I.

Introduction

Urine formation begins with the movement of plasma ultrafiltrate into the kidneys. This plasma
ultrafiltrate is an essentially protein-free fluid which passively passes from the glomerular capillaries into
the Bowman's space. This process is driven by Starling forces. Glomerular filtration is followed by
reabsorption of water and solutes from the different parts of the renal tubules, then by the secretion of
selected solutes into the renal tubules.

URINE FORMATION

The ability of the kidneys to selectively clear waste products from the blood and simultaneously
maintain the body’s essential water and electrolyte balances is controlled in the nephron by the following
functions: renal blood flow, glomerular filtration, tubular reabsorption, and tubular secretion.

RENAL BLOOD FLOW

The renal artery supplies blood to the kidneys. Blood enters the capillaries of the nephron through
the afferent arteriole. It then flows through the glomerulus and into the efferent arteriole. Before returning
to the renal vein, blood from efferent arteriole enters the peritubular capillaries and the vasa recta and
flows slowly through the cortex and medulla of the kidney close to the tubules.

The proximal convoluted tubule provides the immediate reabsorption of essential substances from
the fluid, and final adjustment of the urinary composition in the distal convoluted tubule. The vasa recta
are located adjacent to the ascending and descending loop of Henle in the juxtamedullary nephrons. In
this area, the major exchanges of water and salts take place between the blood and the medullary
interstitium, which maintains the osmotic gradient in the medulla that is necessary for renal concentration.
GLOMERULAR FILTRATION

The glomerulus consists of a coil approximately eight capillary lobes. It serves as a nonselective
filter of plasma substances with molecular weights of less than 70,000. Actual filtration process involves
several factors including cellular structure of the capillary walls and Bowman’s capsule, hydrostatic and
oncotic pressures, and the feedback mechanism of the rennin-angiotensin-aldosterone system.

TUBULAR REABSORPTION

The cellular mechanisms involved in tubular reabsorption are active and passive transport.

SUBSTANCE LOCATION
Active Transport Glucose, amino acids and Proximal convoluted tubule
salts
Chloride Ascending loop of Henle
Sodium Distal convoluted tubule

Passive Transport Water Proximal convoluted tubule,

descending loop of Henle,
and collecting tubules
Urea Proximal convoluted tubule
and ascending loop of Henle
Ascending loop of Henle
Sodium

TUBULAR SECRETION

Tubular secretion serves two major functions: elimination of waste products not filtered by the
glomerulus and regulation of the acid-base balance in the body through the secretion of hydrogen ions.
 Foreign substances which cannot be filtered by the glomerulus because they are bound to plasma
proteins enter the peritubular capillaries, where they dissociate from their carrier proteins because of
strong affinity for tubular cells. The major site for removal of the nonfiltered substances is the proximal
convoluted tubule.

CONCENTRATION AND DILUTION OF URINE

The regulation of plasma osmolarity is accomplished by varying the amount of water excreted by
the kidneys. It is due to the response to water deprivation or to water intake. When the osmolality is too
low, nervous and hormonal feedback mechanisms cause the kidneys to excrete a great excess of water in
urine causing a dilute urine, but removes water from the body to increase the body fluid osmolality back
to normal. When the osmolality of body fluids is too great, the kidneys excrete an excess of solutes to
reduce the body fluid osmolality again back to normal, but at the same time excreting a concentrated
urine.

OSMOLAL CONCENTRATION CHANGES IN THE DIFFERENT SEGMENTS OF THE TUBULES

Proximal Tubule

Osmolality of the fluid remains almost exactly equal to that of the glomerular filtrate, 300
mOsm/L throughout the entire extent of the proximal tubule.

Loop of Henle

The osmolality rises rapidly because of the countercurrent mechanism. During high concentration
of ADH, the loop of Henle osmolality rises much higher than when a dilute urine is being formed because
of large quantity of urea that is passively reabsorbed into the medullary interstitium from the collecting
ducts.

Thick Ascending Limb

The osmolality falls to a very low level usually about 100 mOsm/L.

Late Distal Tubule, Cortical Collecting Duct, and Collecting Duct

The osmolality depends entirely on ADH. In the absence of ADH, very little water is reabsorbed,
osmolality remains less than 100 mOsm/L, very dilute urine is formed.

In the presence of excess ADH, these segments become highly permeable to water, most of the
water is reabsorbed, thus producing a very concentrated urine.

TESTS DONE ON URINE SPECIMENS

VOLUME

Urine volume depends on the amount of water that the kidneys excrete. The amount excreted is
usually determined by the body’s state of hydration. Factors that influence urine volume include fluid
intake, fluid loss from nonrenal sources, variations in the secretion of antidiuretic hormone and the
necessity to excrete increased amounts of dissolved solids, such as glucose or salts. The normal daily
output is usually 1200 to 1500 ml, a range to 600 to 2000 ml is may be considered normal.

SPECIFIC GRAVITY

Defined as the density of a solution compared with the density of a similar volume of distilled
water at a similar temperature. Because urine is actually water that contains dissolved chemicals, the
specific gravity of urine is a measure of the density of the dissolved chemicals in the specimen. The
kidneys produce urine with specific gravity that ranges from 1.005-1.035.

VALUES:

1.106-1.022 normal adults with normal diets and normal fluid intake (24 hr. period)
1.023 random specimen
1.022 no fluids for 12 hrs. overnight
1.026 or higher 24 hrs. without fluid
Less than 1.003 minimum specific gravity after a standard water load.

pH

Along with the lungs, the kidneys are the major regulators of the acid-base content in the body.
They do this through the secretion of hydrogen in the form of ammonium ions, hydrogen phosphate and
weak organic acids, and by the reabsorption of bicarbonate from the filtrate in the convoluted tubules. A
healthy individual will usually produce a first morning specimen with a slightly acidic pH of 5.0 to 6.0.
The pH of normal random samples can range from 4.5 to 8.0. Consequently, no normal values are
assigned to urinary pH, and it must be considered in conjunction with other patient information.

Protein

Of the routine chemical tests performed on urine, the most indicative of renal disease is the protein
determination. The presence of proteinuria is often associated with early renal disease. Normal urine
contains very little protein, usually less than 10mg/dl or 100 mg per 24 hrs. excreted. This protein consist
primarily of low molecular weight serum proteins that have been filtered by the glomerulus and proteins
produced in the genitourinalry tract.

Glucose

Determination of glucose in urine is valuable in the detection and monitoring of diabetes mellitus.

Under normal circumstances, almost all the glucose filtered b the glomerulus is reabsorbed in the
proximal convoluted tubule. Should the blood level of glucose become elevated, the tubular transport of
glucose ceases and glucose appears in urine. The renal threshold for glucose is approximately 160-180
mg/dl.

II. Materials

Wide-Mouth Collecting Bottles (1L capacity)

Graduated Cylinders
1L 0.9% NaCl Solution
1L 5% Glucose
1L Distilled Water
Urine dipstick determination for pH and specific gravity

III. Methodology

Preparation

Four subjects from the group were assigned to do the procedure. They were advised to eat a light
meal the evening prior to the experiment, after which they will be placed on NPO (nothing per orem)
eight hours prior to the start of the experiment. The types of food and fluid taken in prior to the
experiment were noted. The materials used were prepared. Wide mouth collecting bottles or beakers with
one liter capacity were used for urine collection. Graduated cylinders were used for approximation of the
urine volume. Urine reagent strips were used for determination of pH and specific gravity.

Specimen Collection

Specimens must be collected in clean, dry, leak- proof containers. It should have a wide mouth to
facilitate collections from female patients and a wide, flat bottom to prevent overturning. They should be
made of clear materials to allow for determination of color and clarity. All specimens must be properly
labeled with the subject’s name with the date and time of collection in it.

Specimen Handling

The fact that a urine specimen is so readily available and easily collected often leads to laxity in
the treatment of the specimen after its collection. Changes in urine composition take place not only in
vivo but also in vitro, thus necessitating correct handling procedures after the specimen is collected. A
specimen must be tested within two hours.

First Sample

The four subjects were assigned to collect the first urine sample. It was collected at the start of the
laboratory procedure. The first voided morning urine was not used in the experiment. The time of
collection was noted. The first sample of urine collected was subjected for physical and chemical
examination such as: volume determination, specific gravity, pH, sugar content and protein content.

Second Sample

After thirty minutes, the second sample was collected by the same subjects. The samples were also
subjected to physical and chemical examination that was done on the first sample.

As soon as the second sample was collected, the subjects drink one liter of the fluid assigned to
them: distilled water, 5% glucose, 0.9% sodium chloride. The fourth subject will drink nothing.

Third Sample

The third sample was collected thirty minutes after drinking the fluid. The samples were also
subjected to the analysis done in the previous samples.

Fourth Sample
 The fourth sample was collected approximately sixty minutes after the third collection. The
samples were then subjected to the analysis. Results obtained in all samples were recorded and tabulated.

IV. Results and Discussion

Effect of Distilled Water Intake in Urine Output

Table 1. Urine Output of Subject 1 (intake of 1L of distilled water).

Sample # 1: Sample # 2: Sample # 3: after Sample # 4:
NPO after 30 min drinking ditilled water after 1 hour
Volume (mL) 133 12 44 504
Specific Gravity 1.005 1.015 1.005 1.005
pH 6.0 5.0 5.0 5.0
CHO Neg (-) Neg (-) Neg (-) Neg (-)
CHON Neg (-) Trace (+/-) Neg (-) Neg (-)

Maintenance of water balance is tightly regulated by renal functions such that water intake
precisely match water loss from the body. Low water intake or increased water loss results to small
volume of concentrated urine produced by the kidneys to conserve water. Human kidneys have powerful
capability to retain water such that the volume of urine excreted may represent less than 1% of the volume
of glomerular filtrate. In cases of increased water intake or overhydration, such as the subject in this
experiment, large volume of dilute urine is produced. Water reabsorption occurs passively in several parts
of the nephron. Permeability to water by the distal tubule and collecting ducts is regulated by antidiuretic
hormone (ADH). The rate of release of this hormone from the posterior pituitary gland is in turn affected
by changes in fluid intake, or fluid loss from the body, consequently changing the rate of water excretion.

In the proximal tubule (PT), 67% of the filtered water is reabsorbed driven by the transtubular
osmotic gradient established by solute reabsorption generally by NaCl. Na+ is actively reabsorbed along
with organic solutes HCO3 and Cl- through specific channels in the apical membrane and through the Na+-
K+ ATPase pump in the basolateral membrane. These processes thus reduce the osmolality of tubular fluid
and increase the osmolality of the intercellular space. Water then osmotically follows the solutes out of the
tubule, which is highly permeable to it, causing increase in the hydrostatic pressure in the intercellular
space. This causes both water and solutes to ultimately move into the capillaries. Other ions such as K+
and Ca++ are also reabsorbed through solvent drag. It is important to note that in this segment, iso-osmotic
reabsorption of solutes and water occurs such that there is no separation of solutes and water movement.
Thus, the tubular fluid continuing on the later part of the nephron is isoosmotic with the plasma.

Formation of dilute urine begins at the loop of Henle where movement of solute and water
happens independently. As mentioned earlier, the fluid entering this region is isoosmotic with the plasma
brought about by the isoosmotic reabsorption at the PT. The descending thin limb (DTL) of Henle’s loop
is composed of flat cells which are highly permeable to water but not to solutes such as NaCl and urea.
This reabsorption of water is driven by the osmotic gradient set up across the DTL by the thick ascending
loop of Henle. As the fluid descends deeper into the medulla, more water is being reabsorbed due to the
increasing osmolality in this region. The fluid leaving the DTL has greater NaCl and lower urea compared
to the interstitial fluid.

In the ascending thin limb (ATL) of Henle’s loop, NaCl is passively reabsorbed and urea diffuses
into the tubule due to its high permeability to these solutes but water is restricted to any movement in or
out the tubule (direction of movement of the solutes is explained by their initial concentration on the
entering tubular fluid). Consequently, as the tubular fluid ascends ATL, the greater movement of NaCl out
of the tubule decreases the osmolality of the fluid, and without any change in the volume, the tubular fluid
is now diluted. Continuing on, in the thick ascending loop (TAL) of Henle, active pumping of NaCl out of
the tubular fluid, hence reabsorption, occurs while being impermeable both to water and urea. TAL, which
contributes largely in making the urine diluted, is also referred to as the diluting segment of the kidney.
Fluid leaving the TAL can have osmolality as hypoosmotic as 150 mOsm/kg H2O compared to plasma.

In the next segments of the nephron, namely the late distal tubules (DT) and collecting ducts (CD),
ADH controls the permeability and movemet of water thus determining the final amount and osmolality
of urine. In these regions, the reabsorption of salt and water can be controlled independently.

ADH is produced by specialized hypothalamic neurons and is transmitted via their axons to the
posterior pituitary gland where it is released into the blood. The control of its release is mediated by the
baroreceptors and osmoreceptors (see Figure 1). The most common baroreceptors are located in the
carotid sinus and aortic arch but are also present in large veins and cardiac atria. Stimulation of these
receptors sends inhibitory signals to the release of ADH hence increasing the excretion of water.
Osmoreceptors which are found in the hypothalamus act as osmometers which are stimulated by the
increased osmolarity in turn stimulating the release of ADH. This increases the permeability of the DT
and CD to water promoting reabsorption and water retention. Osmoreceptors are more sensitive regulators
of ADH detecting 1% change in osmolality compared to the 10-15% change in plasma volume which is
needed to elicit response of the baroreceptors. Generally, ADH presence during times of dehydration
enhances water permeability in DT and CD to conserve water; when ADH is reduced, less H 2O is
reabsorbed in DT and CD resulting in excretion of large volume of H2O. The most dilute urine possible is
about 50 osmoles/liter.

Figure 1. Plasma volume regulation of ADH. Elevated plasma volume increases left atrial pressures,
stimulating the atrial baroreceptors and inhibiting ADH secretion.

Active reabsorption of NaCl continues in smaller extent in the regions of distal tubule and cortical
collecting duct while being impermeable to urea. When there is large amount of water available in the
body as with increase water intake or decreased osmolality, the absence or the presence of ADH in low
levels further dilutes the tubular fluid to up to 100 mOsm/kg H2O. In the medullary collecting duct, active
reabsorption of NaCl still occurs. Even in the absence of ADH, this region is slightly permeable to water
and urea allowing water reabsorption to a very small degree and influx of urea from the medullary
interstitium. Final concentration of the urine produced with this processes of urine dilution is
approximately 150 mOsm/kg H2O.

The subject in this experiment represents the condition of overhydration. The urine volume (12 ml
– 44 ml) which is the output from just after emptying the bladder and first thirty minutes after drinking a
liter of distilled water showed slight increase in urine output but otherwise not too significant in
consideration with the body size of the subject (~ 80 kg). This time interval may represent the early
processes involving other systems of the body. Because water intake was done orally, it still has to be
processed first in the digestive tract where it should first be absorbed in the intestine before reaching the
extracellular fluid (blood plasma) where it can directly be subjected to renal regulation. After an hour, a
marked increase of urine volume can be observed (44 ml – 504 ml) which reflects renal regulation of
water balance just discuss above. Because of the increase in water intake and water content of the body,
the result is the formation of large volume of dilute urine, which is a consequent of decrease ADH effects
or reduction in the permeability of the distal tubule, cortical and medullary collecting duct to water.

A summary of renal regulation of urine volume in response to excessive water intake as detected
by the osmoreceptors is presented in Figure 2.

Figure 2. Regulation of volume in response to excess water ingestion. Osmoreceptors in the hypothalamus
detect the drop in osmolarity and decrease ADH secretion to allow excretion of excess water.
+
Effect of Glucose Intake in Urine Output
Table 2. Urine Output of Subject 2 (intake of 1L 5% glucose).
Sample # 1: Sample # 2: Sample # 3: after Sample # 4:
NPO after 30 min drinking 5% glucose after 1 hour
Volume (mL) 60 20 50 700
Specific Gravity 1.015 1.015 1.005 1.005
pH 6 6 6 5
CHO Neg (-) Neg (-) Neg (-) Neg (-)
CHON 0.3 (+1) Trace (+/-) Neg (-) Neg (-)

Glucose is one of the essential nutrients needed by the body. It is rarely seen in the urine. It is
completely reabsorbed in the proximal tubule except in cases of Glycosuria where the threshold
concentration of blood glucose is surpassed. This is caused by either lack of insulin, kidney damage due
to toxins and drugs or too much sugar intake. Blood glucose concentration should only be 180-200 mg/dl.

Glucose is actively transported across the apical cell membranes of the epithelial cells. It needs
energy in order to be reabsorbed. The energy is provided by the strong concentration gradient produced by
the downhill movement (influx) of sodium. The glucose-Na+ symport protein uses the sodium gradient to
transport glucose into the cell. Glucose leaves the cell across the basolateral membrane by passive
transport.

Effect of NaCl Intake in Urine Output

Table 3: Urine Output of Subject 3 (intake of 1L 0.9% NaCl).

Sample # 1: Sample # 2: Sample # 3: after Sample # 4:
NPO after 30 min drinking 0.9 NaCl after 1 hour
Volume (mL) 28 9 No urine voided 9
Specific Gravity 1.020 1.020 No urine voided 1.020
pH 5.0 5.0 No urine voided 5.0
CHO Neg (-) Neg (-) No urine voided Neg (-)
CHON Trace (+/-) Trace (+/-) No urine voided Neg (-)

As water is reabsorbed through osmosis in the proximal convoluted tubule from the filtrate,
substances that remain in the renal tubule tend to become more and more concentrated. Water
reabsorption is strongly associated with the active reabsorption of sodium ions. Generally, as sodium
reabsorption increases, water reabsorption increases and vice versa. Therefore, there will be less but more
concentrated urine output .
 Reabsorption of sodium ions in the proximal segment of the renal tubule is about 70 percent of the
total filtrate by active transport known as the sodium pump mechanism. As the sodium ions (Na+) move
through the tubular wall, chloride ions (Cl-), phosphate ions (PO4-3), and bicarbonate ions (HCO3-)
accompany them. The electrochemical attraction between particles of different charges caused the
movement of these negatively charges ions. The concentration of solutes within the peritubular blood
increases as more and more sodium ions are actively transported into the peritubular capillary, along with
various negatively charged ions. The movement of solutes and water into the peritubular capillary causes
the great reduction of the volume of fluid within the renal tubule.

Sodium ions are continuously reabsorbed by active transport as the tubular fluid pass through the
loop of henle the distal convoluted segment, and the collective duct. Because of this, the sodium that
enters the renal tubule as part of the glomerular filtrate maybe reabsorb3ed before the urine is excreted.
Therefore, water is also continuously reabsorbed by osmosis in different segments of the renal tubule.

Through the action of aldosterone and ADH, additional water may be reabsorbed. The cells of the
adrenal cortex secrete aldosterone in response to changes in the concentration of sodium and potassium
ions in the blood. The main effect of aldosterone is retention of sodium ions and water molecules causing
the decrease in urine output. On the other hand, ADH is produced by neurons in the hypothalamus and
released from the posterior lobe of the pituitary gland with decreasing concentration of water in the
blood. In the kidneys, ADH increases the permeability of the epithelial linings of the distal convoluted
tubule and collecting duct which causes the water to move rapidly out of these segments by osmosis,
another reason why urine volume is reduced and urine is concentrated.

In general, ADH stimulates the production of concentrated urine while inhibiting the loss of body
fluids as a compensatory mechanism when dehydration is foreseen.

Effect of No Water Intake in Urine Output

Table 4: Urine Output of Subject 4 (No Fluid Intake)

Sample # 1: Sample # 2: Sample # 3: Sample # 4:
NPO after 30 min after 1 hour
Volume (mL) 17 7 8 13
Specific Gravity 1.015 1.020 1.020 1.020
pH 6.0 5.0 5.0 5.0
CHO Neg (-) Neg (-) Neg (-) Neg (-)
CHON 0.3 (+1) 0.3 (+1) Trace (+/-) 0.3 (+1)
 Our bodies have a finely tuned mechanism for maintaining the proper concentration of salt in our
body fluids such as blood and urine. If the water intake goes down (such as the activity done in the
experiment wherein the subject was prohibited to drink for a certain amount of time), the urine output
goes down and urine salt concentration goes up. Over the long term this can cause a stress on the kidneys
and should be avoided.

Urine production

Urine production is controlled by certain hormones such as ADH (Anti-diuretic hormone) and
aldosterone. They control how much urine can be produced by the body. If the body becomes dehydrated,
the pituitary gland releases ADH. This hormone reduces urine volume by causing the collecting tubules to
allow more water to be reabsorbed into the bloodstream. On the other hand, if too much fluid is in the
body, the pituitary gland stops releasing ADH and the excess water passes out of the body as dilute urine.

Aldosterone enhances sodium reabsorption, which in turn increases water reabsorption into the
blood from the collecting tubules. Because of the effect of aldosterone on the collecting tubules, the
amount of water excreted in the urine decreases and blood volume and blood pressure increase.

In the experiment conducted, the subject was disallowed to drink or eat anything for 8 hours. As
the results show, the volume of urine sample, collected after 30 minutes of initial collection, has greatly
decreased from 17 ml to 7 ml, and has shown to increase gradually by only 5 ml after an hour. This is due
to reabsorption process of the collecting tubules, which are mainly controlled by ADH and aldosterone.
 Osmoreceptors located in the anterolateral hypothalamus measure the Posm. They play major key
roles in water and sodium balance. Changes in Posm result in cell swelling or shrinking, therefore
signaling the release (or inhibition) of ADH. At Posm below 280 mosm/kg, these cells are virtually
inactive and do not stimulate ADH secretion. However, small changes above this Posm (even of only 1%)
will trigger major firing changes in osmoreceptors. At a Posm of 290 mosm/kg, enough ADH would be
secreted (5 pg/mL) to cause a maximum retention of water, yielding Urineosm of 1250 mosm/kg. Any
[ADH] above 5 pg/mL results in this same maximal antidiuresis. Not all osmotically active substances can
stimulate shrinking or swelling of the osmoreceptors; while sodium ions are able to do this, urea cannot
since it can freely cross membranes.

V. Conclusion

Increased water intake or overhydration causes large volume of dilute urine to be produced.
Glucose intake increases volume of urine output as well. NaCl intake causes less volume but more
concentrated urine output. Finally, no water intake causes decreased urine output.

VI. Answer to Questions

1. What is the mechanism of renal concentration of the urine? Of renal dilution?

Renal dilution (water diuresis) follows these steps (Berne, 2004):
1. Fluid entering the descending thin limb of Henle’s loop from the proximal tubule is isoosmotic
with respect to plasma. This isoosmotic fluid reflects the essentially isoosmotic nature of the
solute and water reabsorption in the proximal tubule.
2. The descending thin limb is highly permeable to water but much less so to solutes such as NaCl
and urea. Consequently, as the fluid descends deeper into the hyperosmotic medulla, water is
reabsorbed owing to the osmotic gradient that is set up across the descending thin limb.
 (Osmolality loop = Osmolality interstitial; but composition is different: [NaCl] tubular > [NaCl] interstitial;
[urea] tubular < [urea] interstitial)
3. The ascending thin limb is impermeable to water but permeable to NaCl and urea. Consequently,
as tubular fluid moves up the ascending limb, NaCl is passively reabsorbed, while urea passively
diffuses into the tubular fluid. (Net effect: tubular fluid volume is unchanged, [NaCl] decreases,
[urea] increases; [NaCl] movement out of TAL > [urea] movement into TAL, causing tubular fluid
dilution)
4. The TAL is impermeable to water and urea, but it actively reabsorbs NaCl, causing further dilution
of the tubular fluid. Fluid leaving the TAL is hypoosmotic with respect to plasma.
5. The distal tubule and cortical portion of the collecting ducts actively reabsorb NaCl but are
impermeable to urea. In absence or low levels of ADH, the DT and CD are impermeable to water.
Accordingly, the osmolality of the tubule fluid in these segments is reduced further because NaCl
is reabsorbed without water.
6. The medullary CD actively reabsorbs NaCl. Even in the absence of ADH, this segment is slightly
permeable to water and urea. Consequently, some urea enters the CD from the medullary
interstitium, and a small volume of water is reabsorbed.
7. The urine will have an osmolality of = 50 mOsm/kg H2O and will contain low concentrations of
NaCl and urea.

Urine concentration (antidiuresis) follows these steps:

1-4. The first 4 steps are similar to water diuresis. An important point in understanding concentrated
urine production is while NaCl is reabsorbed by the ascending thin and thick limbs (thereby
diluting the tubular fluid), the reabsorbed NaCl accumulates in the medullary interstitium and
raises its osmolality. This accumulation is important in the production of concentrated urine
because it provides the osmotic driving force for water reabsorption by the CD. This process is
termed countercurrent multiplication.
.5 Due to the NaCl reabsorption by the TAL, fluid reaching the CD is hypoosmotic with respect to
the surrounding interstitial fluid, thus establishing an osmotic gradient. In the presence of ADH,
water diffuses out of the tubule lumen, and tubule fluid osmolality increases, starting the process
of urine concentration. Although the fluid at this point has the same osmolality as the fluid that
entered the descending thin limb, the composition has been altered. NaCl is decreased due to NaCl
reabsorption in the earlier segments. Instead, the tubule fluid osmolality is accounted for by urea
and other non-reabsorbed solutes (e.g. K+, creatinine).
.6 The osmolality of the interstitial fluid in the medulla progressively increases from the
corticomedullary junction (approx. 300 mOsm/kg H2O) to the papilla (approx. 1200 mOsm/kg
H2O). Thus an osmotic gradient exists between the tubular and the interstitial fluid along the entire
medullary CD> In the presence of ADH, the osmolality of the tubular fluid increases as water is
reabsorbed. Because the initial portion of the CD is impermeable to urea, it remains in the tubular
fluid, increasing it concentration in the tubular fluid.
.7 The urine has an osmolality of 1200 mOsm /kg H2O and contains high concentrations of urea and
other non-reabsorbed solutes. Because urea in the tubular fluid equilibrates with urea in the
medullary interstitial fluid, its concentration in the urine will be similar to that of the interstitium.

2. What is the physiologic means of activation of the concentrating and diluting ability of the kidney?

The kidneys are responsible for maintaining water balance. The maintenance of water balance requires
that water intake precisely match water loss from the body. When water intake is low or water loses
increase, the kidneys conserve water by producing a small of volume of urine that is hyperosmotic with
respect to plasma. Conversely, when water intake is high or water loses decrease, the kidneys excrete
more water, producing a large volume of hypoosmotic urine.
 Antidiuretic hormone (ADH), or vasopressin, acts on the kidneys to regulate volume and osmolality
of the urine. When plasma ADH levels are low, large volumes of dilute urine are excreted, while when
plasma ADH levels are elevated, a small volume of concentrated urine is excreted. Regulation of ADH is
controlled by body fluid osmolality and hemodynamics. An increase in plasma osmolality causes an
increase in plasma ADH, thereby creating concentrated urine. A decrease in the percentage of blood
volume, which causes a decrease in blood pressure, causes an increase in plasma ADH, which also causes
production of concentrated urine. These changes occur to limit water loss. Also, ADH increases
permeability of the CD to water, as well as the permeability of the medullary portion of the collecting duct
to urea.

3. What are the standard clinical concentration–dilution tests?

The most widely used concentration and dilution test to test for urinary function is the Fishberg
concentration test apart from the obsolete procecdures which are not of great importance nowadays maybe
because of the limitations of those tests. Thje Fishberg concentration test lies on the principle that given
200 mL of fluid and high protein diet at 6:00 pm (with no other food or fluid until completion of test), a
patient with a well functioning kidney will produce up to 300 mL of urine with a specific gravity of 1.026
or higher by 9:00 am in the next morning. Urine is collected at 7:00, 8:00 and 9:0-0 am. Volume and
specificgravity of each collection is measured. A specific gravity of 1.010 indicates renal impairment.
This test is dangerous for patients with ADH deficiency (patients with diabetes insipidus). Those with
edema, severe sweating, diarrhea and fever may have interferences in the results and thus affect accuracy.

4. How do you account for the difference in the urinary excretion and composition seen in the
experiment?

Please see the corresponding discussions for the different experimental setups.

5. What is the relationship between urinary volume and urinary solute excretion?

An increase in urinary volume causes a decrease of the urinary solute concentration, with respect to
the urinary volume. This is because solute excretion is tightly regulated.
The maintenance of Na balance requires a precise balance between NaCl ingested and NaCl excreted
from the body. (In euvolemia, NaCl intake = NaCl excretion) This balance is regulated by renal sympathetic
nerves, RAAS, ANP and ADH. The combined effect of these actions control NaCl excretion, which
maintains the NaCl balance. Potassium homeostasis is regulated by two mechanisms: 1) regulatory
mechanisms of K+ in the ECF, and 2) renal mechanisms to match renal K+ excretion with K+ intake.
Similar to Na homeostasis, these mechanisms are tightly regulated to maintain the normal K +
concentration. Ca2+ and inorganic phosphate (Pi) homeostasis are also tightly regulated. The kidneys, in
conjunction with the gastrointestinal tract and bone, maintain normal plasma Ca2+ and Pi levels.

If there is an increase of urine solute excretion levels with the increase of urine volume, it might be
indicative of renal failure, and should be verified.

6. What is the significance of specific gravity measurement in the determination of urinary solute
concentration? Are there better measurements possible? Give example.

The volume of excreted urine and its concentration of solute are varied by the kidneys to maintain
homeostasis of body fluid and electrolytes. In order to achieve this, the kidneys produce urine with SG
that ranges from 1.005 to 1.035.
 Measuring the SG is significant because dilute urine is an indication of renal disease or hormonal
deficiency. It will also detect possible dehydration or abnormalities in ADH. Also, useful clinical
information can be obtained from measurement of maximal SG.

In critical circumstances, the measurement of osmolality of urine and plasma is preferred than the
measurement of SG.

Specific gravity depends on the number of particles present in a solution and the density of these
particles, whereas osmolality is affected only by the number of particles present.

When evaluating renal concentrating ability, the substances of interest are small molecules, primarily
sodium and chloride. However, urea, which is of no importance to this evaluation, will contribute more to
specific gravity than sodium and chloride. Because all three molecules contribute equally to the
osmolality of the specimen, a more representative measure of renal concentrating ability can be obtained
by measuring osmolality.

Urine osmolality can be measured by freezing point depression and changes in vapor pressure.
However, these methods are more complicated than USG, so their usage is lessened.

REFERENCES:

Strasinger, SK and Di Lorenzo,MS: Urinalysis and Body Fluids. FA Davis Company, Philadelphia, 2001.

Henry, JB: Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia,
1998.

Guyton, A: Textbook of Medical Physiology. WB Saunders, Philadelphia, 1991.

Berne, R., et. al: Physiology. 5th ed. 2004

http://www.medicinalfoodnews.com/vol09/issue2005/h2o.htm

http://bio1152.nicerweb.com

http://www.georgetownuniversityhospital.org/body.cfm

Kidney Function
Tests.http://www.austincc.edu/kotrla/UALect6AutomationFunctionTests&Calculi.pdf#search='Fishberg%
20concentration%20test'. October 23, 2006 4:35 pm