Introduction

The urinary system consist of two kidneys, two ureters, the urinary bladder and the urethra. A large volume of blood
flows through the kidney, which removes substances from the blood to form urine. The urine contains excess water and ions,
metabolic wastes such as urea, and toxic substances consumed in food. The urine produced by the kidneys flows through the
ureters to the urinary bladder, where it is stored until it is eliminated through the urethra.

The kidneys can suffer extensive damage and still maintain their extremely important role in the maintenance of
homeostasis. As long as about one-third of one kidney remains functional, survival is possible. If the functional ability of the
kidneys fails completely, however, death will result without medical treatment.

1. What changes in the urine output do you expect to see in each of the protocols during the laboratory?
Hypoosmotic Solution Diluted Urine
Isosmotic Solution No Effect
Hyperosmotic Solution Concentrated Urine
2. What is the osmolarity of the fluid in the interstitial space in the renal cortex?
The osmolarity of the interstitial fluid in almost all parts of the body is about 300mOsm/L, which is similar to plasma osmol arity.
The osmolarity of the interstitial fluid in the medulla of the kidney is much higher, increasing progressively to about 1200 to
1400 mOsm/L in the pelvic tip of the medulla. This means that the renal medullary interstitium has accumulated solutes in great
excess of water. Once the high solute concentration in the medulla is achieved, it is maintained by a balanced inflow or outflow
of solutes and water in the medulla.
The major factors that contribute to the buildup of solute concentration into the renal medulla are as follows:
Active transport of sodium ions and co-transport of potassium, chloride and other ions out of the thick portion of Loop
of Henle into the medullary interstitium.
Active transport of ions from the collecting ducts into the medullary interstitium.
Facilitated diffusion of large amounts of urea from the inner medullary collecting ducts into the medullary interstitium.
Diffusion of only small amounts of water from the medullary tubules into the medullary interstitium, far less than the
reabsorption of solutes into the medullary interstitium.
The most important cause of the high medullary osmolarity is active transport of sodium and cotransport of potassium, chloride,
and other ions from the thick ascending loop of Henle into the interstitium. This pump is capable of establishing about a 200-
milliosmole concentration gradient between the tubular lumen and the interstitial fluid. Because the thick ascending limb is
virtually impermeable to water, the solutes pumped out are not followed by osmotic flow of water into the interstitium. Thus,
the active transport of sodium and other ions out of the thick ascending loop adds solutes in excess of water to the renal
medullary interstitium. There is some passive reabsorption of sodium chloride from the thin ascending limb of Henles loop,
which is also impermeable to water, adding further to the high solute concentration of the renal medullary interstitium. The
descending limb of Henles loop, in contrast to the ascending limb, is very permeable to water, and the tubular fluid osmolarity
quickly becomes equal to the renal medullary osmolarity. Therefore, water diffuses out of the descending limb of Henles loop
into the interstitium, and the tubular fluid osmolarity gradually rises as it flows toward the tip of the loop of Henle.
As fluid flows down the descending loop of Henle, water is absorbed into the medulla. The descending limb is highly permeable
to water but much less permeable to sodium chloride and urea. Therefore, the osmolarity of the fluid flowing through the
descending loop gradually increases until it is equal to that of the surrounding interstitial fluid, which is about 1200 mOsm/L
when the blood concentration of ADH is high.When a dilute urine is being formed, owing to low ADH concentrations, the
medullary interstitial osmolarity is less than 1200 mOsm/L; consequently, the descending loop tubular fluid osmolarity also
becomes less concentrated. This is due partly to the fact that less urea is absorbed into the medullary interstitium from the
collecting ducts when ADH levels are low and the kidney is forming a large volume of dilute urine.
The thin ascending limb is essentially impermeable to water but reabsorbs some sodium chloride. Because of the high
concentration of sodium chloride in the tubular fluid, owing to water removal from the descending loop of Henle, there is some
passive diffusion of sodium chloride from the thin ascending limb into the medullary interstitium. Thus, the tubular fluid
becomes more dilute as the sodium chloride diffuses out of the tubule and water remains in the tubule. Some of the urea
absorbed into the medullary interstitium from the collecting ducts also diffuses into the ascending limb, thereby returning the
urea to the tubular system and helping to prevent its washout from the renal medulla. This urea recycling is an additional
mechanism that contributes to the hyperosmotic renal medulla.
The thick part of the ascending loop of Henle is also virtually impermeable to water, but large amounts of sodium, chloride,
potassium, and other ions are actively transported from the tubule into the medullary interstitium. Therefore, fluid in the thick
ascending limb of the loop of Henle becomes very dilute, falling to a concentration of about 100 mOsm/L. The early distal tubule
has properties similar to those of the thick ascending loop of Henle, so that further dilution of the tubular fluid occurs as solutes
are reabsorbed while water remains in the tubule.
In the late distal tubule and cortical collecting tubules, the osmolarity of the fluid depends on the level of ADH.With high levels
of ADH, these tubules are highly permeable to water, and significant amounts of water are reabsorbed. Urea, however, is not
very permeant in this part of the nephron, resulting in increased urea concentration as water is reabsorbed.This allows most of
the urea delivered to the distal tubule and collecting tubule to pass into the inner medullary collecting ducts, from which i t is
eventually reabsorbed or excreted in the urine. In the absence of ADH, little water is reabsorbed in the late distal tubule and
cortical collecting tubule; therefore, osmolarity decreases even further because of continued active reabsorption of ions from
these segments.
3. What is the osmolarity of the fluid in the interstitial space in the renal medulla ?
The concentration of fluid in the inner medullary collecting ducts also depends on (1) ADH and (2) the osmolarity of the
medullary interstitium established by the countercurrent mechanism. In the presence of large amounts of ADH, these ducts are
highly permeable to water, and water diffuses from the tubule into the interstitium until osmotic equilibrium is reached, with
the tubular fluid having about the same concentration as the renal medullary interstitium (1200 to 1400 mOsm/L). Thus, a very
concentrated but small volume of urine is produced when ADH levels are high. Because water reabsorption increases urea
concentration in the tubular fluid, and because the inner medullary collecting ducts have specific urea transporters that greatly
facilitate diffusion, much of the highly concentrated urea in the ducts diffuses out of the tubular lumen into the medullary
interstitium. This absorption of the urea into the renal medulla contributes to the high osmolarity of the medullary intersti tium
and the high concentrating ability of the kidney.
4. What hormome is involved in regulating renal water excretion? What normally inhibits the release of this hormone?
Arginine vasopressin (AVP), also known as vasopressin, or antidiuretic hormone (ADH), is aneurohypophysial hormone found
in most mammals. Vasopressin is responsible for regulating the body's retention of water by acting to increase water absorption
in the collecting ducts of the kidney nephron.[1] Vasopressin increases water permeability of the kidney's collecting duct and
distal convoluted tubule by inducing translocation of aquaporin-CD water channels in the kidney nephron collecting duct plasma
membrane.[2] Vasopressin is a peptide hormone that controls the reabsorption of molecules in the tubules of the kidneys by
affecting the tissue's permeability. It also increases peripheral vascular resistance, which in turn increases arterial blood
pressure. It plays a key role in homeostasis, by the regulation of water, glucose, and salts in the blood. It is derived from
a preprohormone precursor that is synthesized in the hypothalamus and stored in vesicles at the posterior pituitary. Most of it is
stored in the posterior pituitary to be released into the bloodstream. However, some AVP may also be released directly into
the brain, and accumulating evidence suggests it plays an important role in social behavior, bonding, and maternal responses to
stress.
Vasopressin has two effects by which it contributes to increased urine osmolarity (increased concentration) and decreased
water excretion. These are:
Increasing the water permeability of distal tubule and collecting duct cells in the kidney, thus allowing water
reabsorption and excretion of more concentrated urine, i.e.,antidiuresis. This occurs through insertion of water
channels (Aquaporin-2) into the apical membrane of distal tubule and collecting duct epithelial cells. Aquaporins allow
water to move down their osmotic gradient and out of the nephron, increasing the amount of water re-absorbed from
the filtrate (forming urine) back into the bloodstream.
V2 receptors, which are G protein-coupled receptors on the basolateral plasma membrane of the epithelial cells, couple to the
heterotrimeric G-protein Gs, which activatesadenylyl cyclases III and VI to convert ATP into cAMP, plus 2 inorganic phosphates.
The rise in cAMP then triggers the insertion of aquaporin-2 water channels by exocytosis of intracellular vesicles, recycling
endosomes. Vasopressin also increases the concentration of calcium in the collecting duct cells, by episodic release from
intracellular stores. Vasopressin, acting through cAMP, also increases transcription of the aquaporin-2 gene, thus increasing the
total number of aquaporin-2 molecules in collecting duct cells.
Cyclic-AMP activates protein kinase A (PKA) by binding to its regulatory subunits and allowing them to detach from the catalytic
subunits. Detachment exposes the catalytic site in the enzyme, allowing it to add phosphate groups to proteins (including the
aquaporin-2 protein), which alters their functions.
Increasing permeability of the inner medullary portion of the collecting duct to urea by regulating the cell surface
expression of urea transporters,[4] which facilitates its reabsorption into the medullary interstitium as it travels down
the concentration gradient created by removing water from the connecting tubule, cortical collecting duct, and outer
medullary collecting duct.
Atrial natriuretic peptide (ANP), is a powerful vasodilator, and a protein (polypeptide) hormone secreted by heart muscle
cells.[1][2][3] It is involved in the homeostatic control of body water, sodium, potassium and fat (adipose tissue). It is released by
muscle cells in the upper chambers (atria) of the heart (atrial myocytes) in response to high blood pressure. ANP acts to reduce
the water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure.
Dilates the afferent glomerular arteriole, constricts the efferent glomerular arteriole, and relaxes the mesangial cells.
This increases pressure in the glomerular capillaries, thus increasing the glomerular filtration rate (GFR), resulting in
greater excretion of sodium and water.
Increases blood flow through the vasa recta which will wash the solutes (NaCl and urea) out of the medullary
interstitium. The lower osmolarity of the medullary interstitum leads to less reabsorption of tubular fluid and increased
excretion.
Decreases sodium reabsorption in the distal convoluted tubule (interaction with NCC)[9] and cortical collecting duct of
the nephron via guanosine 3',5'-cyclic monophosphate (cGMP) dependent phosphorylation of ENaC
Inhibits renin secretion, thereby inhibiting the renin-angiotensin-aldosterone system.
Reduces aldosterone secretion by the adrenal cortex.
5. In Question 4, you stated what normally inhibits the release of the hormone controlling water excretion. From your own
experience, identify two situations where this inhibition is over-ridden. For each situation, explain the physiological
advantage of over-riding this inhibition.
Antidiuretic hormone:
Released in response to slight increases in tonicity in ECFV
Sodium Concentration is the main determinant of tonicity
Effect: increase water reabsorption; low urine output
Atrial-natriuretic Peptide:
Contributes to volume regulation
Released by the cardiac atrial muscle fibers; stimulus is increased stretch of the atria due to excess blood volume
Acts on the kidney to cause small increase in glomerular filtration rate and decrease in Sodium reabsorption by the
collecting ducts
Increase excretion of salt and water
Effect: decrease Water reabsorption, Decrease Sodium Reabsorption; Increase urine output
SITUATION 1:

Vigorous Exercise


Excessive Sweating


Dehydration




Increased Plasma Sodium Concentration


Increased Osmolarity



Rise in Extracellular Fluid Tonicity




Release of ADH


Thirst



Increased Water Reabsorption


Water Intake



Water Retention



Low Urine Output

When a person goes into vigorous exercise, what happens is there is an excessive sweating. Since there is an excessive sweating,
there is a relative loss of fluid in our body which can put us into a state of dehydration. Since there is loss of water,
concentration of plasma sodium is increased which results to increase in osmolarity.
We have to take note that, the extracellular sodium concentration is the main determinant of plasma tonicity. Therefore, when
tonicity increases, it is generally because the extracellular sodium concentration has increased.
In states of increasing tonicity, the kidney decreases water excretion. The kidney decreases urine that is water excretion by
producing urine that is concentrated relative to the Plasma.
In states of decreasing tonicity, the kidney responds by increasing water excretion by producing urine that is dilute relative to
plasma.
Since there is a rise in the tonicity, ADH will be release noting that Hypertonicity is the main stimulus for thirst and Antidiuretic
(ADH) hormone release, important factors for the regulation of total body water. If the sodium concentration rises, thirst
(leading to water intake) and ADH release (leading to water retention by the kidney) are stimulated. The elevated sodium
concentration tells us that there is too little water relative to sodium.
Upon released of ADH, there will now be increased reabsorption of water in the collecting tubules, hence there is a decrease
water excretion which is explains a low urine output.
Also, since there is a rise in the sodium concentration, Thirst is stimulated leading to water intake as a coping mechanism for the
physiologic dehydration of the body.


SITUATION 2:

INTAKE OF LARGE AMOUNT OF HYPERTONIC
FLUIDS (SPRITE)



Increased Plasma Sodium Concentration


Increased Osmolarity



Rise in Extracellular Fluid Tonicity




Release of ADH


Thirst



Increased Water Reabsorption


Water Intake



Water Retention



Low Urine Output


Intake of large amounts of Hypertonic fluid (e.g.Sprite), would promote ADH release because, again hypertonicity is the main
stimulus for thirst and Antidiuretic (ADH) hormone release, important factors for the regulation of total body water. If the
sodium concentration rises, thirst (leading to water intake) and ADH release (leading to water retention by the kidney) are
stimulated. The elevated sodium concentration tells us that there is too little water relative to sodium.
As ADH is released there will be increased reabsorption of water in the collecting tubules, thereby producing a concentrated and
low output urine.
Inspite of the large fluid intake that can increase the fluid volume into our body, we take into consideration the tonicity of the
fluid, which is a main stimulus for the release of ADH.
If we put into account that ANP must act first before ADH just because of the excess fluid volume, there might be less absorption
of Sodium which would help into its excretion, however, we also should consider the other effect of ANP, which is decreased
water reabsorption, meaning there will be more water excreted in the urine. With the increased sodium concentration, there is
a relative low amount of water which when excreted will produce more dehydration into an individual. Hence, ADH must act
instead of ANP.
6. From your data collected so far, estimate the maximum volume of water that you could drink over 60 minutes while still
remaining in water balance?
Intake = Output
Maximal Flow Rate = 60mL / min x 80 mins = 4800mL
7. What would happen if your water intake over 60 minutes significantly exceeded the maximal volume you could excrete in
the period? Why could this be life threatening?
The kidney has the capacity to excrete excess water in the body. The kidney has the mechanism as follows: this increase in blood
volume decreases the sympathetic stimulation of the arterioles of the nephron particularly the afferent arteriole. This resul ts
then to the dilatation of the vessel and there is an initial rise in the glomerular filtration and increase urine output, excreting the
excess fluid. If a person intake of water exceeds the maximal volume that can be excreted there will be more water in the
plasma. This will dilute solute concentration and plasma concentration will be hyposmotic. This event can be life threatening
because hyposmotic plasma will cause fluid to enter the interstitial spaces and eventually to the cell. It will increase cell volume
than normal causing water intoxication.
8. Provide physiological explanation for the results for the isosmotic sodium chloride solution and hyposmotic solution.
Water moves from intra to extracellular compartment to establish equilibrium. One of the solutes that is capable of such
movement of water is the sodium. Why sodium? Because it remains for the most part in the extracellular space because it is
pumped out of cells by sodium-potasium ATPase. The addition of sodium to extracellular space causes water to move out of the
cell that leads to cell shrinkage. Because of this, the extracellular sodium concentration is considered to be the main
determinant for plasma tonicity. So, if tonicity increases, it is generally because the extracellular sodium concentration has
increased. This control of tonicity determines the normal state of cellular hydration.
Hyposmotic solution (Response to Water Drinking)
When a person drinks water, the ingested water is distributed throughout the body fluids. Since the amount of solute in
the body is unchanged, the added water will dilute the body fluids and cause a decrease in plasma osmolarity.
The decrease in plasma osmolarity inhibits osmoreceptors in the anterior hypothalamus.
Inhibition of the osmoreceptors has two effects. It decreases thirst and suppresses water drinking behavior. It also
inhibits secretion of ADH from the posterior pituitary gland.
When ADH secretion is inhibited, circulating levels of ADH are reduced, and less ADH is delivered to the kidneys. As a
result of the lower ADH levels, there is a decrease in water permeability of the principal cells of the late distal tubule
and collecting ducts.
The decrease in water permeability results in decreased water reabsorption by the late distal tubule and collecting
ducts .The water that is not reabsorbed by these segments is excreted, decreasing urine osmolarity and increasing urine
volume
Since less water is reabsorbed, less water is returned to the circulation. Coupled with the inhibition of thirst and the
suppression of water drinking, plasma osmolarity increases back toward the normal value.
ISOTONIC Solutions
A solution is isotonic when the concentration of dissolved particles is similar to that of plasma. Isotonic solutions have an
osmolality of 250 to 375 mOsm/L.7 With osmotic pressure constant both inside and outside the cells, the fluid in each
compartment remains within its compartment (no shift occurs) and cells neither shrink nor swell. Because isotonic solutions
have the same concentration of solutes as plasma, infused isotonic solution doesn't move into cells. Rather, it remains withi n
the extracellular fluid compartment and is distributed between the intravascular and interstitial spaces, thus increasing
intravascular volume.
Types of isotonic solutions include
0.9% sodium chloride (0.9% NaCl), lactated Ringer's solution,
5% dextrose in water (D5W), and Ringer's solution.
A solution of 0.9% sodium chloride is simply salt water, and contains only water, sodium (154 mEq/L), and chloride (154 mEq/L).
It's often called "normal saline solution" because the percentage of sodium chloride dissolved in the solution is similar to the
usual concentration of sodium and chloride in the intravascular space.
9. Provide physiological explanation for the results for the hyperosmotic glucose solution and hyposmotic solution.
Hypertonic sodium chloride solutions contain a higher concentration of sodium and chloride than that normally contained in
plasma.
To increase ECF tonicity a solute must be confined in an ecf compartment. That is, the solute must be unable to cross from the
extracellular compartment to intracellular compartment, thereby increasing the osmotic pressure and translocating water into
the extracellular compartment.
Hyperosmotic solution (deprived of drinking water)
The circled numbers in the figure correlate with the following steps:
Water is continuously lost from the body in sweat and in water vapor from the mouth and nose (called insensible water
loss). If this water is not replaced by drinking water, then plasma osmolarity increases.
The increase in osmolarity stimulates osmoreceptors in the anterior hypothalamus, which are exquisitely sensitive and
are stimulated by increases in osmolarity of less than 1 mOsm/L.
Stimulation of the hypothalamic osmoreceptors has two effects. It stimulates thirst, which drives drinking behavior. It
also stimulates secretion of ADH from the posterior pituitary gland.
The posterior pituitary gland secretes ADH. ADH circulates in the blood to the kidneys, where it produces an increase in
water permeability of the principal cells of the late distal tubule and collecting duct.
The increase in water permeability results in increased water reabsorption in the late distal tubule and collecting ducts.
As more water is reabsorbed by these segments, urine osmolarity increases and urine volume decreases.
Increased water reabsorption means that more water is returned to the body fluids. Coupled with increased thirst and drinking
behavior, plasma osmolarity is decreased, back toward the normal value. This system is an elegant example of negative
feedback, in which the original disturbance (increased plasma osmolarity) causes a set of feedback responses (secretion of ADH
and increased water reabsorption) that restore plasma osmolarity to its normal values.
10. Reflect on your predictions for the trends of each protocol in the spaces below. Note that your original answers are shown
but not be changed. Explain the relationship, shown in the graph between the urine flow rate & specific gravity.











The hydrated treatment group had a higher flow rate and lower specific gravity in comparison to the dehydrated treatment
group.

Urine Flow Rate
Sample No.
Protocol 1
Flow (mL/min)
Protocol 2
Flow (mL/min)
Protocol 3
Flow (mL/min)
Protocol 4
Flow (mL/min)
Sample 1 0.25 0.50 1.25 1.47
Sample 2 0.50 2.50 1.75 1.25
Sample 3 6.25 1.25 5.00 1.40
Sample 4 0.50 1.15 5.00 7.50
Sample 5 0.60 1.25 3.00 5.25
Sample 6 0.60 1.20 1.25 1.75
Sample 7 0.60 1.00 1.00 0.50
Urine Flow Rate
Sample No.
Protocol 1
Flow (mL/min)
Protocol 2
Flow (mL/min)
Protocol 3
Flow (mL/min)
Protocol 4
Flow (mL/min)
Sample 1 0.25 0.50 1.25 1.47
Sample 2 0.50 2.50 1.75 1.25
Sample 3 6.25 1.25 5.00 1.40
Sample 4 0.50 1.15 5.00 7.50
Sample 5 0.60 1.25 3.00 5.25
Sample 6 0.60 1.20 1.25 1.75
Sample 7 0.60 1.00 1.00 0.50
Urine Specific Gravity
Sample No.
Protocol 1
Specific Gravity
Protocol 2
Specific Gravity
Protocol 3
Specific Gravity
Protocol 4
Specific Gravity
Sample 1 1.029 1.021 1.020 1.030
Sample 2 0.000 1.026 1.017 1.023
Sample 3 0.000 0.000 1.005 0.000
Sample 4 0.000 0.000 1.003 1.002
Sample 5 0.000 0.000 1.005 1.002
Sample 6 0.000 0.000 0.000 0.000
Sample 7 0.000 0.000 0.000 1.008
Urine Specific Gravity
Sample No.
Protocol 1
Specific Gravity
Protocol 2
Specific Gravity
Protocol 3
Specific Gravity
Protocol 4
Specific Gravity
Sample 1 1.029 1.021 1.020 1.030
Sample 2 0.000 1.026 1.017 1.023
Sample 3 0.000 0.000 1.005 0.000
Sample 4 0.000 0.000 1.003 1.002
Sample 5 0.000 0.000 1.005 1.002
Sample 6 0.000 0.000 0.000 0.000
Sample 7 0.000 0.000 0.000 1.008