Diuretics Report MON3F PDF

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PCOL2605: Report Cover SheetTHIS FORM MUST BE THE FRONT PAGE OF YOUR GROUP REPORT.Only 1 submission per group.You must label with your practical day and group number (e. EyeReport_Mon1_GroupA) in the filename of your document.Report Title: The investigation of diuretic drugs and their mechanism of a...

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PCOL2605: Report Cover Sheet THIS FORM MUST BE THE FRONT PAGE OF YOUR GROUP REPORT. Only 1 submission per group. You must label with your practical day and group number (e.g. EyeReport_Mon1_GroupA) in the filename of your document.

Report Title: The investigation of diuretic drugs and their mechanism of action regarding ionic and volume content of urine production and pH.

Due Date:

9/11/20

Submission Date (if after due date)

Group Name (e.g. MON 1A): Mon 3F

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Contribution

e.g. 200030567

e.g. Introduction

480224929

Introduction, Method, References

480424688

Results

480432731

Question 1

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Question 3

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Question 4

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Question 2

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1 PCOL2605 Pharmacology for Pharmacy, Semester 2 2019

The Investigation of Diuretic Drugs and their Mechanism of Action regarding Ionic and Volume Content of Urine Production and pH. Introduction: The human kidney is a vital organ that facilitates the body’s ability to maintain constant homeostasis. The kidney consists of various unique cell types, which enable not only excretion of waste from the body, but also maintenance of overall fluid balance, regulation and filtration of minerals from the blood, creation of hormones that aid to produce red blood cells and promote bone health and regulation of blood pressure (Stephens and Jewell, 2018). Diuretic medications act on the renal tubular system in the nephron by inhibiting the reabsorption of sodium (Na+) ions and increasing urine output which produces greater excretion of both sodium ions and water (Klabunde, 2020). This mechanism of action is integral to the treatment of hypertension, by lowering the amount of fluid in the arteries and veins (Limberg, 2019) and to treat other conditions including renal failure and oedema. This experiment focuses on the efficacy and mechanism of action of three different diuretic classes of drugs including loop diuretics, potassium sparing diuretics and thiazides. Loop diuretics’ function through preventing the reabsorption of Na+ mainly in the loop of Henle compared with thiazide diuretics which inhibit the reabsorption of only Na+ at the distal tubules (Casu and Merella, 2015). By blocking the Na+ channels, potassium sparing diuretics cause retention of K+ in the distal tubule whilst simultaneously excreting both Na+ and water (Chu, 2017). This experiment aimed to investigate how these diuretics affect renal function by administering frusemide 40mg, hydrochlorothiazide (HCT) 50mg and the drug combination of HCT 50mg and amiloride 5mg. This was evaluated by measuring the changes in the urine volume, urinary pH, and in ionic content in relation to the control variable, the lactose placebo. It is hypothesised that frusemide 40mg and the combination of HCT 50mg and amiloride 5mg will produce a significant diuresis. HCT 50mg and frusemide 40mg are also hypothesized to decrease urinary pH whilst amiloride 5mg will produce an increase in urinary pH.

Method: Refer to the experimental method as detailed in Diuretics Practical class notes (Discipline of Pharmacology, 2019)

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Results: The sample size (n) of both the HCT + amiloride and frusemide treatment groups was n=7 compared with n=6 in both the HCT and placebo treatment groups. These sample sizes are consistent throughout the entire experiment. The error bars represent the variability of each dataset and are represented by ± standard error mean (SEM).

Figure 1: Results illustrate after 60 minutes following the administration of HCT + amiloride, HCT and frusemide in subjects, there was a notable decrease in urine pH. In contrast, there was an observed increase in urine pH in subjects administered with the placebo drug. In the subsequent 60 minutes following, the urine pH of subjects treated with HCT + amiloride increased whilst all subjects administered with the other drugs demonstrated an observed decrease in urine pH. Subjects administered with frusemide observed the greatest urine pH difference when compared to patients treated with the placebo. This difference was pH 0.71 ± 0.16 at t=120. In comparison, HCT + amiloride administered subjects noticed the least urine pH difference when compared to placebo subjects. This difference was pH 0.03 ± 0.06 at t=120. The urine pH of HCT administered subjects was lower than the placebo by pH 0.2 ± 0.02 at t=120.

Figure 2: Results demonstrate the total mean volume of urine excreted was greater in all drug treatment groups when compared to the placebo after 120 minutes. Subjects administered with frusemide observed the greatest difference in total mean volume excreted when compared to patients treated with the placebo. This difference was 993.88 ± 50.71 ml at t=120. Whilst subjects who were administered with HCT and HCT + amiloride both produced a greater volume of urine excreted than the placebo, HCT administered patients excreted an additional 33.12 ± 15.39 ml of urine compared with subjects treated with the combination of HCT + amiloride.

Figure 3: Results reveal the total mean sodium (Na+) excreted in urine was greater in all drug treatment groups when compared to the placebo. This notion was also exemplified on a smaller scale when comparing the total mean potassium (K+) excreted in comparison to the placebo. Subjects administered with frusemide observed the greatest difference in total mean Na+ excreted in urine when compared with patients treated with the placebo. This difference was 63.07 ± 11.20 mmol at t=120. Subjects administered with the combination of HCT + amiloride and HCT alone both observed a greater total Na+ excreted in urine compared with 3 PCOL2605 Pharmacology for Pharmacy, Semester 2 2019

the placebo by 35.70 ± 2.97 mmol and 39.22 ± 8.68 mmol respectively. However, when comparing the total amount of Na+ excreted in patients’ administered with HCT + amiloride and HCT, HCT alone demonstrated to excrete an additional 3.52 ± 5.71 mmol of urine than the HCT + amiloride combination. In respect to K+ excretion, subjects administered with frusemide observed the greatest difference in total mean K+ excreted in urine when compared to patients treated with the placebo. This difference was 17.71 ± 5.23 mmol. Subjects administered with the combination of HCT + amiloride and HCT alone both observed a greater total K+ excreted in urine compared with the placebo by 6.54 ± 0.13 mmol and 6.24 ± 0.52 mmol respectively. However, there was minimal variance in the total amount of K+ secreted when comparing subjects administered with HCT + amiloride and HCT. HCT + amiloride demonstrated to excrete an additional 0.30 ± 0.39 mmol of urine compared with HCT alone.

Figure 1. Mean urine pH (± SEM) measured at times 0, 60 and 120 minutes following the administration of HCT 50mg + amiloride 5mg, HCT 50mg, frusemide 40mg and the placebo.

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Figure 2. Mean total urine volume excreted (± SEM), in ml, measured after 120 minutes following the administration of HCT 50mg + amiloride 5mg, HCT 50mg, frusemide 40mg and the placebo.

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Figure 3. Mean total Na+ and K+ excreted from urine (± SEM), in mmol, measured after 120 minutes following the administration of HCT 50mg + amiloride 5mg, HCT 50mg, frusemide 40mg and the placebo. Discussion

1. Describe the mechanism of action of frusemide, hydrochlorothiazide and amiloride. Explain how frusemide, hydrochlorothiazide and hydrochlorothiazide plus amiloride produced the changes you observed in your experiments. Frusemide is a loop diuretic which acts on the thick ascending limb of the loop of Henle inhibiting the sodium/potassium/chloride (Na+ /K+ /2Cl- ) cotransporter in the luminal membrane by binding to its chloride (Cl- ) binding site. It is the most potent diuretic, promoting approximately 15-25% excretion of filtered sodium (Na+ ) in the urine (Ritter et al., 2020). This is displayed in figure 3 as the total mean volume of Na+ and K+ excreted in urine significantly increased for the frusemide treatment group in comparison to the placebo. Additionally, the increase in ion secretion further leads to a greater urine acidity depicted in figure 1. There is a steep decline in pH due to the increase in Na+ excretion in the distal tubule creating a favourable environment for hydrogen (H+ ) and K+ ion secretion (P et al., 1984). Therefore due to its high potency favours and its powerful diuretic capability, Figure 2 displays the total mean volume of urine excreted for the frusemide treatment group as the highest.

HCT is a thiazide diuretic that acts on the early distal convoluted tubule, binding to the Cl- site of the sodium chloride (Na+ / Cl- ) co-transparent system. This inhibits its actions and promotes natriuresis with a decrease in Na+ and Cl- reabsorption into the body. (Ritter et al., 2020) Therefore, the excretion of ions in the urine is qualitatively similar to those of loop diuretics, yet smaller in magnitude which is conveyed in figure 2. Similarly, to the frusemide treatment group, HCT experiences an increase in urine excretion compared to the placebo, due to the vast amount of ions such as Na+ , Cl- and K+ being excreted in the urine. Additionally, this is further denoted in figure 3 as it demonstrates a large increase in Na+ and K+ concentration in the urine for the HCT target group. However, figure 1 displays the least amount of change in pH for the HCT treatment group as only 5-10% of Na+ is reabsorbed in the luminal membrane (Diuretics Lecture, 2020). Overall, these observations signify that HCT is a less powerful diuretic than frusemide in relation to urine production and reabsorption of ions (Ritter et al., 2020).

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Amiloride is a potassium sparing diuretic that acts on the Na+ / K+ exchange of the late distal convoluted tubules and collecting duct. This inhibits the reabsorption of Na+ by blocking the  luminal Na+ channels and indirectly decreases K+ excretion (figure 3). Therefore in figure 3,

amiloride and HCT had the lowest concentration of Na+ excreted in the urine, yet a higher concentration of K+ in comparison to HCT as amiloride decreases K+ absorption (Ritter et al., 2020). As a result, the increase of K+ in the body alkalizes the urine and the pH of the urine rises, shown in figure 1. Additionally, it is the least potent diuretic as only 1-5% of Na+ is reabsorbed in the late distal tube, hence being a weak diuretic (Diuretics Lecture, 2020). This is further supported by figure 2 as the amiloride and HCT treatment group had the lowest volume of urine excreted.

2.

Explain why frusemide produces a greater diuretic effect in comparison to

hydrocholorothiazide. Frusemide produces a greater diuretic effect than HCT due to the different area of the nephron on which it acts. Endogenously, around 20-25% of Na+ is reabsorbed in the loop of Henle and in contrast around 5-10% in the early distal convoluted tubule. Thus, frusemide results in a higher amount of natriuresis in comparison to HCT due to location of action leading to the inhibition of higher amounts of Na+ reabsorption (Diuretics Lecture, 2020), (Tamargo et al., 2014). In addition, due to frusemide inhibiting around double the amount of Na+ reabsorption earlier in the nephron, the Na+/Cl- transporter and Na+/K+ exchange in the early and late distal convoluted tubule is not able to compensate in reabsorption for the extra Na+ that is passed on from the loop of Henle, resulting in high amounts of Na+ retention (Diuretics Lecture, 2020). In general, an increased amount of Na+ retained in the lumen of the nephron will increase osmotic pressure in the tubules and less water will be passively reabsorbed at the collecting duct, leading to a larger volume of urine excreted. Since frusemide causes higher amounts of Na+ retention, the osmotic pressure will be higher than with HCT and result in more water retention and a greater diuretic effect (Tamargo et al., 2014), (Malha et al., 2016)

3. Explain why frusemide and hydrochlorothiazide may produce hypokalaemia. In contrast, explain how amiloride may produce hyperkalemia. Furosemide, a loop diuretics, and HCT, a thiazide diuretics, have the common adverse effect of hypokalemia which refers to the low concentration of K+ in the blood. As furosemide 7 PCOL2605 Pharmacology for Pharmacy, Semester 2 2019

inhibits the Na+/K+ /2Cl- cotransporter, it represses their reabsorption in the loop of Henle (Australian Medicines Handbook, 2019) which leads to an increase of K+ excretion in urine (Kahn, 2006). On the other hand, HCT hinders the reabsorption of Na+ and Cl- in the distal convoluted tubule which in turn increases the Na+ concentration in the collecting tubules (Australian Medicines Handbook, 2019) that triggers the aldosterone-sensitive sodium pump to increase Na+ reabsorption in exchange of K+ resulting in higher K+ excretion (Klabunde, 2020). Due to the excretion of K+ via urine, patients consuming furosemide and HCT may experience hypokalemia. However, K+ sparing diuretics including amiloride may produce hyperkalemia which indicates high K+ concentration in the blood. Amiloride inhibits the sodium channels in the distal tubule suppressing Na+ reabsorption (Australian Medicines Handbook, 2019) and this interference blocks aldosterone-sensitive Na+ reabsorption causing in less K+ to be transported for Na+ (Klabunde, 2020), thereupon, lowering the loss of K+ in the urine. Being a direct inhibitor of K+ secretion, amiloride lowers urinary K+ secretion (Kahn, 2006).

4. Identify limitations of this experiment. What would you do to improve the experimental design? There are a number of different limitations with this experiment resulting from both the experimental design and the methods utilised. Due to human error and the varying heights and weights of all the participants which could result in less accurate results, a more accurate device could be used to make the measurements of the water loads as well as the averages of these measurements to improve the overall accuracy of the data. Another limitation is the sample size and diversity of the subjects. As the experiment only involved a small group of subjects, it only covered a small demographic of the population and is not a good representation of the entire population in showing the different effects of the diuretics on the urine volume, pH and Na+ and K+ levels. Also, different age groups and different ethnicities have different experiences when taking diuretics and therefore further experiments are required with a larger sample size to demonstrate the effects of different classes of diuretics on urine volume, pH, and Na+ and K+ levels.

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References: Australian Medicines Handbook Pty Ltd (2019). Frusemide. Amiloride. Hydrochlorothiazide

Casu, G., and Merella, P. (2015). Diuretic Therapy in Heart Failure – Current Approaches. European Cardiology Review 10: 42. Chu, X. (2017). Potassium-Sparing Diuretic - an overview | ScienceDirect Topics.

The discipline of Pharmacology (2019). The effect of drugs on the human kidney: kuracloud practical notes. The University of Sydney. Kahn, M. (2006). Encyclopedia of heart diseases (Burlington: Elsevier Academic Press). Klabunde, R. (2020). CV Pharmacology | Diuretics. Limberg, J. (2019). What you need to know about water pills (diuretics). Malha, L., Mann, S.J. (2016) Loop Diuretics in the Treatment of Hypertension. Curr Hypertens Rep 18, 27. P, S., C, R., R, C., W, W., J, F., and Arruda, A. (1984). Effect of furosemide on urinary acidification in distal renal tubular acidosis. The Journal Of Laboratory And Clinical Medicine 104: 271-282. Ritter, J., Flower, R., Henderson, G., Loke, Y., MacEwan, D., and Rang, H. (2020). Rang and Dale's pharmacology (Edinburgh: Elsevier). Stephens, C., and Jewell, T. (2018). Kidney: Function and Anatomy, Diagram, Conditions, and Health Tips. Tamargo, J., Segura, J., and Ruilope, L. (2014). Diuretics in the treatment of hypertension. Part 2: loop diuretics and potassium-sparing agents. Journal Expert Opinion On Pharmacotherapy 15: 605-621. University of Sydney (2020) Diuretics Lecture

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