Kidney Function

Balance of Fluid, Na+ and pH, Estimation of GFR

Kirsten Olson
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Introduction

The renal system, in concert with other systems, has primary responsibility for bodily

homeostasis. It produces calcitrol to control calcium levels and erythropoietin to initiate creation

of red blood cells. It controls electrolyte and acid-base balance, manages blood volume and

pressure, and excretes metabolic wastes and xenobiotics (Tortora, 2012, 567-568). This

amazingly complex system works with the help of such disparate actors as the hypothalamus, the

adrenals, and cells of the heart, lungs, and liver (Hall, 2016, 364) (Atherton, 2006, 228, 231).

The kidneys create urine and the ureters transport it to the bladder, to await micturition,

or urination, via the urethra. Inside each kidney, within the cortical and medullary layers, are

approximately one million nephrons and their associated vasculature. A nephron is comprised of:

a glomerulus inside a Bowman’s capsule (renal corpuscle), a proximal convoluted tubule, a loop

of Henle, and a distal convoluted tubule (renal tubule). Multiple distal convoluted tubules

connect to each collecting duct, which all drain through renal papillae and calyxes into the renal

pelvis before reaching the ureters. Approximately 20 percent of blood flow is directed to the

kidneys through the renal arteries, which then branch off into smaller vessels to interact with the

nephrons. The vasculature of a nephron includes: afferent arterioles, which bring blood into the

capillary tangle that makes up the glomerulus, the efferent arteriole, which exists the Bowman’s

capsule, the peritubular capillaries which surround the entire renal tubule, the vasa recta, which

provides blood to the cells and helps maintain the necessary osmotic gradient, and venules that

drain the capillaries (Tortora, 2012, 568-571) (Hall, 2016, 377-378).

The nephrons and collecting ducts perform their homeostatic function through three basic

actions: glomerular filtration, tubular reabsorption, and tubular secretion. Water and small

solutes such as Na+ and creatinine filter out of the glomerular capillaries into the capsular space
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of the Bowman’s capsule via pressure. Specifically, it is driven by net filtration pressure, which

is the difference between capillary hydrostatic blood pressure and the combined blood oncotic

and capsule hydrostatic pressures (Tortora, 2012, 572-573) (Hall, 2016, 337). Nearly all of the

water and much of the solutes in this filtrate are re-admitted to the blood supply through tubular

reabsorption, with specific amounts adjusted as needed. Some of the water is reabsorbed via

osmosis and some through aquaporin channels (Ecelbarger, et al, 2001, 227). Some of the solutes

move by diffusion and some by active transport. To further fine-tune the process, tubular

secretion removes certain substances from the blood into the filtrate. Again, some solutes move

by diffusion and some by active transport (Tortora, 2012, 574-576). H+ and HCO3, for example,

are secreted by intercalated cells of type A and type B, respectively (Hall, 2016, 357).

This is all regulated by a sophisticated system of sensors and hormones. The Renin-

Angiotensin-Aldosterone system (RAAS) is activated when macula densa cells next to the

glomerulus sense a decrease in [Na+]. They secrete renin, which turns circulating

angiotensinogen from the liver into angiotensin I. In capillaries of the kidneys and lungs,

angiotensin converting enzyme activates Angiotensin I into Angiotensin II (ANGII). ANGII

increases thirst, reduces glomerular filtration rate (GFR) by constricting afferent and efferent

arterioles, causes the pituitary gland to secrete more anti-diuretic hormone (ADH) and the

adrenal glands to secrete more aldosterone, and enhances reabsorption of Na+ and water. ADH

causes the kidneys to reabsorb more water by increasing the number of aquaporins and both

ADH and Aldosterone improve reabsorption of Na+ by causing more production of both

facilitated and active transport channels (Atherton, 2006, 228, 231) (Ecelbarger, et al, 2001,

227). Aldosterone also works to regulate blood pressure, volume, and [H+] (Tortora, 2012, 362).

When osmoreceptors in the hypothalamus detect high osmolarity of the blood, they also
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stimulate ADH secretion, and when osmolarity is low, this is inhibited (Tortora, 2012, 354).

Finally, certain cardiac atrial cells act as baroreceptors. When expanded by increased blood

pressure, they secrete atrial naturetic peptide (ANP), which depresses the RAAS and reduces

reabsorption of water and Na+ (Hall, 2016, 364).

The purpose of this experiment is to examine selected aspects of renal function by

looking at the outcomes of drinking different fluids, or no fluid. Outcomes will be compared in

terms of multiple parameters involving water, pH, sodium, and creatinine. Mostly the results will

be compared against the subject’s own baseline and/or the results of other subjects, but

sometimes normal adult reference ranges will also be consulted. For example, urine specific

gravity is expected to range from 1.003 to 1.030, and urine pH ranges from 4.5 to 8 (Simerville,

et al, 2005, 1153-1154).

I hypothesize that Control’s parameters will not change a great deal over the course of the

experiment. I expect that there may be some noticeable effect from exercise and/or from not

drinking anything during lab. However, I expect this effect to be small compared to that of the

challenge drinks upon the other subjects. For the Hypotonic subject, I expect a much greater flow

rate of dilute, less acidic urine, for sodium clearance to drop, and for creatinine clearance to rise.

The Isotonic flow rate, specific gravity, and sodium clearance just a little, pH should stay about

the same, and creatinine clearance should go up. Last, I expect the Alkalotic subject to follow

this pattern: flow rate should decrease to some degree, specific gravity and pH will climb,

sodium clearance, and creatinine clearance will stay about the same.

Methods

This lab evaluated the urine of four subjects, collected five times over a two-hour period,

in terms of multiple parameters. One subject would serve as a control to compare different
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challenge fluids against and each person’s first collected urine would serve as their own baseline.

Control subject was an athletic male, in his 20s, with a body mass of 69.4 kg. He consumed

nothing during the experiment. Hypotonic subject was a slim, 21 y/o female, weighing 48.5 kg.

She consumed 14 ml H2O/kg body mass (679 ml) at the start of the experiment. Isotonic subject

was a slim, 22 y/o female, weighing 53.1 kg. She consumed 14 ml Isotonic saline (0.9%

NaCl)/kg body mass (743 ml) at start. Alkalotic subject was a heavy female, 22 y/o, weighing

84.0 kg. She consumed 2.0 ml NaHCO3- solution (2.5%)/kg body mass (168 ml) at start.

All subjects were asked to drink adequate water and avoid or limit alcohol or caffeine

during the 48 hours prior to lab, and to avoid heavy exercise the day of lab. Some deviations

from these instructions occurred: Control lifted weights the morning of lab (and also ran 6 miles

the day before). All three other subjects had coffee. Hypotonic drank 6 oz. around 1pm the day

before lab. Isotonic had 24 oz. about 9 pm the evening before and 6 oz. about 8 am the morning

of lab. Akalotic had 12 oz. in the morning the day before lab and 12 oz. right before lab.

Subjects noted time of last urination before lab. Test fluids were measured for each and

dispensed in paper cups. Each subject emptied their bladder at start of lab into a collection

container, drank assigned fluid within a specified ten-minute window, measured urine sample

volume, set aside about 50 ml, and labeled it with name and time “0.” Subjects drank nothing

else until after lab. Urine collection steps were repeated every 30 minutes, including and ending

with time “120.” TA announced each collection time.

Between urine collections, measurements were taken to determine: volume, flow rate

(Vurine), specific gravity, pH, Na+ concentration (UNa+), Na+ clearance (CNa+), creatinine

concentration (UCr), creatinine clearance (CCr), and expected versus actual amounts of water, Na+

and H+ excreted. All data was recorded in a table for each subject. Urine volume was measured
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with graduated cylinders, and Vurine was calculated. Specific gravity was measured with a digital

refractometer: The metal tip of the refractometer was placed in the sample and “START”

pressed. The value was noted, and then the refractometer was cleaned. A pH meter was used to

measure pH: Electrode was rinsed with DI water, dried with a Kimwipe, and set into the urine

sample. Cup was gently spun and pH read once stable for a few seconds. Electrode was rinsed,

wiped, and returned to buffer solution. UNa+ was measured with an Na+ meter: The electrode was

rinsed with DI water, dried with a Kimwipe, and set into the urine sample. Cup was gently spun

and value read. Electrode was rinsed with 100 mM NaCl solution, dried with a Kimwipe, and

returned to 100 mM NaCl solution. Electrode reading in mV was converted to UNa+ in mEq/L.

CNa+ was calculated and recorded.

UCr of time 0 and 60 samples was measured by spectrophotometry. Because the

creatinine spectrophotometric-based assay is only linear within concentrations of 0-75 mg/L,

samples had to be diluted. UCr was estimated and appropriate dilution factor determined and

recorded. Diluted solutions were created using micropipettes. For some samples, a single dilution

was carried out (ex: 1 part urine to 9 parts DI for a 10-fold dilution), and for some, serial dilution

was necessary (ex: 1 part urine to 2 parts water for a three-fold dilution, then 1 part this solution

to 9 parts DI for a final 30-fold dilution). 300 μl of each diluted urine solution (“0” and “60”)

were pipetted into labeled tubes, 3.0 ml of alkaline picrate was added to each, and these were

lightly flicked to mix. Mixtures were left to react for 7-8 minutes. Each sample tube was placed

in the machine and its optical density/absorbance value (OD) was read and recorded. OD was

converted to give sample concentration of creatinine and this was multiplied by the proper

dilution factor to give UCr. Then CCr was calculated. Last, actual and expected amounts of

excreted water, Na+, and H+ were calculated and recorded.

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At one point, the Hypotonic subject mixed up two samples. She was using two large

collection containers so she could pour it into the graduated cylinder and then into a discard

container multiple times to measure it all. I believe that she had not emptied the discard container

yet when she went to collect the new sample, so at some point she realized that she could not tell

the new sample from the old discard (perhaps she had also kept the old discard because she was

not done taking measurements on the old sample). She tried to determine by color, but they

looked too similar. I noted that the pH had increased between the time 0 and time 30 samples,

and suggested that she use pH to tell them apart. One sample had a pH that was very similar to

time 0 and time 30, and the other sample had a pH of 7, so we assumed that this must be the

newer sample. I am not entirely sure when this happened, if my pH assumption was correct, or

even if she followed my suggestion, but most of the data seems to support my recollection.

Calculations were done with Excel and/or a TI-83 calculator. Data from subjects were

compared against their own baseline, and against each other. Reference ranges were used for

comparison where applicable. Figures were created in Excel to visualize and help analyze the

data and respond to the question set in the lab manual (Bautista and Korber, 2009, 65-74).

Results

As shown in Figure 1 in ml/min, Hypotonic urine flow rate (Vurine) changed greatly

compared to all other subjects. Control Vurine went down just 23% over the two hours. Hypotonic

Vurine increased about 410% over the first hour, and then fell off just as dramatically over the

second hour, back to baseline. Isotonic Vurine also increased over the first hour and returned to

baseline, but the increase was only 69%. Alkalotic Vurine decreased by 50%.

Figure 2 illustrates the relative changes in subjects’ urine specific gravity as well as how
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each sample compares to normal adult reference ranges and probable relative hydration status.

Control specific gravity varied and slightly declined within a small normal range of about 1.0200

to 1.0100. The other three subjects all started around the same well-hydrated normal specific

gravity (about 1.0080), but quickly diverged: Hypotonic quickly dropped out of the normal range

to 1.0005 within half an hour, began rising back towards normal in the second hour, but only

reached 1.0027 by 120 minutes. Isotonic varied within a small range of 1.0107 to 1.0066.

Alkalotic steadily rose to 1.0222.

Urine Flow Rate

12.0

10.0
Control
Hypotonic
Isotonic
Urine Flow Rate (ml/min)

8.0
Alkalotic

6.0

4.0

2.0

0.0
0 30 60 90 120

Time Point (min)

Figure 1: Change in urine produced, or urine flow rate (Vurine), of each subject over time (ml/min). Vurine
was calculated from ml urine measured over time elapsed since last urination.
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Specific Gravity
1.0300
1.0300
Control
Hypotonic
1.0250
Isotonic (Relative
Alkalotic Dehydration)
1.0200
1.0200
Normal
Specific Gravity

Adult
Reference
1.0150
Range

1.0100
1.0100 (Relative
Hydration)

1.0030
1.0050

1.0000
0 30 60 90 120
Time Point (min)

Figure 2: Change in subjects’ urine specific gravity over time. Normal adult reference range of 1.0250 to
1.0050 and relative probable hydration status above and below certain points of the range are shown for
comparision.

All subjects’ urinary pH varied within reference range, but Alkalotic showed significantly

different behavior (see Figure 3). Control pH overall declined modestly, from around 6.50 to

6.00 pH. Hypotonic varied within a narrow band around 6.75 to 7 pH, rising a bit in the last 30

minutes to 7.07 pH. Isotonic also varied within a narrow, slightly acidic range until the last 30

minutes, when it dropped to 5.85 pH. In comparison, Alkalotic drastically changed from 5.33 up

to 7.71 pH within the first hour, reached a peak of 7.90 pH at 90 minutes, and receded to 7.71 pH

by 120 minutes.
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Urine pH
8.00
8.00
Control
7.50 Hypotonic
Normal
Isotonic Adult
Alkalotic Reference
7.00 Range

6.50 6.50
Urine pH

(Typical
6.00 Range)

5.50

5.50 Normal
Adult
Reference
5.00 Range
4.50

4.50
0 30 60 90 120
Time Point (min)

Figure 3: Change in subjects’ urine pH over time. Normal adult reference range of 4.50 pH to 8.00 pH
and typical range are shown for comparison.

All but the Hypotonic subject had similar response in terms of urine Na+

concentration (UNa+): It went down and then up (see Figure 4). Control went down from 98.1 to

72.7 mEq/L in the first hour and then increased to 177 mEq/L, for an overall increase of 80%.

Hypotonic, on the other hand, sharply declined from 68.4 mEq/L to a low point of 6.2 mEq/L

over the first hour, raising only slightly to 11.8 mEq/L by 120 minutes, for an overall reduction

of 83%. Isotonic dipped from 87.6 and 89.9 mEq/L down to 74.6 mEq/L in the first hour, then

rose to 137 mEq/L, for an overall increase of 56%. Alkalotic dipped from 127 to 115 mEq/L in

the first 30 minutes, and then increased to 231 mEq/L at 90 to 120 minutes, for an overall

increase of 82%.
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Urine Na+ Concentration

250.0

Control

Hypotonic
200.0 Isotonic
Urine Na+ Concentration (mEq/L)

Alkalotic

150.0

100.0

50.0

0.0
0 30 60 90 120
Time Point (min)

Figure 4: Change in subjects’ urine Na+ concentration (UNa+) in mEq/L over time.

As shown in Figure 5, all subjects had a different Na+ clearance (CNa+) response, but the

Hypotonic CNa+ had the most significant change. Control declined from 1.5 to 0.8 ml/min in the

first hour, then shot up to 2.1 ml/min, for an overall increase of 40%. Hypotonic steadily

declined from 1.0 to just 0.2 ml/min, decreasing 80%. Isotonic varied from 0.62 to 1.3 ml/min,

but the overall change was 0.8 to 1.07 ml/min, an increase of 34%. Alkalotic also varied in a

small range, from 1.4 to 0.86 ml/min, and essentially returned to baseline at 1.3 ml.min.
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Na+ Clearance
2.5
Control
Hypotonic
2.0 Isotonic
Alkalotic
Na+ Clearance (ml/min)

1.5

1.0

0.5

0.0
0 30 60 90 120
Time Point (min)
Figure 5: Change in subjects’ Na+ clearance (CNa+) in ml/min over time.

For urine creatinine concentration (UCr) at time 0 and time 60, all subjects but Hypotonic

have a similar response (shown in mg/L in Figure 6). However, the magnitudes are very

different: Control was the largest value to begin with and increased just 17%. Hypotonic, the odd

one out again, began at a smaller value and then decreased markedly, by 82%. Isotonic also

began at a smaller value but increased 28%. Alkalotic began with the intermediate value and

increased 50% to nearly match Control’s final value.

As shown in Figure 7 in ml/min, the Isotonic subject had the most different response for

urine creatinine clearance (CCr). Control again began with the largest value and decreased by just

16%. Hypotonic started with an intermediate rate and then barely decreased, by only 9%. In

comparison, Isotonic started with the smallest value and increased the rate by 110%. Alkalotic

started with an intermediate rate and decreased significantly, by 39%.

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Urine Creatinine Concentration

2500
Urine Creatinine Concentration (mg/L)

2000 2130 2122

1820
1500
1418

1000
920
723 721
500

128
0
0 60
Time Point (min)
Control Hypotonic Isotonic Alkalotic
Figure 6: Urine creatinine concentration (UCr) in mg/L at time 0 versus time 60.

Creatinine Clearance
350
Creatinine Clearance (ml/min)

337
300
284
250

200 220
202
150
159
145 141
100
96
50

0
0 60
Time Point (min)

Control Hypotonic Isotonic Alkalotic

Figure 7: Subjects’ urine creatinine clearance (CCr), in ml/min, at time 0 versus time 60.
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Figure 8 illustrates the difference in expected versus actual amount of urine produced by

each subject during the experiment, in milliliters. Expected water is how much urine would

hypothetically be produced if the initial urine flow rate remained the same for two hours. Actual

water is the total amount of urine collected over two hours under experimental conditions. All

subjects differed from the expected value, but the Hypotonic subject was the most substantial

difference. Control was 77% of expected. Hypotonic was 300% higher than expected. Isotonic

was also higher than expected, but was only 119%. Last, Alkalotic was 58%, which is

significantly less than expected.

Expected vs. Actual Water

900

750 797
Water (ml)

600

450

300
267 264
150 186 206 190
159
108
0
Expected Actual

Control Hypotonic Isotonic Alkalotic

Figure 8: Expected versus actual ml of water (urine) produced by subjects during the two hour lab period.
Expected water is time 0 Vurine multiplied by 120 minutes. Actual water is the combined volumes of the
remaining collections, time 30 through time 120.
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In Figure 9 we see the contrast in expected grams of Na+ excreted by each subject

compared to actual grams collected. Expected Na+ is how much would be excreted if the initial

rate of excretion remained the same for two hours. Actual Na+ is total grams collected under

experimental conditions, from time 30 through time 120. All subjects responded differently from

expectation and each other, but the Hypotonic varied the most, and the Isotonic subject had the

opposite response from all other subjects. Control was 93% of expected, which is nearly the

same. Hypotonic, however, was a much lower 36% of expected. Isotonic had the opposite

response, being 130% higher than expected. Alkalotic had a modest response, being 81% of

expected.

Expected vs. Actual Na+

0.70

0.60
0.60
0.56
0.50 0.54
Na+ (g)

0.40 0.44
0.42 0.41

0.30 0.32

0.20

0.10 0.15

0.00
Expected Actual

Control Hypotonic Isotonic Alkalotic

Figure 9: Expected versus actual grams of Na+ excreted by subjects during the two hour lab period.
Expected Na+ is calculated with a formula, using the time 0 Vurine and UNa+. Actual Na+ also uses a
formula and the combined urine Na+ concentrations and volumes of the remaining collections, time 30
through time 120.
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The final set of results, shown in Figure 10, is a comparison of the expected moles of H+

excreted by each subject versus actual moles collected. Expected H+ is the moles that would be

excreted if the initial rate of excretion remained the same for two hours. Actual H+ is total moles

collected under experimental conditions, from time 30 through time 120. In this case, the

Alkalotic subject was the most unusual, with an opposite and dramatic variance from expected.

Control was a modest 120% of expected. Hypotonic was a significant 200% of expected.

Isotonic was even higher than expected, at 240%. Alkalotic had the opposite response, coming in

at a markedly lower amount, just 16% of expected.

Expected vs. Actual H+

1.0E-06
8.7E-07
9.0E-07

8.0E-07

7.0E-07
H+ (mol)

6.0E-07

5.0E-07

4.0E-07

3.0E-07
1.8E-07
2.0E-07 1.4E-07
1.0E-07
8.8E-08 7.4E-08
4.9E-08 3.1E-08
1.0E-07

0.0E+00
Expected Actual
Control Hypotonic Isotonic Alkalotic

Figure 10: Expected versus actual moles of H+ that subjects produced during the two hour lab period.
Expected H+ is time 0 Vurine multiplied by 120 minutes, multiplied by [H+]. Actual H+ is the sum of the
volumes of the remaining collections multiplied by the respective H+ concentrations, time 30 through time
120.
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Discussion

This experiment involved examining the function of renal systems operating under one of

four challenge fluid conditions: no fluids (control), water (hypotonic), saline (isotonic), or

bicarbonate solution (alkalotic). Subjects were all young and assumed to be healthy and hydrated

at the start of the exercise. Little is known about what the subjects ate or drank before the

exercise commenced. However, the first urine was collected prior to drinking the challenge

liquid (if any) and served as the baseline for each subject. As seen in the preceding section,

experimental results are consistent with some, but not all of the hypotheses.

Based on the physiology and procedures outlined in previous sections, I expected to

observe the following in the four subjects: Control should have experienced normal effects of

circulating basal levels of hormones. His body should have produced normal amounts of normal

urine that stayed similar to his baseline. Some variation would be normal. Because he was not

drinking anything for two hours, it would also make sense for his body to make some effort to

conserve water. Hypotonic should have experienced an increase in blood pressure, which we

would not measure, but that would stretch her cardiac atrial cells and cause them to release ANP

and consequent depression of her RAAS. This, and the increased hydrostatic pressure from the

increased blood volume, would be expected to result in greater production of dilute urine.

Isotonic should have experienced a mixed response, because the increase in both water and Na+

would not work the same as an increase in plain water. The increase in water would increase BP,

but because the fluid was isotonic, much of it would simply pass out through capillaries

everywhere and therefore not increase the hydrostatic pressure in the renal corpuscle as much.

The increase in Na+ would not register at all, because an isotonic solution would not change the

osmolarity of the blood. Therefore, a slight ANP and hydrostatic response might be expected.
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Alkalotic should have activated the RAAS, because the bicarbonate solution was hyperosmolar.

This should have caused her to concentrate her urine/retain water. She should also have been

secreting the excess HCO3 into her urine through her type B intercalated cells.

Urine Flow Rate

The Control flow rate (VUrine) stayed about the same, as expected (Fig 1). There was a

slight drop off, probably due to lack of fluid intake. Hypotonic VUrine was expected to rise

considerably, and it did. The large volume of hypotonic fluid increased plasma volume, which

would increase BP, leading to release of ANP. The ANP reduced reabsorption of water, thereby

increasing production of urine (Hall, 2016, 364). This process was so efficient that a great deal of

excess fluid was eliminated within the first hour, and production of urine returned to baseline

levels by 120 minutes. Isotonic’s modest similar response to the same increase in blood volume

was moderated by the sodium content of the solution. The body controls osmolality far more

strictly than volume, so the body’s response to the increase in volume was blunted by the fact

that the osmolality of the plasma was unchanged (Atherton, 2006, 227-228) (Maher and Mac

Nab, 2018, 247). Alkalotic VUrine decreased twice as much as Control’s, because the increase in

osmolality lead to a release of ADH, causing her body to retain water and therefore produce less

urine (Maher and Mac Nab, 2018, 247-248).

Specific Gravity

Experimental results for specific gravity (SG) (Fig. 2) were as expected, and directly

relate to the mechanisms driving VUrine status. Control and Isotonic SG were fairly stable because

neither of these conditions caused the body to significantly dilute or concentrate urine. Control

was only restricted from fluids for two hours. Isotonic fluid does not produce the same

immediate effects as hypotonic fluid, as explained above. Hypotonic SG dropped because her
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body diluted the urine to get rid of excess water to restore plasma osmolality. Alkalotic SG

climbed for the opposite reason, and because her type B intercalated cells were excreting the

excess bicarbonate ions (Hall, 2016, 357). This agrees with the results in Lindinger, et al: Their

bicarbonate subjects experienced urinary retention and secreted less acid, which was attributed to

secretion of bicarbonate (Lindinger, et al, 2000, 547).

pH

The pH results were largely unsurprising (Fig. 3). Control mostly stayed the same. The

minimal decrease may have been due to normal fluctuation, or lack of fluids plus something the

subject consumed right before lab or lactic acid, because this subject lifted weights the morning

before lab. Liljestrand and Wilson demonstrated that there is a large increase in lactic acid

excretion into urine after exercise. Their subjects eliminated the excess very quickly, but they

also exercised for a much shorter time. In addition, their experiment suggests that merely running

up a flight of stairs would have a noticeable effect within about 20 minutes (Liljestrand and

Wilson. 1925, 774-778). Hypotonic had an even smaller increase in pH, which can be attributed

to simple dilution. Isotonic stayed about the same until the last collection, which showed a

noticeable drop. I am not sure what caused this. It may have simply been because there was less

urine at this point. Alkalotic pH rose significantly, due to secretion of bicarbonate into the urine

(Hall, 2016, 357).

Na+ Concentration

Results for urine Na+ concentration (UNa+) all make sense (Fig. 4). Control’s increase is

larger than expected but can be explained by lack of fluids. Hypotonic UNa+ declined due to

dilution. Isotonic UNa+ did rise, but less than Control. Because this subject consumed both

sodium and water, some ANP was released in response to the increase in BP, which lead to
 19

naturesis (Hall, 2016, 364). Alkalotic’s UNa+ rose because NaHCO3 increases excretion of Na+, as

demonstrated by Lindinger, et al, and because her urine was more concentrated (Lindinger, et al,

2000, 543-544). She produced ADH in response to increased plasma osmolality, which increased

water reabsorption (Maher and Mac Nab, 2018, 247-248).

Na+ Clearance

Subjects’ Na+ clearance (CNa+) also make sense, and relate back to UNa+, but are more

informative (Fig. 4,5). Only the Hypotonic subect showed a significant change, probably because

Na+ balance is regulated over longer periods of time, as demonstrated by Rakova, et al. In their

experiments, they found that subjects with constant daily sodium consumption had weekly and

monthly patterns of sodium excretion (Rakova et al, 2013, 125). Control CNa+ matched his UNa+.

Hypotonic CNa+ dropped, because a small amount of Na+ was being excreted in a very large

amount of urine. Isotonic CNa+ increased a little because of the actions of ANP, as explained

above (Hall, 2016, 364). This increase was small because the response to changes in volume is

less pronounced than responses to changes in osmolality, and the osmolality did not change

(Atherton, 2006, 227-228) (Maher and Mac Nab, 2018, 247). Alkalotic CNa+ did not show a

noticeable change, despite the corresponding increase in UNa+, because her urine was so

concentrated due to the actions of ADH (Maher and Mac Nab, 2018, 247-248).

Creatinine Concentration

Most of the results for urine creatinine concentration (UCr) make sense (Fig. 6). Control

UCr was very high and increased a little. This is likely due to his athletic body type and muscle

breakdown, because he lifted weights in the morning before lab. Higher muscle mass is known to

increase urinary creatinine, as illustrated in previous experiments (Barr, et al, 2005, 197),

(Baxmann, et al, 2008, 360/13). Hypotonic UCr fell dramatically because of her huge increase in
 20

urinary flow in the first hour. Her urine was very dilute in everything at this point. Isotonic also

increased a small amount, but more than Control. I am not sure if this was due to the effects of

her increase in volume, her caffeine intake, or both. Alkalotic UCr increased even more, for the

opposite reason as Hypotonic: Her urine was more concentrated in everything.

Creatinine Clearance

Creatinine clearance can be used to roughly estimate GFR because creatinine is filtered

and only minimally secreted. Therefore, it is expected to overestimate GFR by about 10-20%.

Clearance is the amount of blood that can be cleared of a substance within a unit time. The

creatinine clearance (CCr) results are the least consistent with hypotheses so far (Fig. 7). Control

CCr went down simply because he had more creatinine to clear and slightly less urine to excrete it

in. His response seems to be both related to actual creatinine clearance, and GFR. Hypotonic CCr

(and GFR) were expected to rise, but instead slightly went down. The expectation would be that

the increase in fluids would cause an increase in BP, which would cause cardiac atrial cells to

release ANP, which would depress the RAAS system and reduce water and Na+ reabsorption

(Hall, 2016, 364). The sample mix up is a possible source of error, depending on which samples

were mixed up, what work had already been done at that point, and if my suggestion to resolve

the issue was based on an incorrect assumption, or not followed. I do not think this is the

problem, but it bears mentioning. More likely sources of error include potential differences in the

sample run time and the fact that we estimated creatinine based on one assumed plasma

clearance concentration instead of two measured ones. The CCr was based on a Spec 20

absorbance of a urine sample reacted with an alkaline picrate solution. Perhaps the second

sample did not react long enough. Any colorimetric assay may be susceptible to such error; the

Jaffe reaction is no exception (Cook, 1975, 223-224). Plasma creatinine concentration can differ
 21

within the reference range within the day (for adult females, approximately 5-9 mg/L), but we

just used the figure 10 mg/L for both calculations (Samra and Abcar, 2012, 51), (Pottel et al,

2017, 164-165). If her actual plasma creatinine concentrations were known and the second was

lower than the first, then her CCr could have been more consistent with the hypothesis and the

rest of the data. Isotonic CCr went up, as expected, because of the mechanism described above for

Hypotonic. The expectation is that this relates directly to GFR. Alkalotic CCr went down instead

of staying the same. This makes sense with the values: There was 50% more creatinine to

process at half the rate. But again, there is the possibility that these values are off because of the

limitations of the experiment procedure. Again, this result is likely to relate to her actual GFR.

Expected vs. Actual Water

All of the results for actual versus the hypothetical “expected” urine production are

appropriate (Fig. 8). Control actual is less than “expected,” but I would not actually expect urine

production to remain exactly the same over two hours in which the subject does not drink

anything. This modest reduction is very reasonable. It could have easily varied in the opposite

direction if he had drunk a lot of water before lab, but he apparently did not. Hypotonic actual

was much greater than “expected,” which is consistent with drinking a large volume of water and

the resulting ANP process described above. Isotonic actual rose compared to “expected,” for the

same reason, but by a much smaller degree, due to the fact that water tends to “follow” Na+

osmotically, which makes it harder to remove excess water when lots of sodium is present.

Excess water with excess sodium in the blood tends to pass from the capillaries into the

interstitial spaces due to the increase in hydrostatic pressure, but in a healthy person this will

eventually be returned to the blood and removed by the kidneys through the actions of ANP

(Hall, 2016, 312, 316). Alkalotic actual was much less than “expected” because her
 22

osmoreceptors in her hypothalamus sensed the increase in osmolality caused by the bicarbonate

solution and caused the pituitary to release ADH, which caused her nephrons to open more

aquaporins and reabsorb more water (Maher and MacNab, 2018, 247-248).

Expected vs. Actual Na+

The actual production of Na+ is reasonable (Fig. 9). Control actual only slightly differed

from “expected,” so this is probably just normal variation. Hypotonic was much lower because

until the excess water was eliminated, the body was going to hang on to as much Na+ as possible,

to preserve osmolality. Isotonic increased a little, due to ANP. Alkalotic excreted less Na+

because of ADH.

Expected vs. Actual H+

Most of the H+ results make sense (Fig. 10). Control only went up a little, which is likely

due to normal variation. He was probably excreting some lactic acid from exercise. Hypotonic

seems very surprising at first, but is reasonable. Somehow the actual H+ production went way up

even though the urine produced became more alkaline. However, this works out because of the

huge volume of urine produced. The second and fourth collections were below neutral, and much

more volume than the fifth collection, which was the only one above 7 pH (Fig. 1). Isotonic

produced a lot more H+ than expected, but I do not have a reason for this. It is possible that her

caffeine consumption had something to do with this. Alkalotic was the most dramatic and has the

most obvious explanation: Initial urine happened to be rather acidic, but after subject consumed

the bicarbonate solutions, the body conserved H+ ions to balance out pH.
 23

References

Atherton, J. 2006. Regulation of fluid and electrolyte balance. Anaesthesia and Intensive Care

Medicine. 7(7): 227-233.

Bautista, E. and J. Korber. 2009. NPB 101L Systemic Physiology Lab Manual. Cengage

Learning: 65-74.

Barr, D., L.C. Wilder, S.P. Caudill, A.J. Gonzalez, L.L. Needham, and J.L. Pirkle. 2005. Urinary

Creatinine Concentrations in the U.S. Population: Implications for Urinary Biologic

Monitoring Measurements. Environmental Health Perspectives. 113(2): 192-200.

Baxmann, A.C., M.S. Ahmed, N.C. Marques, V.B. Menon, A.B. Pereira, G.M. Kirsztajn, and

I.P. Heilberg. 2008. Influence of muscle mass and physical activity on serum and urinary

creatinine and serum cystatin C. Clinical Journal of the American Society of Nephrology.

3(2): 348-354.

Cook, J.G.H. 1975. Factors influencing the assay of creatinine. Annals of Clinical Biochemistry.

12: 219-232.

Ecelbarger, C., G.H. Kim, J.B. Wade, and M.A. Knepper. 2001. Regulation of the abundance of

renal sodium transporters and channels by vasopressin. Experimental Neurology. 171:

227–234

Hall, J. 2016. Guyton and Hall Textbook of Medical Physiology, 13th Ed. Elsevier: 337, 357,

364, 377-378.

Liljestrand, S. and D. Wilson. 1925. The excretion of lactic acid in the urine after muscular

exercise. Journal of Biological Chemistry. 65: 773-782

 24

Lindinger, M., T. Franklin, L. Lands, P. Pedersen, D. Welsh, and G. Heigenhauser. 2000.

NaHCO3 and KHCO3 ingestion rapidly increases renal electrolyte excretion in humans.

Journal of Applied Physiology. 88: 540-550

Maher, W. and R. MacNab. 2018. Regulation of fluid and electrolyte balance. Anaesthesia and

Intensive Care Medicine. 19(5): 245-248

Pottel, H., L. Dubourg, E. Schaeffner, B.O. Eriksen, T. Melsom, E.J. Lamb, A.D. Rule, S.T.

Turner, R.J. Glassock, V. De Souza, L. Selistre, K. Goffin, S. Pauwels, C. Mariat, M.

Flamant, S. Bevc, P. Delanaye, N. Ebert. 2017. The diagnostic value of rescaled renal

biomarkers serum creatinine and serum cystatin C and their relation with measured

glomerular filtration rate. Clinica Chimica Acta. 471: 164-170.

Rakova, N., K. Jüttner, A. Dahlmann, A. Schröder, P. Linz, C. Kopp, M. Rauh, U. Goller, L.

Beck, A. Agureev, G. Vassilieva, L. Lenkova, B. Johannes, P. Wabel, U. Moissl, J.

Vienken, R. Gerzer, K. Eckardt, D.N. Müller, K. Kirsch, B. Morukov, F.C. Luft, and J.

Titze1. 2016. Long-term space flight simulation reveals infradian rhythmicity in human

Na+ balance. Cell Metabolism. 17: 125–131.

Samra, M. and A.C. Abcar. 2012. False estimates of elevated creatinine. Permanente Journal.

16(2): 51-52.

Simerville, J.A., W.C. Maxted, and J.J. Pahira. 2005. Urinalysis: A Comprehensive Review.

American Family Physician. 71(6): 1153-1162.

Tortora, G. Introduction to the Human Body, 9th Ed. Wiley Higher Ed. Kindle Edition: 362, 354,

567-576.
 25

Raw Data

Control Subject
Time 0 30 60 90 120
Urine Volume (ml) 100 60 48 46 52

Time Elapsed (min) 45 30 30 30 30

Vurine - Urine Flow Rate

2.2 2.0 1.6 1.5 1.7
(ml/min)

Specific Gravity 1.0203 1.0160 1.0112 1.0172 1.0124

Urine pH 6.48 6.41 6.00 5.81 6.10

Na+ Meter Reading (mV) -45.6 -47.2 -51.4 -39.0 -34.2

UNa+ - Urine Na+ Concentration

98.1 90.3 72.7 138 177
(mEq/L)

CNa+ - Na+ Clearance (ml/min) 1.5 1.2 0.80 1.5 2.1

Estimated Urine Creatinine

675 938
Concentration (mg/L)

Dilution Factor 20 25

OD from Spec20 0.675 0.622

Sample Creatinine
91.0 85.2
Concentration (mg/L)

UCr - Urine Creatinine

1820 2130
Concentration (mg/L)

CCr - Creatinine Clearance

337 284
(ml/min)

Expected Actual
Water (ml) 267 206

Na+ (g) 0.60 0.56

H+ (mol) 8.8E-08 1.8E-07

 26

Hypotonic Subject
Time 0 30 60 90 120
Urine Volume (ml) 44 258 340 130 69

Time Elapsed (min) 20 30 30 30 30

Vurine - Urine Flow Rate

2.2 8.6 11 4.3 2.3
(ml/min)

Specific Gravity 1.0074 1.0005 1.0005 1.0018 1.0027

Urine pH 6.73 6.79 7.00 6.75 7.07

+
Na Meter Reading (mV) -52.6 -90.5 -99.0 -92.0 -86.7

UNa+ - Urine Na+ Concentration

68.4 9.68 6.24 8.96 11.8
(mEq/L)

CNa+ - Na+ Clearance (ml/min) 1.0 0.57 0.49 0.27 0.19

Estimated Urine Creatinine

568 110
Concentration (mg/L)

Dilution Factor 10 3

OD from Spec20 0.505 0.235

Sample Creatinine
72.3 42.7
Concentration (mg/L)

UCr - Urine Creatinine

723 128
Concentration (mg/L)

CCr - Creatinine Clearance

159 145
(ml/min)

Expected Actual
Water (ml) 264 797

Na+ (g) 0.42 0.15

H+ (mol) 4.9E-08 1.0E-07

 27

Isotonic Subject
Time 0 30 60 90 120
Urine Volume (ml) 186 30 66 60 34

Time Elapsed (min) 140 30 30 30 30

Vurine - Urine Flow Rate

1.3 1.0 2.2 2.0 1.1
(ml/min)

Specific Gravity 1.0078 1.0099 1.0095 1.0107 1.0066

Urine pH 6.71 6.95 6.83 6.67 5.85

+
Na Meter Reading (mV) -47.8 -47.3 -50.9 -47.2 -39.2

UNa+ - Urine Na+ Concentration

87.6 89.9 74.6 90.3 136
(mEq/L)

CNa+ - Na+ Clearance (ml/min) 0.80 0.62 1.1 1.2 1.1

Estimated Urine Creatinine

941 568
Concentration (mg/L)

Dilution Factor 15 30

OD from Spec20 0.284 0.126

Sample Creatinine
48.0 30.7
Concentration (mg/L)

UCr - Urine Creatinine

721 920
Concentration (mg/L)

CCr - Creatinine Clearance

96 202
(ml/min)

Expected Actual
Water (ml) 159 190

Na+ (g) 0.32 0.41

H+ (mol) 3.1E-08 7.4E-08

 28

Alkalotic Subject
Time 0 30 60 90 120
Urine Volume (ml) 186 44 20 20 24

Time Elapsed (min) 120 30 30 30 30

Vurine - Urine Flow Rate

1.6 1.5 0.7 0.7 0.8
(ml/min)

Specific Gravity 1.0083 1.0140 1.0152 1.0199 1.0222

Urine pH 5.33 5.50 7.71 7.90 7.71

+
Na Meter Reading (mV) -40.6 -42.5 -33.2 -29.0 -29.0

UNa+ - Urine Na+ Concentration

127 115 186 231 231
(mEq/L)

CNa+ - Na+ Clearance (ml/min) 1.4 1.2 0.86 1.1 1.3

Estimated Urine Creatinine

806 1875
Concentration (mg/L)

Dilution Factor 30 50

OD from Spec20 0.277 0.233

Sample Creatinine
47.3 42.4
Concentration (mg/L)

UCr - Urine Creatinine

1418 2122
Concentration (mg/L)

CCr - Creatinine Clearance

220 141
(ml/min)

Expected Actual
Water (ml) 186 108

Na+ (g) 0.54 0.44

H+ (mol) 8.7E-07 1.4E-07

 29

Sources
30
31
32
33
34
35
36
37
38
39
40
 41

Calculations

All example calculations are for Control subject at time 0, unless noted otherwise.

Body mass in kg:

1 kg
Body mass (lbs) × 2.205 lbs = body mass (kg)

153 lb
Example: = 69.39 kg
2.205 lb⁄kg

Quantity of challenge fluid to drink:

ml fluid
Subject ′ s body mass (kg) × = amount to drink (ml)
kg

14 ml
Example (Hypotonic subject): 48.5 kg × = 679 ml
kg

Urine Flow Rate (ml/min):

Urine Volume (ml) ml

= Urine Flow Rate ( )
Time Elapsed since last urination (min) min

100 ml
Ex: 45 min = 2.2 ml/min

Urine Na+ Concentration (mEq/L):

This involved using a calibration equation to convert the Na+ meter reading, given in millivolts,

into a urine concentration in milliequivalents per liter.

Calibration equation: y = 19.387 ln(x) − 134.51; y = mV reading and x = urine [Na+ ]

Na+ Meter Reading (mV)+134.51

e( 19.387
)
= Urine Na+ Concentration (mEq/L)
−45.6(mV)+134.51
Ex: e( 19.387
)
= 98.1 mEq/L
 42

Na+ Clearance (ml/min):

mEq ml
Urine Na+ Concentration ( L ) × Urine Flow Rate (min) ml
= Na+ Clearance ( )
mEq min
Plasma Na+ Concentration ( L )

mEq
(Plasma Na+ Concentration = 145 )
L

mEq ml
98.1 ( )×2.2 ( )
L min
Ex: mEq = 1.5 ml/min
145 ( )
L

Est. UCr - Estimated Urine Creatinine Concentration (mg/L):

ml mg
Creatinine Clearance (min) × Plasma Creatinine Concentration( L ) mg
= Est. UCr ( )
ml L
Urine Flow Rate min

ml
Creatinine Clearance = 125 ;
min
mg mg
Plasma [Creatinine] = 12 (males) or 10 (females)
L L
ml mg
125 ×12
min L
Ex: ml = 675 mg/L
2.2
min

Dilution Factor:

mg
Estimated Urine Creatinine Concentration ( L )
mg = Approximate Dilution Factor
Desired Concentration ( L )

mg
Desired Concentration had to be between 25 − 50 ;
L

Approximate Dilution Factor was then rounded.

mg
675( )
L
Ex: mg = 19, which was then rounded to 20
35 ( )
L
 43

Sample Creatinine Concentration (mg/L):

This involved using a calibration equation to convert the absorbance value from the Spec 20

(OD) into a creatinine concentration in mg/L.

Calibration equation: y = 0.0091x − 0.5132; y = OD and x = sample [creatinine] in mg/L

OD + 0.1532 mg
= Sample Creatinine Concentration ( )
0.0091 L
0.675+0.1532
Ex: = 91 mg/L
0.0091

Urine Creatinine Concentration (mg/L):

mg mg
Sample Creatinine Concentration ( ) × Dilution Factor = Urine [Creatinine]( )
L L

Ex: 91 × 20 = 1820 mg/L

Creatinine Clearance (ml/min):

mg ml
Urine [Creatinine] ( L ) × Urine Flow Rate(min) ml
mg = Creatinine Clearance ( )
Plasma Creatinine Concentration ( L ) min

mg mg
Plasma [Creatinine] = 12 (males) or 10 (females)
L L
mg ml
1820( )×2.2 ( ) ml
L min
Ex: mg = 337 (min)
12 ( )
L

Expected Water (ml):

ml
Urine Flow Rate at time 0 ( ) × 120 minutes = Expected Water (ml)
min
ml
Ex: 2.2222 (min) × 120 min = 267 (ml)
 44

Actual Water (ml):

This is simply the sum of the volumes of the time 30 through time 120 collections.

Ex: 60 ml + 48 ml + 46 ml + 52 ml = 206 ml

Expected Na+ (g):

g ml
2.76 min Urine Flow Rate at time 0 (min) mEq
× × Urine [Na+ ] ( ) = Expected Na+ (g)
mEq 1000 ml L
L
g ml
2.76 2.2 ( ) mEq
min min
Ex: × 1000 ml × 98.1 ( ) = 0.60 (g)
mEq L
L

Actual Na+ (g):

g mEq Urine Volume (ml)

∑ [0.023(mEq) × Urine [Na+ ] ( )× 1000 ml ] for time 30 through time 120 yields
L
L

Actual Na+ in grams.

g mEq 60 (ml) g mEq 48 (ml)

Ex: (0.023(mEq) × 90.3 ( )× 1000 ml ) + (0.023(mEq) × 72.7 ( )× 1000 ml )+
L L
L L

g mEq 46(ml) g mEq 52(ml)

(0.023(mEq) × 138 ( )× 1000 ml ) + (0.023 (mEq) × 177 ( )× 1000 ml ) = 0.56 g
L L
L L

Expected H+ (mol):

ml
Urine Flow Rate at time 0 (min)
[H + ] × × 120 min = Expected H + (mol)
1000 ml
L

[H + ] = 10−pH
ml
mol 2.2
Ex: 10−6.48 × min
1000 ml × 120 min = 8.8 × 10−8 mol
L
L
 45

Actual H+ (mol):

Urine Volume (ml)

∑ [[H + ] × 1000 ml ] for time periods 30 through 120 yields Actual H+ (mol).
L

mol 60 ml mol 48 mol 46

Ex: (10−6.41 × 1000 ml ) + (10−6.00 × 1000 ml ) + (10−5.81 × 1000 ml )+
L L L
L L L

mol 52
(10−6.10 × 1000 ml ) = 1.8 × 10−7
L
L