LAB 5 BIO

Lab 5 The Renal System Investigating Cell Osmolarity, Tonicity and Conducting Urinalysis 1. The data presented in the table represents the spectrophotometric analysis of red blood cells lysis. Hemolysis is the process through which the membranes of red blood cells or erythrocytes get ruptured and cause the release of Hemoglobin. A spectrophotometer determines how much a substance absorbs light at a particular wavelength, by measuring

the light intensity as it passes through the sample. The absorbance at 540 nm indicates absorbance of hemoglobin or the amount of hemoglobin while 640 nm indicates the intact red blood, which when dissolved in solution will form a cloudy mixture that will absorb light. Decrease in absorbance at 540 nm indicates hemolysis. At 640 nm, light dispersion of RBC suspension was measured. Decrease in absorbance at 640 nm indicates decrease in light dispersion. At 5% NaCl, the absorbance was 0.723, which is the second highest. The highest was 2% NaCl and was 1.3% at 540 nm. This is so because when the surrounding solution is concentrated, the water by osmosis leaves the cell and it leads to crenation and has a higher absorbance since there is less or no absorbance of hemoglobin. At 640 nm, 2% NaCl, the absorbance is highest 0.620, which shows most of the red blood cells are intact. the Moreover, in the solution with a concentration of 0.9% NaCl, the absorbance at 540 nm was low, 0.048, which can be due to some error since the normal osmolarity of the blood is around 275 to 295 milliosmole/kg and that is equivalent to 0.9%. At this osmolarity the red blood cells stay intact to give an opaque solution. At 0.2 % NaCl, the absorbance is lowest indicating that there is lysis of red blood cells at 0.028. During lysis, the solution has a pretty low osmolarity, causing the red blood cells to take up water and burst, releasing all of the intracellular contents. 0.1% Tween 20, 0.5 M glucose, 0.2% NaCl, 0.4% NaCl and 0.6% NaCl are hypo- osmotic compared to red blood cells. Hypo-osmotic solution causes an increase in red blood cell volume as water enters the red blood cell, thereafter the cell swells and eventually ruptures. 0.9% NaCl is iso-osmotic to red blood cells. 2% NaCl and 5 % NaCl are hyper-osmotic compared to red blood cells. Hyperosmotic solutions cause decrease in

protein to be passed down in urine but in table 2, the results are negative, which might be due to some error. The ketones levels, however, were large. Lastly, in the dehydrated individual urine sample, the level of ketones, nitrites were high and the pH was alkaline, 8.5. Loss of water leads to loss of minerals and electrolytes and increases blood pH. High levels of nitrite can indicate UTD infection. The hormone that is secreted in response to dehydration is called ADH or vasopressin. The role of vasopressin is to maximize water reabsorption and to maintain the osmolarity. It is produced by the hypothalamus and is transported to the posterior pituitary. ADH is released in response to increased osmolarity of plasma and decreased arterial blood volume. It stimulates the addition of aquaporins into the membrane of kidney tubules. These channels transport solute free water through tubules and eventually back into blood, leading to increase in osmolarity of urine. The volume of urine is also reduced and it results in the increase in Glomerular Filtrate rate. 3. The Glomerulus is a cluster of capillaries, known as tuft, located between two resistance vessels (Pollak et al., 2014). These capillaries are embedded in the Bowman’s capsule and are the only capillaries in the body not surrounded by interstitial tissue. The Bowman's capsule is a cup-like structure of the nephrons in the kidneys. It is connected to the tubular part of the nephrons. The glomerulus is inside this capsule. The blood is filtered in the Bowman's capsule as it passes through the glomerular capillaries. The factors affecting renal filtration are the hydrostatic pressure in the glomerulus , which pushes filtrate out of the capillaries and into the nephron. Increased blood volume and blood pressure will increase glomerular filtration rate. Vasodilation in the afferent arteriole and vasoconstriction in the efferent arteriole will increase blood flow

(and hydrostatic pressure) in the glomerulus and will increase the filtration rate. Osmotic pressure also plays a role and is the pressure exerted by proteins. Increased osmotic pressure decreases the glomerular filtration rate. If a crystal becomes lodged in the left ureter of a person, due to Urolithiasis, the passage of urine from the left kidney into the bladder will be obstructed. This will put stress on the nephrons because the glomerular filtrate will exit the kidneys at a slower rate due to the obstruction in the ureter. The glomerular filtration rate will slow down and if this condition is not treated, it will lead to left kidney failure. Appendix Table 1. Assessing Red Blood Cell Lysis using Spectrophotometry at two different wavelengths. The table below shows red blood cell lysis at a wavelength of 540 nm and 640 nm. The highest absorbance recorded at 540 nm was 1.300 at 5% NaCl and the lowest was recorded 0.028 at

progression toward diabetes. The individual with Glomerulonephritis had 300 (++) mg/dl of protein and was positive, unlike the control. The pH was within normal range, with a value of 6.0. Furthermore, the Leukocytes count was also recorded as Large, along with high levels of protein which were >2000 (++++) mg/dl. The urine sample of a dehydrated person had the highest recorded pH of 8.5 and was positive for nitrites. Lastly, the urine sample with nephrotic syndrome was negative for nitrites, protein, blood and bilirubin. However, Nephrotic syndrome causes protein to be passed down in urine usually. Test Control Glomerulonephritis Type 2 Diabetes Mellitus Dehydration Nephrotic Syndrome Leukocytes small Moderate (++) 125 Large (+++) small Small (+) 70 Nitrite Negative Positive Negative positive Negative Urobilinogen 8 Normal 0.2 8 8 8 Protein Trace 300 (++) >2000 (++++) 110 negative Negative pH 5.0 6.0 6.5 8.5 6.5 Blood Negative Moderate (++) negative negative Negative Specific gravity 1.010 1.015 1.025 1 1.025 Ketone Large (160) 16 negative Large (80) large Large (80) 8.0 Bilirubin Negative Moderate (++) negative large Negative Glucose 250(+) 15 250(+) 15 1000 (+++) 60 Negative 100 (5)

Figure. 1 Histological slide of a kidney glomerulus. The figure above shows a cross section of a kidney where tubular structures can be visible running through the cortex. The space between or in the middle of the cell can be the collecting duct.

● Nephrotic syndrome - Symptoms and causes. (2022, February 23). Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/nephrotic-syndrome/symptoms- causes/syc-20375608 ● Nephrotic Syndrome in Adults. (2022, August 29). National Institute of Diabetes and Digestive and Kidney Diseases. https://www.niddk.nih.gov/health-information/kidney- disease/nephrotic-syndrome-adults ● Cameron, M. A., Maalouf, N. M., Adams-Huet, B., Moe, O. W., & Sakhaee, K. (2006). Urine Composition in Type 2 Diabetes: Predisposition to Uric Acid Nephrolithiasis. Journal of the American Society of Nephrology, 17(5), 1422–1428. https://doi.org/10.1681/asn.2005121246 ● Cuzzo B, Padala SA, Lappin SL. Physiology, Vasopressin. [Updated 2022 Aug 22]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK526069/ ● Pollak, M. R., Quaggin, S. E., Hoenig, M. P., & Dworkin, L. D. (2014). The Glomerulus: The Sphere of Influence. Clinical Journal of the American Society of Nephrology, 9(8), 1461–1469. https://doi.org/10.2215/cjn.09400913 ● Bowman’s Capsule: Anatomy, Function & Conditions. (n.d.). Cleveland Clinic. https://my.clevelandclinic.org/health/body/22964-bowmans-capsule ● Libretexts. (2020, August 14). 24.3C: Regulation of Glomerular Filtration Rate. Medicine LibreTexts. https://med.libretexts.org/Bookshelves/Anatomy_and_Physiology/Book:_Anatomy_and_ Physiology_(Boundless)/24:__Urinary_System/24.3:_Physiology_of_the_Kidneys/24.3C :_Regulation_of_Glomerular_Filtration_Rate ●