Abstract

The countercurrent multiplier of the loop of Henle in a mammalian kidney is a challenging concept for instructors to teach and for students to learn. We developed an interactive game to support student learning of this dynamic process with a team of faculty as part of the 2014 HHMI/NAS Summer Institute on Undergraduate STEM Education. Students move game pieces representing fluid osmolarity on a game board representing the loop of Henle. They follow rules and answer questions as they "Grow the Gradient", establishing a salt concentration gradient in the medulla. The game was used by multiple instructors teaching an introductory biology laboratory over the course of three semesters and can be extended for use in more advanced classes. In our small sample, no differences in learning gains were detected between students doing the activity and those receiving only a passive lecture on the topic. However, students preferred the activity to a passive lecture, finding it more fun and feeling as if it improved their understanding of the concepts.

Citation

Lesson Learning Goals

• Students will understand the structure and function of the loop of Henle.
• Students will understand the membrane transport processes in the loop of Henle.
• Students will understand the countercurrent multiplier of the loop of Henle.

Lesson Learning Objectives

• Students will be able to simulate the movement of water and sodium at each region of the loop of Henle.
• Students will be able to associate osmosis and active transport with movement of water/solutes at each region of the loop of Henle.
• Students will be able to model how the descending and ascending limbs of the loop of Henle maintain a concentration gradient within the medulla.
• Students will be able to predict the effects of altering normal water and salt movement out of the loop of Henle on the salt concentration of the medulla, urine concentration, and urine volume.

• Students will be able to predict the impact of the length of the loop of Henle on the magnitude of the concentration gradient within the medulla.
• Students will be able to predict the length of the loop of Henle in organisms from different habitats.

INTRODUCTION

The countercurrent multiplier of the loop of Henle is a challenging process to teach. It is a dynamic process involving the movement of water via osmosis and salt via active transport. Osmosis is a challenging topic for students with many students holding misconceptions about the process (1). In addition, movement of these molecules is not consistent along the loop of Henle (i.e., the descending limb is permeable to water, not salt; the ascending limb is permeable to salt, not water). Indeed,given its complex nature, Katz (2) advocates that the countercurrent multiplier of the loop of Henle be taught only to medical or graduate students. For those who choose to teach the countercurrent multiplier to undergraduates, published approaches include a description of a conceptual model to be used during passive lecture (3), guiding students through a classic paper from the primary literature (4), and having students take an "inside view" of the process (5). While each approach is useful in its own way, we were interested in developing an interactive approach to engage students with this dynamic physiological process.

The Grow the Gradient activity was developed by a team of faculty at the 2014 National Academies/HHMI Northeast Summer Institute on Undergraduate STEM Education. In the activity, students explore the function of the countercurrent multiplier with a focus on the membrane transport processes of the loop of Henle. Students follow a set of rules as they move game pieces (representing fluid osmolarity) around a game board (representing the loop of Henle). The rules focus on movement of filtrate through the loop of Henle and of water and salts out of the loop of Henle. Students interact with each other and the game pieces as they document development of a salt concentration gradient within the medulla. This activity provides a structured, interactive approach to learn about the countercurrent multiplier.

INTENDED AUDIENCE

This activity was designed for undergraduate introductory biology students. The activity was led by faculty and graduate students at a liberal arts institution for introductory biology lab sections of 22 students each. Over the course of three semesters, approximately 500 first- and second-semester freshmen with majors in biology, psychology, health sciences, engineering, chemistry, exercise science, math and others, completed the activity. The activity can also be used with more advanced anatomy and physiology students, particularly if used in conjunction with the extensions described later. Though we ran the activity in lab, it can also be used in a 50 to 60 minute lecture period.

REQUIRED LEARNING TIME

This activity requires 50 minutes. If a lecture is included, the activity is extended to 60 minutes. See Lesson Plan Timeline (Table 1) for a breakdown of time spent on each part.

PRE-REQUISITE STUDENT KNOWLEDGE

Students should be able to meet the following objectives before beginning the lesson:

• Identify the basic macroscopic (e.g., kidney, ureter, bladder) and microscopic (e.g., nephron, Bowman's capsule, proximal and distal convoluted tubules, loop of Henle) structures of the mammalian excretory system.
• Explain osmosis and active transport.

PRE-REQUISITE TEACHER KNOWLEDGE

The instructor should be able to meet the following objectives before teaching the lesson:

• Identify the structure and function of the macro- and microscopic parts of the mammalian excretory system.
• Describe the processes involved with the countercurrent multiplier (e.g., where and why osmosis occurs, why water does not move out of the ascending limb, where and why salt moves).
• Anticipate common student misconceptions (e.g., that urine is concentrated in the loop of Henle, that water and salt move out of all parts of the loop of Henle).

SCIENTIFIC TEACHING THEMES

ACTIVE LEARNING

Students engage with the processes of water and salt movement within the countercurrent multiplier to "Grow the Gradient," creating a salt concentration gradient within the medulla. They are provided with a game board (Supporting File S1) representing the loop of Henle, game pieces (Supporting File S2) representing the osmolarity of fluids in the nephron, a Rule Sheet (Supporting File S3) to guide their movement of game pieces, and a Score Card (Supporting File S4) to document the results of their work. Students work in groups of three to four with the instructor serving as facilitator. The activity can also be extended for upper level students to include concepts such as concentration of urine in the collecting duct, effects of diuretics and antidiuretics, hypertension, etc.

ASSESSMENT

Learning is measured via formative assessment as students record their results and answer questions. The instructor (and teaching assistants, if present) should circulate among groups asking students what is moving and by what process it is moving as students manipulate the osmolarity game pieces. The instructor facilitates student learning by encouraging students to reason through their responses and to explain processes to their peers. The instructor may also choose to give a summative quiz at the end of the activity. Suggested questions are provided in Supporting File S6.

INCLUSIVE TEACHING

Students work in small groups, developing teamwork skills with a diverse peer group. Students are exposed to multiple learning modalities as they manipulate pieces on a game board, record data, discuss with peers, and answer written questions.

LESSON PLAN

The lesson plan includes the materials required, preparation, and a description of the lesson. A detailed timeline including all materials is included (Table 1). Preparation the first time the activity is taught takes at least one hour. Preparation for subsequent iterations takes approximately 15 minutes.

MATERIALS REQUIRED

Grow the Gradient Game Board

Game boards (Supporting File S1) can be prepared ahead of time on large (approximately 25 x 30.5 inches) paper or Post-It self-stick table top pads. Alternatively the instructor or students could draw the game board on a white/chalkboard or the table top pads instead of making game boards ahead of time. We initially used the table top pads but subsequently made sturdier game boards from 25 x 30 inch paper that we laminated and attached to magnetic whiteboards with adhesive-backed magnets.

Game Pieces

Post-It sticky notes (3 x 3 inches) can be used as game pieces on any surface (paper, table top pads, white/chalkboard). If the surface is magnetic, game pieces can be printed on cardstock and adhesive-backed magnets applied. The list of game pieces is for three rounds of play. Ready-to-print pieces are available in Supporting File S2. The number of game pieces of each osmolarity will need to be increased if more rounds or longer Loops of Henle are used.

Rule Sheet

The Rule Sheet (Supporting File S3) guides students through each step, instructs students to stop and check their answers at designated points, and asks students to answer questions.

Score Card

The purpose of the Score Card (Supporting File S4) is for students to record the values on their game board at each step of the game. This is helpful when troubleshooting why a group's values are incorrect.

Other Materials

Dry Erase Markers (red, blue, and black)

INSTRUCTOR PREPARATION

The time to prepare depends on which type of game board/pieces are used. Using self-stick table top pads and sticky notes takes an hour to set up six games. Printing and laminating the game boards and cutting game pieces and attaching them to magnets can take 3-4 hours if done alone. However, the process is much faster if others (e.g., faculty, graduate students, teaching assistants) help.

LESSON DESCRIPTION

If students are not familiar with the mammalian excretory system, instructors may choose to give a short (approximately 10 minute) passive lecture to introduce students to basic macro- and microscopic anatomy of the mammalian excretory system. A suggested lecture outline is provided in Supporting File S7. Online activities could also be assigned to students before the activity to familiarize them with kidney anatomy and nephron function (e.g., those available through the online component of textbooks offered by publishers).

Each group of three to four students needs a game board, dry erase markers, Rule Sheets, Score Cards, and set of game pieces. Students initially populate the game board with yellow game pieces of 300 osmolarity at each of six game squares within the loop of Henle and pink game pieces of 300 osmolarity in three squares in the medulla. We chose a value of 300 to reflect the osmolarity of blood. Students are told that the kidneys in a healthy adult mammal are never in this state, however, the idea is for the students to use the countercurrent multiplier to "Grow the Gradient" in the medulla, so the game begins with no gradient.

Students are directed to use dry erase markers to label their game boards with the following: proximal convoluted tubule, distal convoluted tubule, to collecting duct, descending limb, ascending limb, loop of Henle. Students should be able to complete the labeling based on their prerequisite knowledge.

Students then draw arrows of different colors to indicate movement of water and salt out of the descending and ascending limbs of the loop of Henle, respectively. Blue arrows represent passive movement of water via osmosis and are drawn indicating water movement out of the descending limb. Red arrows represent movement of salt and are drawn indicating movement of salt out of the ascending limb. Black arrows are drawn within both limbs of the loop of Henle indicating direction of filtrate flow (Figure 1).

If students do not have the prerequisite knowledge to complete this step on their own, they may label the game boards as the instructor describes each process and where it occurs.

To avoid students arriving at the misconception that water and salt simply move across cell membranes, the instructor can query students regarding how these molecules move. Students can draw blue or red channels around the shafts of the respective arrows to indicate the protein channels through which the molecules move. They should note that the descending limb is permeable to water (not salt) and the ascending limb is permeable to salt (not water) due to the presence or absence of the respective channels.

Once the game boards are set up, the instructor facilitates group work, checks answers, and answers student questions. Students work in groups to complete each step listed on the Rule Sheet, stopping to answer questions on the Rule Sheet and fill in the Score Card. It is useful to require all groups to stop at designated points in the game to double check their answers with the instructor to correct any mistakes or misconceptions.

The first step is the Filtrate Pump. Students follow two rules: Rule 1: Pump the filtrate through the loop of Henle by moving each filtrate game piece one space through the loop of Henle in the direction of filtrate flow. They fill in the now empty first space with a yellow 300 game piece, as incoming filtrate always enters at 300 mOsm (the osmolarity of the blood). The 300 game piece that exited the Loop of Henle can be placed back in the pool of game pieces to be used again later. Students stop to answer a question about what causes the filtrate to advance through the loop of Henle.

Rule 2: Dilute the interstitial fluid. Students check their game board. If the game pieces in the descending limb of the loop of Henle do not equal the values on the adjacent game pieces in the interstitial fluid, students decrease the interstitial concentration to match the adjacent filtrate concentration in the descending limb. Students must answer a question about what moves (water or salt) and how it moves (e.g., osmosis, passive diffusion, active transport). In this case, water moves passively out of the descending limb into the interstitial fluid. While this would simultaneously increase the osmolarity of the filtrate, we explain that this is a dynamic system that we are modeling. Given that it is a model, we are breaking the process into discrete steps. The filtrate will ultimately become concentrated as they progress through each round of play. During the first round of play, all values are 300 so no game pieces change at this point.

Students stop, record their values on the Score Card and check with the instructor that their values are correct. One option to facilitate checking answers in a large course is for instructors to project a correct game board on an overhead screen when all groups are ready so all students may check their values simultaneously.

Students then move on to Step 2: Membrane Transport. Again, they follow two rules. Rule 1: Transport salt out of the ascending limb. Here students must do simple calculations. They are instructed that a) there is no change in the sum of the filtrate in the ascending limb and the adjacent interstitial concentration and b) the interstitial concentration becomes 200 units greater than the filtrate concentration (e.g, if the values are 300 and 300 to start, totaling 600, for the interstitial concentration to be 200 units greater than the filtrate concentration, the interstitial concentration becomes 400 and the filtrate 200, still totaling 600). Having the interstitial concentration become 200 units greater than the filtrate concentration was chosen to keep the math relatively simple and to reflect values used in some textbook figures explaining the loop of Henle.

Students are provided with a set of steps to help them with the math: a. average the starting adjacent interstitial fluid and ascending limb filtrate concentrations, b. the final interstitial concentration is the average + 100, c. the final filtrate concentration is the average - 100. They also have a designated space on the Score Card to do the calculations. We provide this much support for the calculations as we repeatedly found that students got stuck on the math, shifting their focus away from the processes we were trying to teach.

In addition to the calculations, students answer a question about what moves (water or salt) and how it moves (e.g., osmosis, passive diffusion, active transport). In this case, salt is transported out of the ascending limb, decreasing the osmolarity of the filtrate and increasing the osmolarity of the interstitial fluid.

Rule 2: Concentrate the filtrate in the descending limb. If adjacent values in the descending limb and interstitial fluid are not equal, students increase the filtrate concentration in the descending limb to match the interstitial concentration. Students answer a question about what moves (water or salt) and how it moves (e.g., osmosis, passive diffusion, active transport). In this case, water moves out of the descending limb via osmosis.

Students are directed to stop, each student records the values on the Score Card, and students check with the instructor that their values are correct. Students also calculate the difference in salt concentration in the interstitial fluid near the top and bottom of the loop of Henle and enter that value on the Score Card.

Students complete two more rounds following the same procedure. They discover that the salt concentration gradient increases with continuing rounds.

TEACHING DISCUSSION

INSTRUCTOR CONSIDERATIONS

It is critical that the instructor be very comfortable with the countercurrent multiplier. We found that while graduate teaching assistants who understood the basic concepts but who were not confident were able to guide students to the "right" answers, they were unable to ask students questions about why certain things were happening. Moving game pieces around a board is not useful unless it is tied to deep learning about which processes are occurring and why they are occurring. We found we needed to teach the graduate assistants the game, then have them teach us so that we could gauge their comfort level and give advice regarding the types of questions to ask to facilitate student learning.

PRACTICAL CONSIDERATIONS

Laminating the game boards and using game pieces printed on card stock was a worthwhile time and monetary investment as sticky notes lose their stickiness and the self-stick table top pads get damaged after just a few uses. Laminating the game boards also allows students to write on them with dry erase markers and makes them more durable.

It is useful if the game boards can be propped up, rather than lying flat on a table or benchtop, as it is easier for students to gather around and participate as a group. It also facilitates the instructor looking around the room to assess at what point students are and which groups might be struggling. We achieved this by attaching the game boards to magnetic white boards (we used adhesive-backed magnets, but large binder clips would also work) and using adhesive-backed magnets on the card stock game pieces.

STUDENT EVALUATIONS

We used identical pre- and post-tests (using questions 1-9 and 13 in Supporting File S6) to assess student learning gains and a survey to assess student attitudes during Fall 2015. Only responses from students who signed a waiver and completed both the pre- and post-tests (n=92) are reported here. The research was declared exempt by the Institutional Review Board.

Students enrolled in six lab sections of an introductory biology course completed the pre- and post-tests and attitudes survey. The six sections were taught by three instructors (each instructor taught two sections) allowing us to account for any "instructor effects" and compare student learning gains and attitudes based solely on their lab experience.

Pre-tests were administered two weeks prior to the lab. Students were not provided with results of the pre-test. The week of the lab, each instructor gave a passive lecture on the loop of Henle and led an interactive activity on hormonal regulation of the ovarian cycle in one of their sections. In their other section, they gave a passive lecture on hormonal regulation of the ovarian cycle and led the interactive Grow the Gradient activity. We selected the ovarian cycle as the counterpoint because we traditionally cover the excretory and reproductive systems in this particular lab. This design allowed us to spend equal amounts of time covering the lab material in both sections. However, the interactive activities take longer than passive lecture so students were exposed to the concepts in the interactive activities for a longer period than concepts presented in the passive lecture.

The post-test and attitudes survey were administered immediately following the lab. Only students who answered all questions on both the pre- and post-tests were included in the analysis. Approximately half of the students (n=48) received the passive loop of Henle lecture and an interactive activity on hormonal regulation of the ovarian cycle. The remainder (n=44) received a passive lecture on hormonal regulation of the ovarian cycle and completed the Grow the Gradient activity. Normalized learning gains (NLGs) (6) on loop of Henle-related pre- and post-test questions were calculated as the difference in post- and pre-test scores divided by the difference between the total possible score of 10 and the pre-test score. We chose to use NLGs since pre-test scores affect the possible learning gain (6). There was no effect of approach (Grow the Gradient activity vs passive lecture on loop of Henle) on normalized learning gains, however, there was an effect of instructor and an approach x instructor interaction (two- way ANOVA for ranked data (SPSS v23); Happroach=1.34, df=1, p>0.1; Hinstructor=8.87, df=2, p<0.01; Hinteraction = 8.39, df=2, p<0.01; Figure 2).

Students who completed the Grow the Gradient activity (n=44) were also asked to choose which pedagogical approach (the passive lecture on the ovarian cycle or active learning with the Grow the Gradient activity) they felt improved their understanding of the respective material more, was more fun, more challenging, more interesting, and which approach they preferred. Only students who completed all questions on the pre-/post-tests and all questions on the attitudes survey were included in the analysis (n=38). Binomial tests (SPSS v23) were used to determine if the proportion of students choosing either approach differed from the null hypothesis of 0.5. A Bonferroni adjusted alpha of 0.01 was used to control for multiple comparisons. More students preferred the Grow the Gradient activity (p<0.001) and thought the activity was both more fun (p<0.001) and did a better job increasing their understanding (p<0.001) compared to the passive ovarian cycle lecture. Students did not find the Grow the Gradient activity more challenging (p=0.871) or more interesting than the passive ovarian cycle lecture (p=0.626; Figure 3).

MODIFICATIONS/ADAPTATIONS

STUDENT RESPONSES DURING THE ACTIVITY

Instructors may choose to have students submit their responses to questions or their game board numbers online using a student response system or a Google Sheet. A Google Sheet could work well for larger classes so the instructor can receive real-time information from all groups without having to walk to each group individually.

ADDITIONAL FORMATIVE AND SUMMATIVE ASSESSMENT

We include several questions (Supporting File S6) that can be used as follow up questions. These could be delivered via a student response system (e.g., "clickers", Socrative, Learning Catalytics).

INCREASING THE NUMBER OF ROUNDS PLAYED

The activity can be extended by having students complete more than three rounds of play. We use three rounds because it is enough to establish a salt concentration gradient without taking too much time. Note that if more rounds are conducted, students need additional game pieces with different osmolarity values than provided in the activity. Alternatively, students could write in values on blank game pieces for values not provided to them.

EXTENDING THE NEPHRON

If one game board can be attached to a large whiteboard (larger than the width of the game board, e.g., a wall-mounted whiteboard at the front of the room), students can extend the nephron by drawing the distal convoluted tubule and the collecting duct. Students can move filtrate game pieces through the collecting duct and predict the movement of water as the collecting duct travels through the salt concentration gradient they just produced in the medulla.

If paper game boards are being used, a second sheet of paper can be positioned to the right of the game board and the distal convoluted tubule and collecting duct drawn there.

ALTERING THE LENGTH OF THE GAME BOARD

Game boards can also be made with longer loops allowing the addition of more spaces down the length of the descending and ascending limbs and within the medulla. Additional game pieces would be needed. Alternatively, students could be asked to predict what would happen to the salt concentration gradient if the loops were longer (without the need for extended game boards). Students can also be guided to discuss the adaptive advantages of longer loops.

FOR ADVANCED PHYSIOLOGY STUDENTS

For advanced physiology students, pores, hormones, and aquaporins can be added to the game. At each point that a question is asked about what moves/how it moves, advanced students can be asked for additional information about the transport proteins involved and the effects of hormones on the transport processes.

This lesson could also be used in conjunction with the primary literature approach described by Tauck (4) in which students read and discuss a classic paper regarding the studies that confirm the predictions of the countercurrent model.

FOR A DIFFERENT PERSPECTIVE

This lesson can be used in conjunction with the reporter from within technique (5). In brief, students take on the role of "reporters" with designated "vantage points." They then describe what is happening around them from this vantage point.

FOR A 3-HOUR LAB SESSION

This lesson can be used in conjunction with a kidney dissection and one of the approaches outlined by Tauck (4) or Modell (5) to create a 3-hour laboratory session focused on different aspects of the structure and function of the mammalian kidney.

SUPPORTING MATERIALS

• S1. Grow the Gradient: Game Board Template (25in x 30in) 
• S2. Grow the Gradient: Game Pieces
• S3. Grow the Gradient: Rule Sheet
• S4. Grow the Gradient: Score Card
• S5. Grow the Gradient: Completed Score Card
• S6. Grow the Gradient: Suggested Assessment Questions and Answers
• S7. Grow the Gradient- Slides for Lecture and Activity

ACKNOWLEDGMENTS

This activity was developed at the 2014 National Academies Summer Institutes on Undergraduate STEM Education by Peter Daniel, Ashley Martino, Vladimir Poltorastky, Jessica Santangelo, and Claire Ting with support from Oyenike Olabisi and Christov Roberson. We thank the Hofstra University Department of Biology for funding to upgrade from sticky notes to magnetic game pieces and game boards. We thank the faculty and graduate students who helped teach the activity and the undergraduates who participated.

References

1. Odom AL. 1995. Secondary & college biology students' misconceptions about diffusion & osmosis. The American Biology Teacher:409-415.
2. Katz SA. 1998. Some teaching tips on the mechanisms of urinary concentration and dilution: countercurrent multiplication be damned. Adv. Physiol. Educ. 275:S195-S205.
3. Kurbel S, Dodig K, Radi? R. 2002. The osmotic gradient in kidney medulla: A retold story. Advances in Physiology Education 26:278-281.
4. Tauck DL. 2006. Using a classic paper by Gottschalk and Mylle to teach the countercurrent model of urinary concentration. Advances in Physiology Education 30:63-66.
5. Modell HI. 2007. Helping students make sense of physiological mechanisms: the "view from the inside." Advances in Physiology Education 31:186-192.
6. Slater SJ, Slater TF, Bailey JM. 2011. Discipline-based science education research: A scientist's guide. New York, NY:W.H. Freeman.