Urine Concentration and Dilution; Regulation of Extracellular Fluid Osmolarity and Sodium Concentration. Chapter 28 Lecture-7 7/4/2015 Yanal A. Shafagoj MD. PhD

One of the most important tests is to look if the kidney can concentrate urine which is considered to be a tubular function - Urine specific gravity test ( it tests the ability of the kidneys to concentrate urine) High specific gravity means concentrated urine Low specific gravity means diluted urine This is the last function to be retained following acute renal failure

Minimal urine concentration Concentration and Dilution of the Urine Maximal urine concentration = 1200 - 1400 mOsm / L (specific gravity ~ 1.030) Minimal urine concentration = 50 - 70 mOsm / L (specific gravity ~ 1.003)

Relationship between urine osmolarity and specific gravity Influenced by glucose in urine protein in urine antibiotics radiocontrast media S.G versus osmolality: One mole of any solution contains the same number of particles (Avogadro’s number: 6.02 X 1023). The freezing point of any solution with a concentration of one Mole/kg water is –1.86 C. This solution is called osmolal solution. The change in temperature is called molal freezing point constant (Kf) this is equal one Osmole. To calculate the osmolality of a solution you use an osmometer. It calculate the freezing point of the solution and compare it to Kf. For instant the freezing point for plasma equals –0.53 C. Therefore the osmolality of plasma equals –0.53/-1.86 X 1000=285 mOsm. S.G depends on the mass of the particles present in a given weight of urine (mass/mass) and is a traditional measure of urine concentration (very simple test). 1.003 diluted urine (compared to plasma) 1.010 isotonic urine (the same as plasma). 1.040 concentrated urine (compared to plasma) The urine osmolality is determined by the number of particles in the solution. In contrast, the urine specific gravity, which is a measure of the weight of the solution compared to that of an equal volume of distilled water, is determined by both the number and size of particles in the solution. In most cases, the urine specific gravity varies in a relatively predictable way with the osmolality, with the specific gravity rising by .001 for every 35 to 40 mosmol/kg increase in osmolality. Thus, a urine osmolality of 280 mosmol/kg (which is isosmotic to plasma) is usually associated with a specific gravity of 1.008 or 1.009. To predict osmolality from S.G (e.g. 1.006) as follows: Take the last 2 digits (for example: 06) and multiply by 40 06 X 40 = 240 mOsm/L Normal urine osmolarity is  600 mOsm/lit (SG 1.015) (Fig. 28-1). Osmolality of the urine varies from as low as 30 mOsm/L to as high as 1400 (30-fold difference). (30 mOsm/L concentration means moreBut if all these are absent, we can predict osmolality from S.G (e.g. 1.006) as follows:   Osmolality of the urine varies from as low as 30 mOsm/L to 1400 (30-fold difference). (30 mOsm/L concentration means at least 20 liters of urine per day to remove 600 mOsm/day) This relationship, however, is altered when there are appreciable quantities of larger molecules in the urine, such as glucose, radiocontrast media, or some antibiotic s. In these settings, the specific gravity can reach 1.030 to 1.050 (falsely suggesting a very concentrated urine), despite a urine osmolality that may be only 300 mosmol/kg.

Making concentrated urine means conservation of body water. - Making diluted urine mean eliminating excess water. Observation: Only those species that have juxtamedullary nephrons (long loop of Henle) can make concentrated urine (desert rodents). While beaver with short loop of Henle make less concentrated urine

To make diluted urine is somehow easy to understand To make diluted urine is somehow easy to understand. The kidneys needs just to actively transport NaCl at the collecting duct and end up with diluted urine=hypo-osmolar urine. To make concentrated urine was an enigma for 100 years (Active transport of water is not known). The micropuncture technique came up with the answer. Using the micropuncture technique scientists measured the osmolarity of inner medullary interstitium and found to be hyperosmolar.

Introduction Before we start the lecture, we ask the following question: Why we must drink water? Normally, we must excrete 700 mOsm\day as a minimum amount (in a complete bed rest individual), or 1000 mOsm/D at normal daily activity. The kidney can make a concentrated urine up to 1400 mOsm/l Therefore, 0.5 l is the minimum daily obligatory volume of urine (MDOVU): (700/1400) or 714 ml (1000/1400) during normal daily activity MDOVU is: Infants < 1 ml/kg/h Children < 0.5 ml/kg/h Adults <0.3 ml/kg/h In general MDOVU= 300ml\m2 BSA/D

Water Balance Output Intake GIT 100 ml Metabolism 200 ml 1. Skin   GIT 100 ml Metabolism 200 ml 1. Skin 600 ml Drinking 1600 ml 2. Respiratory 300 ml Food 700 ml 3. Urine 1500 4. 2500

In order to form Concentrated Urine, two conditions must be met: 1. Hyperosmolar interstitium around the medullary collecting ducts 2. ADH to permit H2O permeability.

How to generate hyperosmolar interstitium? By the so called “single effect”. In thick ascending loop of Henle, Na+-K+ -2Cl – transporter without H2O reabsorption, making interstitium hyperosmolar with 700 mOsm/L - The rest is made by urea - Na+ that is reabsorbed must not be removed from the area, and therefore blood flow in Vasa Recta must be low (2% of renal blood flow). High blood flow does not allow this hyperosmolar media to build up.

ADH Both ADH and oxytocin are composed of 9 aa (small peptides) ADH: 5/6th from supraoptic neuclei and 1/6th from paraventricular nuclei. ADH V2 receptor (AQP V2) cAMP  intracellular vesicles aquaporins  in luminal side (takes 5-10 min). AQP V3 and V4 do not respond to ADH. ADH receptors are found in last one third of DT and further. Functions of ADH: 1.  H2O reabsorption from last one third of DT and further. 2. Enhances active NaCl reabsorption from last one third of DT and further. 3. Enhances urea reabsorption from IMCD. The last two factors make medulla hyperosmolar.

Control of Extracellular Osmolarity (NaCl Concentration) ADH Thirst ] ADH -Thirst Osmoreceptor System Mechanism: increased extracellular osmolarity (NaCl) stimulates ADH release, which increases H2O reabsorption, and stimulates thirst (intake of water)

Water diuresis in a human after ingestion of 1 liter of water.

Formation of a dilute urine Continue electrolyte reabsorption Decrease water reabsorption Mechanism: Decreased ADH release and reduced water permeability in distal and collecting tubules

Continue electrolyte reabsorption Increase water reabsorption Formation of a Concentrated Urine Continue electrolyte reabsorption Increase water reabsorption Mechanism : Increased ADH release which increases water permeability in distal and collecting tubules High osmolarity of renal medulla Countercurrent flow of tubular fluid

Formation of a Concentrated Urine when antidiuretic hormone (ADH) are high.

Obligatory Urine Volume Again: The minimum urine volume in which the excreted solute can be dissolved and excreted Example: If the max. urine osmolarity is 1400 mOsm/L, and 700 mOsm of solute must be excreted each day to maintain electrolyte balance, the obligatory urine volume is: 700 mOsm/d 1400 mOsm/L = 0.5 L/day

Obligatory Urine Volume In renal disease the obligatory urine volume may be increased due to impaired urine concentrating ability Example: If the max. urine osmolarity = 300 mOsm/L, If 700 mOsm of solute must be excreted each day to maintain electrolyte balance obligatory urine volume = ? 700 mOsm/d = 2.3L/day 300 mOsm/L

Active transport of Na+, Cl-, K+ and other ions from thick Factors That Contribute to Buildup of Solute in Renal Medulla - Countercurrent Multiplier Active transport of Na+, Cl-, K+ and other ions from thick ascending loop of Henle into medullary interstitium Active transport of ions from medullary collecting ducts into interstitium Passive diffusion of urea from medullary collecting ducts Diffusion of only small amounts of water into medullary interstitium

Summary of Tubule Characteristics Permeability Tubule Active NaCl Segment Transport H2O NaCl Urea Proximal ++ +++ + + Thin Desc. 0 +++ + + Thin Ascen. 0 0 + + Thick Ascen. +++ 0 0 0 Distal + +ADH 0 0 Cortical Coll. + +ADH 0 0 Inner Medullary + +ADH 0 +++ Coll.

Countercurrent multiplier system in the loop of Henle. Figure 28-4 Page 394

Net Effects of Countercurrent Multiplier 1. More solute than water is added to the renal medulla. i.e solutes are “trapped” in the renal medulla 2. Fluid in the ascending loop is diluted 3. Most of the water reabsorption occurs in the cortex (i.e. in the proximal tubule and in the distal convoluted tubule) rather than in the medulla 4. Horizontal gradient of solute concentration established by the active pumping of NaCl is “multiplied” by countercurrent flow of fluid.

Recirculation of urea absorbed from medullary collecting duct into interstitial fluid.

Urea recycling Urea toxic at high levels, but can be useful in small amounts. Urea recycling causes buildup of high [urea] in inner medulla. This helps create the osmotic gradient at loop of Henle so H2O can be reabsorbed.

Urea Recirculation Urea is passively reabsorbed in proximal tubule (~ 50% of filtered load is reabsorbed) In the presence of ADH, water is reabsorbed in distal and collecting tubules, concentrating urea in these parts of the nephron The inner medullary collecting tubule is highly permeable to urea, which diffuses into the medullary interstitium ADH increases urea permeability of medullary collecting tubule by activating urea transporters (UT-1)

The Vasa Recta Preserve Hyperosmolarity of Renal Medulla The vasa recta serve as countercurrent exchangers Vasa recta blood flow is low (only 1-2 % of total renal blood flow) Figure 28-7

Changes in osmolarity of the tubular fluid Figure 28-8 Page 352

Summary of water reabsorption and osmolarity in different parts of the tubule Proximal Tubule: 65 % reabsorption, isosmotic Desc. loop: 15 % reasorption, osmolarity increases Asc. loop: 0 % reabsorption, osmolarity decreases Early distal: 0 % reabsorption, osmolarity decreases Late distal and coll. tubules: ADH dependent water reabsorption and tubular osmolarity Medullary coll. ducts: ADH dependent water reabsorption and tubular osmolarity

If: Uosm > Posm, CH2O = - “Free” Water Clearance (CH2O) (rate of solute-free water excretion) CH2O = V - Uosm x V Posm where: Uosm = urine osmolarity V = urine flow rate P = plasma osmolarity Cosm is osmolar clearance Quantifying Renal Urine Concentration and Dilution: FREE WATER AND OSMOLAR CLEARANCE:   Osmolar Clearance =Cosm FREE WATER CLEARANCE CH2O = V - Cosm Normally is equal to (1 – 2.1) = -1.1 ml/min If the number is positive, then we excrete water more than solute (solute-free water excretion). If: Uosm < Posm, CH2O = + If: Uosm > Posm, CH2O = -

Question Given the following data, calculate “ free water” clearance : urine flow rate = 6.0 ml/min urine osmolarity = 150 mOsm /L plasma osmolarity = 300 mOsm / L Is free water clearance in this example positive or negative ?

Answer Uosm x V Posm CH2O = V - = 6.0 - ( 150 x 6 ) 300 = 6.0 - 3.0 = + 3.0 ml / min (positive)

Disorders of Urine Concentrating Ability Failure to produce ADH : “Central” diabetes insipidus Failure to respond to ADH: “nephrogenic” diabetes insipidus - impaired loop NaCl reabs. (loop diuretics) - drug induced renal damage: lithium, analgesics - malnutrition (decreased urea concentration) - kidney disease: pyelonephritis, hydronephrosis, chronic renal failure

Maximum urine osmolarity Development of Isosthenuria With Nephron Loss in Chronic Renal Failure (inability to concentrate or dilute the urine) 300 600 1200 Maximum urine osmolarity Osmolarity (mOsm / L) Plasma Osmolarity Minimum urine osmolarity 100 75 50 25 0 Nephrons (% normal)

Total Renal Excretion and Excretion Per Nephron in Renal Failure 75 % loss of nephrons Normal Number of nephrons 2,000,000 Total GFR (ml/min 125 GFR per nephron (nl/min) 62.5 Total Urine flow rate (ml/min) 1.5 Volume excreted 0.75 per nephron (nl/min) 500,000 40 80 1.5 3.0

Osmoreceptor– antidiuretic hormone (ADH) feedback mechanism for regulating extracellular fluid osmolarity.

ADH synthesis in the magnocellular neurons of hypothalamus, release by the posterior pituitary, and action on the kidneys

Stimuli for ADH Secretion Increased osmolarity Decreased blood volume (cardiopulmonary reflexes) Decreased blood pressure (arterial baroreceptors) Other stimuli : - input from cerebral cortex (e.g. fear) - angiotensin II - nausea - nicotine - morphine

ADH is released upon: 1. Increased osmolarity (Osmoreceptors in hypothalamus). An increase of 1% (2-3 mOsm/l) is enough to stimulate ADH secretion. 2. Decreased blood pressure (arterial baroreceptor reflex (high-pressure regions such as aortic and carotid sinus, and low-pressure regions such as cardiac atria and cardiopulmonary reflexes), 3. Decreased blood volume. We need a decrease of blood volume by 10% to stimulate ADH release.  

The effect of increased plasma osmolarity or decreased blood volume. Figure 28-11

Factors That Decrease ADH Secretion Decreased osmolarity Increased blood volume (cardiopulmonary reflexes) Increased blood pressure (arterial baroreceptors) Other factors : - alcohol - clonidine (antihypertensive drug) - haloperidol (antipsychotic, Tourette’s)

Stimuli for Thirst Increased osmolarity Decreased blood volume (cardiopulmonary reflexes) Decreased blood pressure ( arterial baroreceptors) Increased angiotensin II Other stimuli: - dryness of mouth

Factors That Decrease Thirst Decreased osmolarity Increased blood volume (cardiopulmonary reflexes) Increased blood pressure ( arterial baroreceptors) Decreased angiotensin II Other stimuli: -Gastric distention

Sodium Intake ( mEq/day) Effect of Changes in Sodium Intake on Plasma Sodium After Blocking ADH-Thirst System 136 140 144 148 152 ADH-Thirst blocked Plasma Sodium Conc. (mEq/L) normal 30 60 90 120 150 180 Sodium Intake ( mEq/day)

Effect of Changes in Sodium Intake on Plasma Sodium After Blocking Aldosterone or Ang II System 136 140 144 148 152 Plasma Sodium Conc. (mEq/L) Ang II Blocked normal aldosterone system blocked 30 60 90 120 150 180 Sodium Intake ( mEq/day)