The central goal of the human body is to maintain homeostasis which is a state of stability within the body. The renal system is critical in regulating homeostasis as it performs a plethora of functions. It is responsible for maintaining water balance, blood osmolarity, appropriate plasma volume, excreting waste as urine and so on.
This fluid balance and homeostasis is maintained throughout the body via a myriad of pathways. The hormones ADH and aldosterone which makes up the Renin-Angiotensin-Aldosterone system (RAAS) respond to changes in plasma volume and mean arterial pressure to keep them within acceptable ranges. Diuresis, the increase of urine volume production, antidiuresis (the exact opposite) and production of varying concentrations of urine are defense mechanisms to counteract homeostasis disturbances.
Blood flows through the glomerulus, a vascular region of capillaries that sifts through blood plasma to remove plasma proteins and filters substances such as water, solutes and different substances into Bowman’s capsule. Filtrate that enters Bowman’s capsule undergoes one of three fates. It can be reabsorbed which is the process by which substances are moved from the kidney tubules into the circulation if they are needed by the body. Secretion is another process where substances are transferred from the peritubular capillary blood into the tubular lumen. It is a method of quickly removing essential substances from the plasma to add it to the amount of the substance that is already in the tubule due to filtration (Sherwood, 2008, p. 515). Finally, excretion is the elimination of constituents from the body as urine.
Clearance is the amount of volume from which a substance is removed per unit time. GFR is the amount of filtrate that is pushed into Bowman’s capsule from the glomerular capillaries and can be estimated by a substance that is not absorbed or secreted. If clearance of a substance is equal to the GFR, then the substance is only filtered. If it is greater than GFR, then the substance is secreted and if clearance is lower than GFR, then it is reabsorbed. For this reason, it is essential to avoid fluctuations in GFR could have adverse effects on the reabsorption, secretion and excretion of essential substances.
This lab investigated renal function by determining GFR and different characteristics of urine such as flow rate, specific gravity, pH, sodium and creatinine clearance as well as sodium, water and H+ ion excretion values. These values were determined for four different subjects: a 164 pound male control that did not drink, a 118 pound female hypotonic (distilled water), a 160 pound male isotonic (saline solution) and a 120 pound female alkalosis (sodium bicarbonate solution) subject. The control should produce a low volume of concentrated urine in an attempt to retain water. The control really should not see a significant change in any of the other aforementioned parameters. The other three subjects should see an increase in flow rate due to increased volume intake. The sodium clearance should spike for these three subjects too since they are hypervolemic and will rid the body of excess water and sodium. The other three subjects should experience a drop in specific gravity with the most dramatic drop being by the hypotonic subject. The urine pH should increase for the three non-controls especially for the alkalosis subject due to the intake of base and the dilution of H+ ion by the solutions. Creatinine clearance should stay the same for all subjects since GFR is tightly controlled via autoregulation.
Materials and Methods
For a more detailed protocol, refer to NPB 101L Systemic Physiology Lab Manual (Bautista et al., 2008, p. 65-74). Before the lab, the subjects were instructed to stay hydrated and not consume caffeine (24 hours prior to lab) and to not exercise heavily the day of. They were also told to completely empty their bladder one to two hours prior to lab and to note the exact time.
There were four subjects in this experiment:
1. Control ‘” Does not consume any fluids in lab. Since the subject is dehydrated, the control serves as the hypertonic scenario.
2. Hypotonic ‘” Consumes 14 ml of water per kilogram of body mass. Results in plasma volume increase and a decrease in plasma osmolarity.
3. Isotonic ‘” Drinks 14 ml isotonic saline solution relative per 1 kilogram of body weight. Increase in amount of volume without changing the plasma osmolarity.
4. Alkalosis ‘” Intakes a small (2 ml/kg body mass), but significant amount of sodium bicarbonate, a base.
Urine was collected at t=0, 30, 60, 90 and 120 minutes for all four subjects. They were supposed to consume their respective solutions immediately after emptying their bladder at t=0 minutes. The urine was used to determine volume, flow rate, specific gravity (measure of osmolarity) using a refractometer, pH (indicator of [H+]) via a pH meter, sodium concentration using Na+ electrodes and creatinine concentration using a spectrophotometer.
The flow rate for the control subject remained somewhat constant throughout the two hours after starting at .78 ml/min. It peaked at 0.80 ml/min at the 30 minute mark before dropping to .53 at t= 60 minutes, reaching a low of .46 ml/min at t= 90 min and jumping back up to .53 ml/min at 120 minutes. The hypotonic subject’s flow rate fluctuated by dropping down to .32 ml/min at t= 30 min before leaping up to 5.47 at t= 60 min; flow rate proceeded to drop to 4.5 by t=120 min. The isotonic subject’s flow rate changed most drastically skyrocketing from .71 ml/min initially to 6.67 ml/min at t=60 min and then plummeted to .83 ml/min by the end. The alkalosis subject’s flow rate quickly jumped up to 2.13 ml/min at t=30 min before eventually dropping to .33 ml/min.
The control’s specific gravity rose from 1.021 at t=0 min to 1.027 at t=60 minutes before leveling off at 1.028 by the end (at t=120 min). The hypotonic’s specific gravity was very steady starting at 1.004 before slightly rising to 1.005 30 minutes later and dropping to 1.002 for the remainder of the time. The other two subjects, however, displayed large oscillations in their data. The isotonic’s specific gravity sharply dropped from 1.018 to 1.002 at t=60 min before climbing back up to 1.018 at t=120 min. The alkalosis subject showed an almost parabolic distribution as it dropped from the initial 1.026 value to 1.005 at t=30 min and dipped very slightly to 1.003 at t=60 min before increasing to 1.022 at the end.
Urine pH stayed pretty constant initially for the control at 5.09 at t=0 min and 5.10 at t=30 min before continuing to rise the rest of the way to 5.66 at t=120 min. The hypotonic subject had the most neutral urine pH. It was also the most stable increasing by .11 to 7.04 at t=30 min before dipping to 6.7. The alkalosis subject’s urine pH bounced all over the place from 5.38 at the start to 6.47 at t=60 min before eventually dropping to finish at 5.64. The isotonic subject’s urine pH crept up to 6.62 at the 30 min mark before trending downwards to finish at 5.86.
The urine sodium concentration (Una+) started at 82.1 mEq/L for the first hour until it rose to 126.6 mEq/L at t=90 min before dropping to 62.4 mEq/L. The Una+ initially rose from 23.3 to 29.5 mEq/L in the hypotonic subject before falling to 11.5, 10.6 and then 9.1 mEq/L. The bicarbonate subject’s Una oscillated wildly starting at 67.5 mEq/L before hitting rock bottom at 5.2 mEq/L at the hour mark before leapfrogging to 33.2 mEq/L at t=90 minutes and concluding at 37.4 mEq/L. The isotonic subject’s urine sodium concentration fluctuated down and up at each time point. It started at 96.1 mEq/L before nearly halving to 53.3 mEq/L (t=30 min) and increased to 82.1 mEq/l (t=60 min). It then proceeded to plummet to 20.7 mEq/L before finishing at 126.6 mEq/L..
The control subject initially displayed consistent sodium clearance (Cna+) staying close to .45 ml/min at t=30 min compared to .43 ml/min initially before dropping to .30 ml/min. It jumped to .40 ml/min and then proceeded to .23 ml/min, almost half the previous value by the end. The hypotonic subject’s sodium clearance progressively dropped from .72 ml/min initially to .28 ml/min at the end. The isotonic subject actually saw a very sharp increase at t=60 min to 3.8 ml/min (after having dipped low at t=30 minutes), but tumbled far down to .52 ml/min at t=90 min. Alkalosis sodium clearance was very linear all the way throughout staying at .09 ml/min at t=30, 90 and 120 min and never exceeding the initial Cna+ of .14 ml/min.
Creatinine clearance (Ccr) was relatively constant an hour later for the control subject, only decreasing by 273.7 ml/min or about 18.4%. The hypotonic subject saw only a small increase (relatively) from 24,809 to 26,858 ml/min. The alkalosis subject saw a mass surge in Ccr as it almost quadrupled from 1,212 to 4,580 ml/min. The isotonic Ccr increased way more dramatically increasing by almost eight times from 3,461 ml/min to a staggering 26,619 ml/min.
Kidney anatomy is very complex, but it is these complexities that enable the kidney to be such a good regulator of the body’s internal environment. The functional unit of the kidney is the nephron and each kidney is composed of about one million of these tiny urine producing units that are bound together by connective tissue. Nephrons all contain a hollow tubular component. The first segment is the double layered Bowman’s capsule which wraps around the glomerulus to bring in filtrate from the glomerular capillaries. The vascular component of Bowman’s capsule is the glomerulus which is a clump of capillaries that filters proteins out of plasma. The filtrate then moves into the convoluted proximal tubule (which is almost entirely in the cortex) which is known for its high uncontrolled secretion and absorption of substances (Sherwood, 2008, p. 513-514). The Loop of Henle descends into the highly concentrated medulla and has a descending and ascending limb, which ascends back into the cortex, that are permeable to different substances. This loop of Henle is the highly important countercurrent multiplier that establishes the high osmolarity of the medullary interstitium which drives water reabsorption by the distal tubule and collecting duct, the final two structures of the nephron. The vasa recta wraps around the loop of Henle and is composed of the vascular peritubular capillaries (only in juxtamedullary nephrons). Peritubular capillaries provide blood to the renal tissue and transport reabsorbed material to the renal vein which is where blood leaves the kidneys. The vasa recta are comprised of efferent blood vessels that return solute to the blood (Sherwood, 2008, p. 515). After the loop of Henle, the distal tubule and collecting duct function to further reabsorb water and sodium while secreting potassium and hydrogen ions. The fluid that exists in the collecting duct is moved down from the cortex to the medulla and sent into the renal pelvis where it drains into the bladder and can be excreted via the urethra as urine.
The loop of Henle, which has a descending and ascending limb, is critical in maintaining homeostasis by acting as a countercurrent multiplier. The descending limb and the proximal tubule allow for the reabsorption of water since they are permeable to water. Water leaves the descending limb to enter the highly concentrated medulla. The medulla has a low water concentration which creates a favorable high to low gradient for water to travel down (Musso et al., 2010). The ascending limb on the other hand, is impermeable to water and permeable to solute. As a result, sodium and other ions passively move down this concentration gradient into the medulla where solute concentration is now lower. This allows for the reabsorption of solute. The proximal tubule, on the other hand, has another mode of action for the reabsorption of materials. Water moves out of the tubules and lumenal membrane via paracellular transport (between epithelial cells) and brings other solutes along with it via bulk flow. This process is referred to as solvent drag and doesn’t utilize Na+/K+ ATPase which is highly advantageous since there is no limitation or transport maximum to limit reabsorption.
The Na+/K+ ATPase or Na+/K+ pump acts as a catalyst that creates the gradient seen in the nephron by creating a more highly concentrated medullary region than the cortex. Sodium-Potassium ATPase actively pumps sodium out of the ascending Loop of Henle into the medulla creating an osmolarity of up to 1200 mOsm. It is an active gradient for reabsorption and set up gradients for co-transporters (Su±© et al, 2010, p. 5). Secondary active reabsorption of glucose and amino acids is driven by these pumps as well as the passive reabsorption of water, chloride and urea. These pumps power the countercurrent multipliers that enable humans to produce urine of varying concentrations especially as water travels down the collecting duct to produce more concentrated urine to conserve water.
The Renin-Angiotensin-Aldosterone complex helps maintain homeostasis by regulating fluid balance and blood pressure. The kidney’s juxtaglomerular macula densa cells (which are aligned along the distal tubule too) are activated to release renin in response to three factors: decreased arterial blood pressure, decreased extracellular fluid volume and decreased NaCl blood concentration. Renin cleaves the inactive peptide angiotensinogen, which is produced in the liver, to form angiotensin I. Angiotensin I is then converted to angiotensin II via ACE, angiotensin-converting enzyme which is primarily secreted by the lungs. Angiotensin II acts as a vasoconstrictor to constrict blood vessels and increase blood pressure. Angtiotensin II then stimulates the release of the aldosterone mineralcorticoid hormone from the zona glomerulosa of the adrenal gland. Aldosterone secretion leads to increased reabsorption of sodium and water into the blood by acting on the distal tubule and collecting ducts to increase the number of sodium channels (Sherwood, 2008, p. 527-528). Water is passively reabsorbed by following sodium down its concentration gradient. This increase in blood volume subsequently leads to an increase in blood pressure.
Arginine Vasopressin (AVP) or ADH is a peptide hormone that is released from the posterior pituitary gland of the hypothalamus where it is made. AVP responds to dehydration and low plasma volume to help the body retain water. It does this by concentrating the urine so it has a higher solute concentration and releasing water from the tubules so less water is excreted from the body. ADH allows for the reabsorption of water (which results in less excretion of water) by increasing water permeability in the distal tubule and collecting ducts, increasing water reabsorption and eventually plasma volume (Krag et al, 2010, p. 3). After ADH binds to receptors in the distal and collecting tubules, the cyclic AMP second-messenger system within the tubular cells is activated. This increases water permeability by promoting insertion of aquaporins in the tubules via the fusion of vesicles (that contain aquaporins) with the membrane (Bouley et al, 2010, p. 3). ADH also stimulates aldosterone and operates more rapidly than the multi-step RAAS.
In terms of sodium clearance, the control remained relatively constant and only the isotonic subject’s clearance increased. Since the non-control subjects ingested significant amounts of fluid, they were considered hypervolemic and were all expected to see an increase in clearance as well as flow rate. Sodium clearance dropped for the hypotonic patient which is the exact opposite of what should have occurred especially considering that the hypotonic subject excreted 3.8% more sodium than expected. Sodium clearance was expected to rise most drastically for the hypotonic patient since osmoreceptors and volume receptors were activated to decrease circulating levels of ADH and inhibit aldosterone secretion. Volume receptors are detectors of stretch that are activated in response to a change in fluid balance. The isotonic and bicarbonate subjects were also expected to see an increase, but not as high as the hypotonic subject although this was not the case as the isotonic subject saw the largest increase in clearance and excreted 298% more sodium than expected. This is because even though ADH levels also drop, only volume receptors are affected in these subjects, not osmoreceptors since the isotonic subject’s blood plasma has the same osmolarity as the solution. The control and alkalosis subjects experienced significantly lower sodium excretion than expected as seen in Table 1 which correlates with the decreased sodium clearance in those two subjects even though clearance was supposed to increase for alkalosis. Specific gravity, which is the measure of urine osmolarity, was expected to drop most for the hypotonic subject for the same aforementioned reasons since both volume and osmoreceptors worked to decrease ADH and inhibit the RAAS. However, none of the three subjects saw a decrease at all five time points even though all three saw a decrease at 120 minutes.
As alluded to above, flow rate also should increase for the three non-control subjects especially for the hypotonic subject since it had the highest sodium clearance due to osmoreceptor activity as well as volume receptor activity. The water actual that was excreted was only a little off from the water expected for the control and the hypotonic subjects, but was considerably higher for the isotonic (by about 4.5 times) and alkalosis subject (by almost 8 times) as shown in Table 1. The isotonic subject may have excreted the most water because he could not finish his isotonic saline solution within ten minutes (took thirty minutes) and had to drink the largest volume of solution as he was the only male and weighed the most of the three. The hypotonic subject should have lost water at a faster rate than the other two non-controls since the hypotonic subject is supposed to lose her medullary gradient for reabsorption since she is losing sodium as well.
The control retained water as shown in Table 1 and saw an overall decreased flow rate as hypothesized. This matches up with physiological expectations as this subject did not drink anything yet was still asked to urinate at the five time points. To compensate, the control went into a state of antidiuresis, decreasing urine volume. Physiologically, this subject experienced an increase in ADH and aldosterone secretion to assist in the reabsorption and conservation of water. The hypotonic subject experienced diuresis, a homeostatic mechanism to produce high volumes of urine to compensate for high fluid intake. The idea is to dilute tubular fluid by reabsorbing solutes if needed while increasing urine volume output.
In addition, urine pH did not follow the physiological trend. The alkalosis subject was expected to see the largest increase in urine pH and was the only one (besides the control surprisingly) that experienced a rise although it proceeded to drop. The alkalosis urine pH was expected to increase the most since the body cannot reabsorb all of the excess base; this resulted in a decrease in bicarbonate reabsorption so it was excreted out in large amounts in the urine. The hypotonic and isotonic patients should have seen increases in urine pH as well since the additional volume of fluid should dilute out the hydrogen ion concentration in the body. This should result in more dilute urine when compared to the control and an increase in dilute hydrogen ion should have produced less acidic urine resulting in a pH increase. However, only the control and alkalosis subjects saw a lower than expected amount of hydrogen ion (mol) excreted which is very odd considering how the alkalosis subject is essentially drinking base.
The vascular component of the nephron is instrumental in filtration, filtrate movement into the Bowman’s capsule and GFR. This process of filtration is determined by glomerular capillary blood pressure and Bowman’s capsule oncotic pressure (to a much lower extent), the two forces that favor filtration, and plasma-colloid osmotic pressure and Bowman’s capsule hydrostatic pressure, both of which oppose filtration. Capillary blood pressure is pressure blood puts on the glomerular capillaries. The capillary blood pressure is around 55 mm Hg which is so high due to the large diameter of the afferent arteriole that traps blood in the glomerular capillaries. The efferent arteriole’s diameter is much smaller than the afferent’s resulting in the pooling of blood driving the pressure up. The extremely high resistance created by the efferent arterioles prevents a decrease in pressure as blood flows through the capillaries unlike capillaries in the rest of the body. This results in the movement of the filtrate out of the glomerulus into the Bowman’s capsule so it can move through the Loop of Henle and so on (Sherwood, 2008, p.519). Bowman’s capsule oncotic pressure is basically negligible since there is no solute in Bowman’s capsule to provide any driving force.
Plama-colloid osmotic pressure results from the presence of plasma proteins in the glomerular capillaries that cannot be filtered into Bowman’s capsule due to their large size. This results in a water gradient since there is a higher concentration of water in Bowman’s capsule and water tries to move to the glomerular capillaries via osmosis due to their lower water concentration creating a force that opposes filtration. The plasma-colloid osmotic pressure is higher here than in other capillaries at about 30 mm Hg because they have glomerular capillaries with the most plasma proteins due to the high filtration of water out of glomerular blood. Bowman’s capsule hydrostatic pressure, about 15 mm Hg, pushes fluid back out of Bowman’s capsule because of pressure in the beginning of the tubule (Sherwood, 2008, p. 519). Adding up these four pressures, the sum of 55 mm Hg + 0 mm Hg ‘” 30 mm Hg ‘” 15 mm Hg nets a 10 mm Hg filtration pressure exemplifying how the body favors filtration.
Clearance is used to make sure the kidneys are properly functioning. Creatinine clearance is used because creatinine is a metabolic muscle waste product that is produced via the breakdown of creatine, a natural process within the body (Sherwood, 2008, p. 539). As a result, creatinine is used because although it is secreted, it is not absorbed and can be used a reasonably accurate measure of GFR despite overshooting it by about 10-20%. The lab results however show creatinine clearances for all four subjects that are ridiculously far outside the normal range of 125 ml/min (in the tens of thousands as seen in Figure 6). The data also shows that urine flow rates increased as creatine clearance increased at t=60 minutes for all subjects except the control which saw a corresponding decrease in Ccr after urine flow rate dropped. It is also important to note that water follows sodium and that sodium clearance is less than GFR. Sodium is always being reabsorbed but with varying rates. There is some sodium secretion, but there is always net reabsorption of sodium.
GFR is controlled via autoregulation by the myogenic and tubuloglomerular feedback mechanisms. Myogenic feedback occurs when smooth muscle contracts in response to stretching in vessels triggered by increased arterial pressure. The afferent arteriole automatically vasoconstricts when stretched to prevent excess blood flow into the glomerulus and relaxes when there is low pressure in the afferent arteriole to increase blood flow in. Tubuloglomerular feedback is activated by the macula densa cells. They are receptors that detect changes in sodium concentration and act as an indicator of flow rate (Sherwood, 2008, p. 521). The flow rate is adjusted by a change in resistance of the glomerulus which maintains a physiologically acceptable mean arterial pressure. These branches of autoregulation help keep GFR relatively constant and arterial blood pressure within the range of 80-180 mm Hg.
The data problems may have been due to a multitude of factors. Many of the errors in the data may be due to the subjects being unable to completely void their bladders at t=0 minutes which may have led to the expectation of a lower flow rate than actually seen from t=30 to t=120 minutes. From t=30 minutes and on the, the subjects may have indeed fully emptied their bladders resulting in an elevated water, sodium and H+ actual excreted in comparison to the lower expected values. Furthermore, the unusually high water actual excreted by the isotonic subject was probably because he was unable to drink the isotonic saline solution within the allotted ten minutes. It took him roughly thirty minutes to finish the solution which definitely could have altered parameters such as flow rate. It should be noted though that none of the subjects drank caffeine or any stimulants prior to the lab and did not exercise heavily the day of. The calculated creatinine clearance values are physiologically impossible and this may be due to miscalibration of the spectrophotometer or dilution series that were incorrectly done since the spectrophotometer yielded extremely high spec readings of up to 57.6 O.D.
In conclusion, the kidneys and renal system are vital to maintaining homeostasis and concentrating urine to match the physiological needs of the body. The balance between salt and water concentrations in blood, tight autoregulation of GFR and ADH and RAAS ensures the proper amount of substances are absorbed and excreted and that blood pressure and plasma volume are adequate for survival. In light of the decreasing water supply around the world, it is crucial to note that drinking seawater is a hopeless alternative since the ingested seawater is four times more concentrated than the medulla and the gradient that moves water out of the descending loop of Henle into the medulla would be reversed, completely screwing up our renal system.
Works Cited Page
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