (8) Kobori H, Nangaku M, Navar LG, Nishiyama A. The intrarenal reninangiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacological reviews 2007 Sep;59(3): pp. 251-287. (9) Rozansky DJ. The role of aldosterone in renal sodium transport. Seminars in nephrology 2006 Mar;26(2): pp. 173-181. (10) Rieg T, Steigele H, Schnermann J, Richter K, Osswald H, Vallon V. Requirement of intact adenosine A1 receptors for the diuretic and natriuretic action of the methylxanthines theophylline and caffeine. Journal of Pharmacology & Experimental Therapeutics 2005 Apr;313(1): pp. 403-409. (11) Fredholm BB, Hedqvist P, Vernet L. Effect of theophylline and other drugs on rabbit renal cyclic nucleotide phosphodiesterase, 5-nucleotidase and adenosine deaminase. Biochemical pharmacology 1978;27(24): pp. 2845-2850.