Using a classic paper by Gottschalk and Mylle to teach the countercurrent model of urinary concentration
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《生理学进展》
Department of Biology, Santa Clara University, Santa Clara, California Abstract
Most undergraduates lack the scientific background to read and appreciate much of the primary literature in physiology. Even when the underlying concepts are elegantly simple, the inherent complexity of contemporary papers often makes the work inaccessible to them. However, with a little help, they can be guided to an understanding of the creative thought processes that underlie the research and to appreciate its significance. This is especially true of many classic papers in physiology that often rely on easily comprehensible techniques. Moreover, the American Physiological Society (APS) has invited prominent scientists to select important papers in their fields and to write essays that both put the work into historical context and explain why it is scientifically important. The APS Legacy Project makes these classic papers freely available online. One such paper by Gottschalk and Mylle presents data from a series of micropuncture studies that confirm all of the predictions of the countercurrent exchange model of concentrated urine production (2). The included handout of questions for discovery learning and teaching points suggest ways to use the paper as an instructional resource.
Key words: renal; kidney; education
RENAL PHYSIOLOGY challenges even the best students. Many will memorize relative solute concentrations in different segments of the nephron and never actually understand the physiology of the kidney. Working through the paper by Gottschalk and Mylle (2) makes the most fundamental concepts of renal physiology comprehensible to students. In my class, they begin by learning the context in which the research was done by reading the American Physiological Society Legacy Project essay written by Dr. James A. Schafer (7). He explains that the mechanism by which mammalian kidneys produce a concentrated urine was unclear until Hargitay and Kuhn (3) first proposed the countercurrent exchange model in the 1940s and Kuhn and Kyffel (4) elaborated on it in the early 1950s. However, at this time, many of the most respected scientists did not believe in the countercurrent hypothesis. For example, when Gottschalk asked the legendary physiologist Homer Smith what he thought of the model, he replied that "the smart boys don't believe in it" (9).
Despite this prevailing attitude, Gottschalk and Mylle had the courage and determination to pursue the technically demanding experiments required to test the countercurrent hypothesis. Their measurements indicated that urine is isosmotic to systemic plasma in the proximal tubule and hyposmotic at the beginning of the distal tubule, regardless of the final osmolality of the urine. They also demonstrated that urine is more concentrated at the tip of the loop of Henle than in the proximal tubule. Most importantly, Gottschalk and Mylle showed that both the blood in the vasa recta and the fluid in the descending limbs of the loop of Henle are isosmotic to urine in adjacent collecting ducts at the same level of the papilla. These data validated the countercurrent hypothesis.
Even introductory textbooks now explain that when mammals need to conserve water, their kidneys withdraw it osmotically from the urine. This requires that interstitial fluid in the medulla of the kidney be hyperosmotic to plasma and that the walls of the collecting duct be permeable to water. Of course, antidiuretic hormone regulates the water permeability of the collecting duct. Gottschalk and Mylle showed that solute reabsorption from the ascending limb of the loop of Henle is the "single effect" that drives the countercurrent exchanger, establishing and maintaining an osmotic gradient in the medulla of the kidney.
Before giving students this classic paper, I briefly describe the kidney's ability to independently control the excretion of water and solutes. I also remind them that water moves by osmosis, not by active transport. With this introduction, students read several annotated sections of the paper, which illustrate some of the most fundamental processes of renal function. An on-line course management system presents the excerpts sequentially. Students answer questions and write a brief summary of each section before gaining access to the next. In each of the excerpts, I provide links to a medical dictionary as well as to explanations of the techniques, jargon, and complex concepts that might confuse them. For example, in this paper, one of the most crucial links leads to a brief description of the micropuncture technique that allows one to remove samples of fluid from distinct regions of living nephrons. In addition to my own explanation, I include a link to a paragraph from the classic 1924 paper by Wearn and Richards (10) that first describes this important method of obtaining fluid from nephrons to analyze chemically.
After reading each short section of the paper, students answer a series of questions that focus their attention on the main ideas. For example, in the METHODS, Gottschalk and Mylle indicated that they used golden hamsters (Mesocricetus auratus) and a kangaroo rat (Dipodomys spectabilis) to collect blood and urine from the papilla of the kidney. I ask students what motivated the authors to include these animals in their experimental design because most of the data were collected from rats. This makes the students think about the role of the vasa recta in the countercurrent multiplier. It also provides me with an opportunity to touch on some unusual behavioral adaptations of the kangaroo rat to life in an arid environment. The mention of coprophagia always lightens any classroom discussion!
In terms of the RESULTS, I pose several questions that address not just countercurrent exchange but also two of the main processes of renal function. For example, I bring up glomerular filtration by asking the students why Gottschalk and Mylle merely tested for the presence of protein to determine whether a sample of fluid had been collected from a nephron or a blood vessel. This forces them to recall that despite its relatively high concentration in plasma, protein is not normally filtered through the glomerulus into the tubule. The data also provide an opportunity to consider the role of reabsorption. The observation that an intravenous infusion of a hypertonic solution of mannitol causes a diuresis in hydropenic animals initially seems counterintuitive to many students. Because they are unlikely to know that mannitol is filtered but essentially not reabsorbed, students have to integrate what they know about how the kidney produces urine to deduce the mechanism of action of osmotic diuretics like mannitol. Obviously, this promotes critical thinking. These data also segue to a discussion of diabetes mellitus and its effects in the kidney.
All physiology textbooks explain that urine is concentrated in the collecting duct, but most students have never considered how this fact was discovered. I ask them to cite the evidence in the Gottschalk and Mylle paper showing that urine is concentrated after it passes through the distal convoluted tubule. The authors demonstrated that even in rats that are producing highly concentrated urine, fluid in the early distal tubule is always dilute and that it is never hyperosmotic to systemic plasma in the late distal tubule. This emphasizes the importance of the longitudinal osmotic gradient established by the countercurrent mechanism in withdrawing water from urine across the walls of the collecting duct. It can also initiate a discussion of the role of antidiuretic hormone and the effects of diabetes insipidus on renal function.
In addition to providing solutions to many of the questions that I ask students to answer as they progress through the excerpts from the paper, the DISCUSSION also adds new dimensions to some of the topics. For example, before it enters the distal tubule, fluid could be made hypotonic to systemic plasma either by the reabsorption of solute from the ascending limb of the loop of Henle or by the secretion of water into the tubule. Gottschalk and Mylle distinguished between these two possibilities by comparing the osmolality of fluid entering the distal tubule after an intravenous infusion of an aqueous solution of either sodium chloride or sugar (mannitol or glucose). The data showed that fluid entering the distal tubule is more dilute if the animal had been infused with a solution of sodium chloride than with one of the sugars. One would predict this result if sodium and chloride are reabsorbed to a greater extent than glucose or mannitol in the loop of Henle. In contrast, if water were secreted into the ascending limb of the loop of Henle, one would expect the urine to be equally dilute under both experimental conditions. The fact that it is not suggests that the reabsorption of solute from the tubule rather than the secretion of water into it is responsible for the low osmolality of urine entering the distal tubule. I ask the students to explain how to obtain a more definitive answer. This requires them to imagine the existence of a nonreabsorbable solute such as inulin. If its concentration were to remain constant as it moved up the ascending limb of the loop of Henle, then the dilution must be the result of solute reabsorption rather than the addition of water to the tubule fluid.
Writing helps students organize their thinking. In addition to writing answers to the questions, I include two short writing assignments with this paper. First, students write a few sentences that summarize the main points of each section of the paper before getting access to the next one; by the time they finish the paper, they have written a brief summary of the work. The last exercise is a short writing assignment on Calibrated Peer Review, a free on-line instructional tool funded by the National Science Foundation and Howard Hughes Medical Institute (8). After submitting a short essay on the interaction between the vasa recta and loop of Henle, students are then presented with three "calibration" essays of varying quality. Answering a series of questions forces them to critically evaluate the essays and teaches them how to assign a grade. Once students master the calibration exercise, they anonymously evaluate three essays written by their peers and then apply the same criteria to their own work. This sort of peer review of problem-based writing assignments has been shown to improve academic performance in physiology courses (6). I then give them the opportunity to revise their essays before submitting them to me for the final grade.
Teaching Points
The following teaching points highlight some of the important physiological principles that can be explored using this classic paper.
Data collected from animals that were excreting concentrated urine confirmed several predictions of the countercurrent model.
The osmolality of fluid increases as it moves down into the loop of Henle and then decreases as it moves up toward the distal tubule.
Urine is not concentrated until it reaches the collecting duct.
Fluid collected from the tip of the loop of Henle is more concentrated than fluid in the proximal tubule.
The osmolality of blood in the vasa recta and fluid in the tip of the loop of Henle is essentially equal to that of urine in the collecting duct at the same level of the papilla.
Although this paper is not about glomerular filtration, it is obviously key to understanding renal physiology. Gottschalk and Mylle use the fact that protein is not normally filtered through the glomerulus to distinguish between fluid collected from a nephron and that taken from a blood vessel.
Because osmotic diuretics are filtered by the glomerulus but essentially not reabsorbed, they act by retaining some water in the urine. Data in this paper show that an intravenous infusion of a hypertonic solution of mannitol causes a diuresis even in hydropenic animals. One could easily introduce the renal effects of diabetes mellitus here.
Urine is not concentrated until after it passes through the distal convoluted tubule. Even in rats that are producing concentrated urine, fluid in the early distal tubule is always dilute. Furthermore, it is never hyperosmotic to systemic plasma in the late distal tubule. Concentration of the urine requires the longitudinal osmotic gradient established by the countercurrent mechanism. Water is withdrawn from urine across the walls of the collecting duct in the presence of antidiuretic hormone. A discussion of diabetes insipidus would interest some students.
Tubular fluid is isosmotic to systemic plasma when it enters the loop of Henle but hyposmotic by the time it gets to the distal tubule. This could have been achieved either by the reabsorption of solute or by the secretion of water into the loop of Henle. Gottschalk and Mylle distinguished between these two possibilities by comparing the osmolality of fluid entering the distal tubule in rats that had either been infused with an aqueous solution of sodium chloride or with a sugar solution. The data showed that fluid entering the distal tubule is more dilute if the animal had been infused with a solution of sodium chloride than with mannitol or glucose. If water were secreted into the ascending limb of the loop of Henle, one would expect the urine to be equally dilute under both experimental conditions. The fact that it is not suggests that the reabsorption of solute rather than the secretion of water is responsible for the low osmolality of urine entering the distal tubule. At this point, one could introduce the properties of inulin and ask students to design a more definitive experiment to determine whether fluid is made more dilute in the loop of Henle by the removal of solutes or by the addition of water.
In conclusion, this classic paper by Gottschalk and Mylle provides an outstanding framework for learning renal physiology. At its core, their model for countercurrent exchange, summarized in Fig. 13, is nearly identical to those described in modern textbooks and journal articles (5). Although it focuses on the fundamental processes of establishing an osmotic gradient in the kidney, students can learn other aspects of renal physiology more deeply, too, by reading and thinking about this short paper. Answering the discovery questions (Table 1) will lead students not just through the idea of countercurrent exchange but also through two of the main processes of urine formation: glomerular filtration and reabsorption. These study questions and the paper could be given to students all at once. Alternatively, excerpts from the paper and their respective discovery questions could be given sequentially in a problem-based learning format (1). Regardless of how the content is delivered to them, working through this classic paper will help students appreciate that physiology is much more than a collection of facts in their textbook.
GRANTS
This work was supported by a Technology Innovation Fellowship awarded by Santa Clara University.
Received for publication October 12, 2005. Accepted for publication January 14, 2006.
REFERENCES
Allen DE and Duch BJ. Thinking Toward Solutions: Problem-Based Learning Activities for General Biology. Fort Worth, TX: Saunders College Publishing, 1998, p. 1–11.
Gottschalk CW and Mylle M. Micropuncture study of the mammalian urinary concentrating mechanism: evidence for the countercurrent hypothesis. Am J Physiol 196: 927–936, 1959. http://ajplegacy.physiology.org/cgi/reprint/196/4/927 [11 October 2005].
Hargitay B and Kuhn W. Das Multiplikationsprinzip als Grundlage der Harnkonzentrierung in der Niere. Z Elektrochem 55: 539–558, 1951.
Kuhn W and Ryffel K. Herstellung konzentrieter Losungen aus Verdunnten durch blo?e Membranwirkung. Ein Modellversuch zur Funktion der Niere. Z Phys Chem 276: 145–147, 1942.
Pallone TL, Turner MR, Edwards A, and Jamison RL. Countercurrent exchange in the renal medulla. Am J Physiol Regul Integr Comp Physiol 284: R1153–R1175, 2003.
Pelaez NJ. Problem based writing with peer review improves academic performance in physiology. Adv Physiol Educ 26: 174–184, 2002.
Schafer JA. Experimental validation of the countercurrent model of urinary concentration (Online). http://www.the-aps.org/publications/classics/schafer.pdf .
University of California. CPR. Calibrated Peer Review (Online). http://cpr.molsci.ucla.edu .
Valtin H and Carl W. Gottschalk's contributions to elucidating the urinary concentrating mechanism. J Am Soc Nephrol 10: 620–627, 1999.
Wearn JT and Richards AN. Observations on the composition of glomerular urine, with particular reference to the problem of reabsorption in the renal tubules. Am J Physiol 71: 209–227, 1924. http://ajplegacy.physiology.org/cgi/reprint/71/1/209 .(David L. Tauck)
Most undergraduates lack the scientific background to read and appreciate much of the primary literature in physiology. Even when the underlying concepts are elegantly simple, the inherent complexity of contemporary papers often makes the work inaccessible to them. However, with a little help, they can be guided to an understanding of the creative thought processes that underlie the research and to appreciate its significance. This is especially true of many classic papers in physiology that often rely on easily comprehensible techniques. Moreover, the American Physiological Society (APS) has invited prominent scientists to select important papers in their fields and to write essays that both put the work into historical context and explain why it is scientifically important. The APS Legacy Project makes these classic papers freely available online. One such paper by Gottschalk and Mylle presents data from a series of micropuncture studies that confirm all of the predictions of the countercurrent exchange model of concentrated urine production (2). The included handout of questions for discovery learning and teaching points suggest ways to use the paper as an instructional resource.
Key words: renal; kidney; education
RENAL PHYSIOLOGY challenges even the best students. Many will memorize relative solute concentrations in different segments of the nephron and never actually understand the physiology of the kidney. Working through the paper by Gottschalk and Mylle (2) makes the most fundamental concepts of renal physiology comprehensible to students. In my class, they begin by learning the context in which the research was done by reading the American Physiological Society Legacy Project essay written by Dr. James A. Schafer (7). He explains that the mechanism by which mammalian kidneys produce a concentrated urine was unclear until Hargitay and Kuhn (3) first proposed the countercurrent exchange model in the 1940s and Kuhn and Kyffel (4) elaborated on it in the early 1950s. However, at this time, many of the most respected scientists did not believe in the countercurrent hypothesis. For example, when Gottschalk asked the legendary physiologist Homer Smith what he thought of the model, he replied that "the smart boys don't believe in it" (9).
Despite this prevailing attitude, Gottschalk and Mylle had the courage and determination to pursue the technically demanding experiments required to test the countercurrent hypothesis. Their measurements indicated that urine is isosmotic to systemic plasma in the proximal tubule and hyposmotic at the beginning of the distal tubule, regardless of the final osmolality of the urine. They also demonstrated that urine is more concentrated at the tip of the loop of Henle than in the proximal tubule. Most importantly, Gottschalk and Mylle showed that both the blood in the vasa recta and the fluid in the descending limbs of the loop of Henle are isosmotic to urine in adjacent collecting ducts at the same level of the papilla. These data validated the countercurrent hypothesis.
Even introductory textbooks now explain that when mammals need to conserve water, their kidneys withdraw it osmotically from the urine. This requires that interstitial fluid in the medulla of the kidney be hyperosmotic to plasma and that the walls of the collecting duct be permeable to water. Of course, antidiuretic hormone regulates the water permeability of the collecting duct. Gottschalk and Mylle showed that solute reabsorption from the ascending limb of the loop of Henle is the "single effect" that drives the countercurrent exchanger, establishing and maintaining an osmotic gradient in the medulla of the kidney.
Before giving students this classic paper, I briefly describe the kidney's ability to independently control the excretion of water and solutes. I also remind them that water moves by osmosis, not by active transport. With this introduction, students read several annotated sections of the paper, which illustrate some of the most fundamental processes of renal function. An on-line course management system presents the excerpts sequentially. Students answer questions and write a brief summary of each section before gaining access to the next. In each of the excerpts, I provide links to a medical dictionary as well as to explanations of the techniques, jargon, and complex concepts that might confuse them. For example, in this paper, one of the most crucial links leads to a brief description of the micropuncture technique that allows one to remove samples of fluid from distinct regions of living nephrons. In addition to my own explanation, I include a link to a paragraph from the classic 1924 paper by Wearn and Richards (10) that first describes this important method of obtaining fluid from nephrons to analyze chemically.
After reading each short section of the paper, students answer a series of questions that focus their attention on the main ideas. For example, in the METHODS, Gottschalk and Mylle indicated that they used golden hamsters (Mesocricetus auratus) and a kangaroo rat (Dipodomys spectabilis) to collect blood and urine from the papilla of the kidney. I ask students what motivated the authors to include these animals in their experimental design because most of the data were collected from rats. This makes the students think about the role of the vasa recta in the countercurrent multiplier. It also provides me with an opportunity to touch on some unusual behavioral adaptations of the kangaroo rat to life in an arid environment. The mention of coprophagia always lightens any classroom discussion!
In terms of the RESULTS, I pose several questions that address not just countercurrent exchange but also two of the main processes of renal function. For example, I bring up glomerular filtration by asking the students why Gottschalk and Mylle merely tested for the presence of protein to determine whether a sample of fluid had been collected from a nephron or a blood vessel. This forces them to recall that despite its relatively high concentration in plasma, protein is not normally filtered through the glomerulus into the tubule. The data also provide an opportunity to consider the role of reabsorption. The observation that an intravenous infusion of a hypertonic solution of mannitol causes a diuresis in hydropenic animals initially seems counterintuitive to many students. Because they are unlikely to know that mannitol is filtered but essentially not reabsorbed, students have to integrate what they know about how the kidney produces urine to deduce the mechanism of action of osmotic diuretics like mannitol. Obviously, this promotes critical thinking. These data also segue to a discussion of diabetes mellitus and its effects in the kidney.
All physiology textbooks explain that urine is concentrated in the collecting duct, but most students have never considered how this fact was discovered. I ask them to cite the evidence in the Gottschalk and Mylle paper showing that urine is concentrated after it passes through the distal convoluted tubule. The authors demonstrated that even in rats that are producing highly concentrated urine, fluid in the early distal tubule is always dilute and that it is never hyperosmotic to systemic plasma in the late distal tubule. This emphasizes the importance of the longitudinal osmotic gradient established by the countercurrent mechanism in withdrawing water from urine across the walls of the collecting duct. It can also initiate a discussion of the role of antidiuretic hormone and the effects of diabetes insipidus on renal function.
In addition to providing solutions to many of the questions that I ask students to answer as they progress through the excerpts from the paper, the DISCUSSION also adds new dimensions to some of the topics. For example, before it enters the distal tubule, fluid could be made hypotonic to systemic plasma either by the reabsorption of solute from the ascending limb of the loop of Henle or by the secretion of water into the tubule. Gottschalk and Mylle distinguished between these two possibilities by comparing the osmolality of fluid entering the distal tubule after an intravenous infusion of an aqueous solution of either sodium chloride or sugar (mannitol or glucose). The data showed that fluid entering the distal tubule is more dilute if the animal had been infused with a solution of sodium chloride than with one of the sugars. One would predict this result if sodium and chloride are reabsorbed to a greater extent than glucose or mannitol in the loop of Henle. In contrast, if water were secreted into the ascending limb of the loop of Henle, one would expect the urine to be equally dilute under both experimental conditions. The fact that it is not suggests that the reabsorption of solute from the tubule rather than the secretion of water into it is responsible for the low osmolality of urine entering the distal tubule. I ask the students to explain how to obtain a more definitive answer. This requires them to imagine the existence of a nonreabsorbable solute such as inulin. If its concentration were to remain constant as it moved up the ascending limb of the loop of Henle, then the dilution must be the result of solute reabsorption rather than the addition of water to the tubule fluid.
Writing helps students organize their thinking. In addition to writing answers to the questions, I include two short writing assignments with this paper. First, students write a few sentences that summarize the main points of each section of the paper before getting access to the next one; by the time they finish the paper, they have written a brief summary of the work. The last exercise is a short writing assignment on Calibrated Peer Review, a free on-line instructional tool funded by the National Science Foundation and Howard Hughes Medical Institute (8). After submitting a short essay on the interaction between the vasa recta and loop of Henle, students are then presented with three "calibration" essays of varying quality. Answering a series of questions forces them to critically evaluate the essays and teaches them how to assign a grade. Once students master the calibration exercise, they anonymously evaluate three essays written by their peers and then apply the same criteria to their own work. This sort of peer review of problem-based writing assignments has been shown to improve academic performance in physiology courses (6). I then give them the opportunity to revise their essays before submitting them to me for the final grade.
Teaching Points
The following teaching points highlight some of the important physiological principles that can be explored using this classic paper.
Data collected from animals that were excreting concentrated urine confirmed several predictions of the countercurrent model.
The osmolality of fluid increases as it moves down into the loop of Henle and then decreases as it moves up toward the distal tubule.
Urine is not concentrated until it reaches the collecting duct.
Fluid collected from the tip of the loop of Henle is more concentrated than fluid in the proximal tubule.
The osmolality of blood in the vasa recta and fluid in the tip of the loop of Henle is essentially equal to that of urine in the collecting duct at the same level of the papilla.
Although this paper is not about glomerular filtration, it is obviously key to understanding renal physiology. Gottschalk and Mylle use the fact that protein is not normally filtered through the glomerulus to distinguish between fluid collected from a nephron and that taken from a blood vessel.
Because osmotic diuretics are filtered by the glomerulus but essentially not reabsorbed, they act by retaining some water in the urine. Data in this paper show that an intravenous infusion of a hypertonic solution of mannitol causes a diuresis even in hydropenic animals. One could easily introduce the renal effects of diabetes mellitus here.
Urine is not concentrated until after it passes through the distal convoluted tubule. Even in rats that are producing concentrated urine, fluid in the early distal tubule is always dilute. Furthermore, it is never hyperosmotic to systemic plasma in the late distal tubule. Concentration of the urine requires the longitudinal osmotic gradient established by the countercurrent mechanism. Water is withdrawn from urine across the walls of the collecting duct in the presence of antidiuretic hormone. A discussion of diabetes insipidus would interest some students.
Tubular fluid is isosmotic to systemic plasma when it enters the loop of Henle but hyposmotic by the time it gets to the distal tubule. This could have been achieved either by the reabsorption of solute or by the secretion of water into the loop of Henle. Gottschalk and Mylle distinguished between these two possibilities by comparing the osmolality of fluid entering the distal tubule in rats that had either been infused with an aqueous solution of sodium chloride or with a sugar solution. The data showed that fluid entering the distal tubule is more dilute if the animal had been infused with a solution of sodium chloride than with mannitol or glucose. If water were secreted into the ascending limb of the loop of Henle, one would expect the urine to be equally dilute under both experimental conditions. The fact that it is not suggests that the reabsorption of solute rather than the secretion of water is responsible for the low osmolality of urine entering the distal tubule. At this point, one could introduce the properties of inulin and ask students to design a more definitive experiment to determine whether fluid is made more dilute in the loop of Henle by the removal of solutes or by the addition of water.
In conclusion, this classic paper by Gottschalk and Mylle provides an outstanding framework for learning renal physiology. At its core, their model for countercurrent exchange, summarized in Fig. 13, is nearly identical to those described in modern textbooks and journal articles (5). Although it focuses on the fundamental processes of establishing an osmotic gradient in the kidney, students can learn other aspects of renal physiology more deeply, too, by reading and thinking about this short paper. Answering the discovery questions (Table 1) will lead students not just through the idea of countercurrent exchange but also through two of the main processes of urine formation: glomerular filtration and reabsorption. These study questions and the paper could be given to students all at once. Alternatively, excerpts from the paper and their respective discovery questions could be given sequentially in a problem-based learning format (1). Regardless of how the content is delivered to them, working through this classic paper will help students appreciate that physiology is much more than a collection of facts in their textbook.
GRANTS
This work was supported by a Technology Innovation Fellowship awarded by Santa Clara University.
Received for publication October 12, 2005. Accepted for publication January 14, 2006.
REFERENCES
Allen DE and Duch BJ. Thinking Toward Solutions: Problem-Based Learning Activities for General Biology. Fort Worth, TX: Saunders College Publishing, 1998, p. 1–11.
Gottschalk CW and Mylle M. Micropuncture study of the mammalian urinary concentrating mechanism: evidence for the countercurrent hypothesis. Am J Physiol 196: 927–936, 1959. http://ajplegacy.physiology.org/cgi/reprint/196/4/927 [11 October 2005].
Hargitay B and Kuhn W. Das Multiplikationsprinzip als Grundlage der Harnkonzentrierung in der Niere. Z Elektrochem 55: 539–558, 1951.
Kuhn W and Ryffel K. Herstellung konzentrieter Losungen aus Verdunnten durch blo?e Membranwirkung. Ein Modellversuch zur Funktion der Niere. Z Phys Chem 276: 145–147, 1942.
Pallone TL, Turner MR, Edwards A, and Jamison RL. Countercurrent exchange in the renal medulla. Am J Physiol Regul Integr Comp Physiol 284: R1153–R1175, 2003.
Pelaez NJ. Problem based writing with peer review improves academic performance in physiology. Adv Physiol Educ 26: 174–184, 2002.
Schafer JA. Experimental validation of the countercurrent model of urinary concentration (Online). http://www.the-aps.org/publications/classics/schafer.pdf .
University of California. CPR. Calibrated Peer Review (Online). http://cpr.molsci.ucla.edu .
Valtin H and Carl W. Gottschalk's contributions to elucidating the urinary concentrating mechanism. J Am Soc Nephrol 10: 620–627, 1999.
Wearn JT and Richards AN. Observations on the composition of glomerular urine, with particular reference to the problem of reabsorption in the renal tubules. Am J Physiol 71: 209–227, 1924. http://ajplegacy.physiology.org/cgi/reprint/71/1/209 .(David L. Tauck)