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1PHY 423 University of British Columbia Linda.firstname.lastname@example.org Proximal Nephron Na coupled transport systems Diabetes Mellitus, Nephrogenic glucosuria Diabetes InsipidusPHY 423University of British Columbia
2What effect does the disorder have on renal glucose excretion? Group 1: Diabetes MellitusGroup 2: Nephrogenic GlucosuriaGroup 3: Diabetes InsipidusWhat effect does the disorder have on renal glucose excretion?What is the underlying cause?Are there any changes in NaCl, K or water excretion?
3What is the primary defect in each of these disorders? Primary underlying cause of the problemDefective Nephron SegmentAltered transport moleculeNephronSegment(s)affectedDiabetes MellitusNephrogenic glucosuriaDiabetes Insipidus
4What effect does the disorder have on the following What effect does the disorder have on the following? Complete the table, compare to normal and other disordersConditionGlucose excretion*NaCl and water excretion*K excretion*ECF loss* Urine 24 hrNormalDiabetes Mellitus(Untreated)Nephrogenic glucosuriaDiabetes Insipidus (water intake = water loss)In the case of Diabetes Insipidus we are looking at the situation where ADH is absent but water losses are replaced by increased water intake. So even though ADH is absent, there is no net impact on total body water and therefore body fluids, ECF, effective circulating volume and BP are normal. We would not call it treated DI because that would entail giving the hormone.Complete the normal- glucose trace, excretion = intake, same for K, no loss and 1-2 liters.Diabetes mellitus ? Should be able to do a lot of it.Nephrogenic glucosuria someDI some but don’t spend more than 5-10 on this – say they can fill in as we go along.* vs normal
5Ron is 20 years old and has just gone to his family doctor for a health assessment since he is applying to the fire department. Urine dipstick showed the presence of more than a trace of glucose in his urine. His plasma glucose concentration is normal. Is this OK?
6Length of the Proximal Tubule Glucose ReabsorptionShows the normal disapearance of glucose along the length of the PT- notice it starts at the concentration of glucose in plasma and then declines to nearly Zero by about 25% of the length of the PT how does this happen?Length of the Proximal Tubule
7Na-glucose reabsorption-lumen becomes negative- then Cl- and water are reabsorbed too! NaCl +glucoseH2O-2 mVLumenSGLUT2H2OglucoseglucoseAQP1gGlucoseIn this cell in the first segment of the PCT, the Na-K ATPase pump creates the low intracellular Na and the K diffusion gradient creates the negative cell interior. This provides the driving force for Na to enter the cell.Because these cells express the gene for the Na-glucose co-transport molecules, if both of these molecules are present in the lumen of the proximal tubule, then both can bind and the energy of the Na electrochemical concentration (EC) gradient is sufficient to bring both Na and glucose into the cell.When Na leaves the lumen of the proximal tubule, since glucose is uncharged, a charge separation occurs making the lumen negative. This process is electrogenic.The lumen negative potential difference provides the driving force for Chloride ions to move between the cells (paracellular) transport.Na ions that entered via SGLUT2 leaves the cell via the Na-K ATP ase pump,As glucose enters the cell, its concentration increases. This creates the driving force for glucose molecules that entered via SGLUT2 leave the cell via the GLUT2 carrier. The GLUT proteins mediate faciiated diffusion.The net effect is that NaCl and glucose are moved from the lumen to the lateral spaces and interstitial space beneath the basolateral membrane.As the molecules of glucose, Na and Cl move from the lumen into interstitial fluid on the blood side of the cells, it creates an osmotic gradient for water to follow. Water enters and leaves the PCT cells via the AQP1 channels.Normally the reabsorption of NaCl glucose with water following is fairly temporarily linked. For the longest time it was thought that all water reabsorption was paracellular but now it clear that almost all water movement along the nephron is through the transcellular path and occurs through AQP water channels.The SGLUT2 cotransporter is a high capacity but low affinity transporter. This means that it has a high speed of transport but its affinity to bind glucose is low. As glucose concentration starts to fall in the lumen due to the active transport, then the rate of transport by SGLUT2 begins to decline. This transporter can not reduce the concentration to zero which is what happens as we move down the length of the Proximal tubule. Proximal tubule cells express another Na-glu transporter which can bind glucose at very low concentrations and is able to reduce the luminal concentration to near zero. This is SGLUT1.Blood SideGLUT2H2O
8NaCl +glucose H2O Lumen Blood Side -2 mV High affinity but low capacity 2Na-glucose cotransporter- last 80% PT Reduces glucose concentration nearly to zero!NaCl +glucoseH2O-2 mVLumenSGLUT1H2O2glucoseglucoseAQP1gGlucoseIn this cell which appears about 20% of the proximal tubule length expresses a different Na-glu cotransporter i.e. SGLUT1.All of the steps in transport are exactly the same as for the SGLUT2 system. Except that SGLUT1 binds two Na ions and one glucose molecule.The affinity of this transporter is very high for glucose which means it will bind glucose when glucose concentrations are very low.This transporter accounts for the reduction of glucose concentration to zero as we see under normal circumstances.These cotransporters are expressed along the length of the proximal tubule but are seldom called into play because all the glucose is reabsorbed so early in the PT. This represents reserve capacity to reabsorb glucose when glucose load increases.As glucose enters the cell, its concentration increases. This creates the driving force for glucose molecules that entered via SGLUT1 leave the cell via the GLUT1 carrier. The GLUT proteins mediate faciiated diffusion. See notes.The net effect is that NaCl and glucose are moved from the lumen to the lateral spaces and interstitial space beneath the basolateral membrane.As the molecules of glucose, Na and Cl move from the lumen into interstitial fluid on the blood side of the cells, it creates an osmotic gradient for water to follow. Water enters and leaves the PCT cells via the AQP1 channels.There are other carriers that bind Na and other nutrients e.g. acidic amino acids, or basic amino acids. Etc. these are separate gene products and all take advantage of the electrochemical gradient for Na to drive the reabsorption of these nutrients. Mutations can and do occur in these transporters causing specific abnormalities/disorders.Blood SideGLUT1H2O
9GLUT2 and GLUT1 mediate facilitated transport of glucose out of proximal tubular cells GLUT 2 is present on the basolateral membrane of cells in the early part of the PCT that also express SGLUT2GLUT1 is expressed in cells of the remaining part of the proximal tubule that express SLUT1 on the luminal surface.
10Facilitated Diffusion Facilitated Diffusion is the simplest from of carrier mediated transportFacilitated DiffusionCarrier mediated transport- there is never an open path through the membraneThese proteins are embedded in membrane. They move ions and/or other molecules. The substrate binds to the carrier and the carrier undergoes a series of conformational changes to bring about the translocation of the molecules from one side of the membrane to the other.Normally only one type of molecule is carried. The direction of movement is determined by the EC concentration gradient. The movement can be reversed if the concentration gradient is reversed. This is an example of passive transport.The best example of a carrier that mediates facilitated diffusion is the family of Glucose transporters called GLUT GLUT transporters are expressed in most cells of the body. This includes the GLUT 1 and 2 transporters found in the basolateral membrane of the proximal tubule. Net reabsorption of glucose can not occur from lumen to blood if the facilitated transporters were not present.Is this active or passive transport?
11So what does Ron have?Ron is 20 years old and has just gone to his family doctor for a health assessment since he is applying to the fire department. Urine dipstick showed the presence of more than a trace of glucose in his urine. His plasma glucose concentration is normal. Is this OK?
13Why is glucose excretion fairly low in patients with nephrogenic glucosuria? Carrier mediated transport systems in the PT are not operating at Vmax i.e. if [S] increases then v increasesIf a patient has a defect in SGLUT2 (e.g. Vmax is less than normal) then [glucose] does not decrease as rapidly as it should. So more glucose is shifted to the next PT segment which has SGLUT1. The higher luminal [glucose] will drive an increase in velocity of transport in this segment- reducing the amount that escapes into the urine. What if the defect was in SGLUT1?Also the patient is able to take advantage of the entire length of the PT to reabsorb what would normally disappear in the first third of the segmentRemember in these patients , glucose delivery to the PT is normal, its rate of reabsorption is impaired. The reductions in transport efficiency usually only cause a small but measurable increase in glucose excretion.
14Why is glucose excretion in by patients with untreated Diabetes Mellitus so high? Part 1 Remember, glucose delivery to the PT is 2-3 times higher than normal, there are no defects in glucose reabsorption.The SGLUTs (2 and 1) which normally are not operating at Vmax become saturated by the steady high delivery of glucose (2-3 time normal).In this situation, [glucose] does not decrease as it normally does, the higher luminal [glucose] drives an increase in velocity of transport of all the SGLUTs.Despite saturation of all SGLUT2 and 1 carriers along the length of the PT, glucose concentration fails to decline and spills into the loop of Henle.
15Why is glucose excretion in by patients with untreated Diabetes Mellitus so high? Part 2 The TAL and DCT, CCD and CD have essentially no ability to reabsorb glucose if it is delivered to these segmentsThus large amounts are excreted in the urineWe will see that the presence of large amounts of un-reabsorbed glucose in the PT and downstream nephron segments will disrupt NaCl , water and potassium transportWe need to look at the mechanism NaCl and water reabsorption that is not coupled to glucose reabsorption to understand this. Coming soon!
16Summary The amount of glucose excretion and the underlying reason differ between diabetes mellitus and nephrogenic glucosuriaDisorderPrimary underlying cause of the problemDefective Nephron Segment(s)Altered transport moleculeNephronSegment(s)affectedDiabetes MellitusLack Insulin- raises plasma [glucose]NAPT, and all downstream segmentsNephrogenic glucosuriaDefective glucose reabsorptionProximal TubuleSGLUT2, SGLUT1, or GLUT2 or GLUT1PT
17Summary Comparison of the primary defect in the three disorders Primary underlying cause of the problemDefective Nephron Segment(s)Altered transport moleculeNephronSegment(s)affectedDiabetes MellitusLack Insulin- raises plasma [glucose]NAPT, and all downstream segmentsNephrogenic glucosuriaDefective glucose reabsorptionProximal TubuleSGLUT2, SGLUT1, or GLUT2 or GLUT1PTDiabetes InsipidusLack Antidiuretic hormone ADHNA: AQP2 expression is absent due to absence of ADHEntire collecting duct system i.e. CCD and downstream .
18Recap-First third of the proximal tubule Proximal Convoluted TubuleRecap-First third of the proximal tubuleThe lumen potential is negative because of electrogenic Na glucose and some types of Na-amino acid reabsorption- which ones?Any Cl that is reabsorbed is going through the paracellular pathway driven by the lumen negative potentialThere is NaHCO3 reabsorption which you will learn about in the acid base section of the course
19Proximal Convoluted Tubule Are there other mechanisms of reabsorbing NaCl and water in the last half of the PT?Yes**Carrier mediated reabsorption of NaCl- MOST is going this way- ACTIVE TRANSPORTPassive reabsorption of Cl driven by its chemical concentration gradient and passive reabsorption of Na driven by the lumen + potential through the paracellular pathway – THIS IS PASSIVEWater follows all- i.e. isosmotic NaCl and water reabsorption
20Last Half Proximal Tubule Most Na and Cl transport is transcellular and some is paracellularCl-Cl-+The early reabsorption of Na with other molecules besides Cl, causes the increase in chloride concentration. This chemical concentration gradient provides the driving force for Cl diffusion through the tight junctions in the last third of the Proximal tubule. These tight junctions have a high Cl permeability. As Cl moves through the tight junctions without Na, it creates a diffusion potential with the lumen developing a slight positive potential of +2-3mV. This electrical gradient is sufficient to drive Na movement through the tight junction. Thus some NaCl is reabsorbed passively through the tight junctions (about 10% of the amount reabsorbed by the proximal tubule. Most of the NaCl in this segment is reabsorbed transcellularly by carrier mediated active transport. Water follows the movement of Na and Cl by traversing the cell through AQP1 channels.Note the primary active NaKATPase pump, the secondary active Na-H antiporter and tertiary active Cl –base exchanger. The net effect is that NaCl moves through the apical membrane. The protons and base keep recycling.. Very clever process. Water will follow as NaCl accumulates in the basolateral spaceBring up point about primary, secondary and tertiary active transport- atpase, NaH exchanger, drives the Cl Base exchangerTranscellular NaCl transport is active
21Why is NaCl and water reabsorption in the PT disrupted in patients with untreated Diabetes Mellitus? NaCl & water reabsorption in the PT is disrupted by the creation of a concentration gradient for Na to leak back into the lumen. Thus a large amount of NaCl and water is delivered downstream
22How unreabsorbed glucose decreases NaCl and water reabsorption in the PT Read noteThe example given here is mannitol which is a substance used to induce an osmotic diuresis in some clinical settings. It is the same size as glucose and is the alcohol derived from glucose. In your mind replace Mannitol in the figure with Glucose. In patients with untreated Diabetes Mellitus, their plasma glucose concentration is about 5-10 times higher than normal and sometimes higher. Normal plasma [glucose] is 5 mM. For discussion purposes and to make the figure easier to use, lets say we are dealing with a patient whose glucose is 10x normal. That would make it 50 mM. So this is the glucose concentration as it enters the first part of the PT. As described in the slides, despite the fact that the SGLUTs transporting glucose, NaCl, at maximal rates with water following initially, a huge amount of glucose will remain unreabsorbed because the load is so high. Then as Na is reabsorbed by the other mechanism i.e. Na with HCO3, and NaCl with water following initially, the concentration of glucose will rise as show in the slide. The presence of these unreabsorbed glucose molecules prevents water from following Na as it is reasborbed by the transport systems. As Na is pumped out and water does not follow, its concentration starts to fall. We mentioned that the PT is capable of transporting large amounts of Na but can not pump against a Na gradient. The tight junctions are too leaky to Na. When the Na concentration drops to about 110, then the back leak of Na becomes very large. Thus the Na backleak undermines the effectiveness of the carrier mediated transport system and NET NaCl and water reabsorption will STOP. All of this unreabsorbed NaCl, glucose and water will now enter the loop of Henle where it will disrupt transport in the distal parts of the nephron.Gennari and Kassirer, NEJM 291: , Oct 3, 1974 with permission
23Osmotic Diuresis in DMIncreased glucose load to the PT exceeds NaGlu reabsorptive capacity. Initially water follows the NaGlucose, Cl reabsorption thus the glucose concentration in the PT starts to increase.Osmotic pressure of unreabsorbed Glu causes water to remain in the PTRetained water causes [Na] to decrease in the lumen as NaCl is pumped out by the mechanism previously described- this creates a [Na] gradient between blood and lumenDespite maximal rates of NaCl reabsorbed via NaGlu and NaCl coupled mechanism, the gradient causes a back leak of Na Cl and water through the tight junctions. Now the backleak of NaCl into the tubule equals the rate of NaCl pumpted out. Thus net transport stopsThis net decrease in NaCl and water reabsorption leads to increased delivery to the distal nephron which exceed the capacity of these downstream segments.The increase in delivery of NaCl to the cortical collecting duct (CCD) stimulates K secretion.Consequently you will loose NaCl and water = ECF and K into the urine. More on this laterThe increase in plasma [glu] in DM exceeds the capacity of the PT to reabsorb the load. The unreabsorbed glucose causes water to remain in the lumen i.e. its does not follow NaCl reabsorption. This causes the lumen Na concentration to fall. Thus a gradient is created between the blood and the lumen i.e. lower in the lumen than in the blood.Even though NaCl is being reabsorbed at maximal rates via NaGlu and NaCl coupled mechanism, the gradient causes a back leak of NaCl and water through the tight junctions. Remember the PT can not maintain a NaCl concentration gradient between the lumen and the blood due to the leakiness of the tight junctions. When Na concentration reaches 110 mM, then maximal transport of Na out of the lumen is equal to Na backflux into the lumen between the tight junctions. This is refered to as an osmotic diuresis. This net decrease in NaCl and water reabsorption leads to increased delivery to the distal nephron which exceed the capacity of these downstream segments.
24What are the changes in glucose excretion, electrolyte ConditionGlucose excretion*NaCl and water excretion*K excretion*ECF loss* Urine 24 hrNormal0 -traceNaCl excretion = NaCl intakeWater balance is maintainedK excretion = K intakelitersDiabetes Mellitus(Untreated)+++++literselectrolyte richNephrogenic glucosuria+normal1-2 litersDiabetes Insipidus (water intake = water loss)Free water excretion little NaCl loss0 virtually no K losslitersmostly waterIn the case of Diabetes Insipidus we are looking at the situation where ADH is absent but water losses are replaced by increased water intake. So even though ADH is absent, there is no net impact on total body water and therefore body fluids, ECF, effective circulating volume and BP are normal. We would not call it treated DI because that would entail giving the hormone.* vs normal
25Discussion Questions- next class See Group Assignments Sheet Three working groupsGroup 1 Bartter’s Syndrome/Disease and FurosemideGroup 2 Gitelman’s Syndrome/Disease and ThiazidesGroup 3 Liddle’s Syndrome/Disease and Amiloride
26Proximal Tubule Length Cl concentration is increased because it lags behind Na glucose reabsorption and because Na is reabsorbed with bicarbonate in the early part of the PT. Cl diffusion down its concentration gradient through the paracellular pathway creates the lumen + TEPThis figure shows the changes in the ratio of tubular fluid concentration to plasma concentration from the beginning of the proximal tubule i.e. right as fluid comes across the glomerulus, to almost the end of this nephron segment (about ¾). By comparing the TF/P ratio for a given molecule we can learn something about what is happening to it. For the case of glucose and amino acids, this is very easy. Since the concentration falls essentially to zero in the first quarter of the PT, it has to mean that it is being reabsorbed. We can also say that bicarbonate is being reabsorbed but it looks like to a lesser degree. The whole picture can only be understood by looking at the concentration of a marker that it is not either reabsorbed or secreted. In this case inulin is that marker. Please note that the concentration ratio of inulin is 1 at the beginning of the PT just like all the other substances that are being tracked but then it increases to 2! What does that mean? Since we know the properties of inulin, it means that half of the water has been reabsorbed. This is the fact that we must consider in looking at the changes in the TF/P for all the other molecules. So the steady decline in TF/P of glucose and aminio acids means that they are virtually completely reabsorbed. The Na and osmolality TF/P ratios are 1.0 since 50% of the volume has been reabsorbed, it means that 50% of the Na and 50% of the osmotically active solutes have been reabsorbed but by a process that is isosmotic. You can see that Cl is less avidly reabsorbed than Na or HCO-, glucose or amino acids.What we see is at the end of the PT that about 80% of the filtered bicarbonate has been reabsorbed and most of the Na entering the loop of Henle is accompanied by Cl.This figure also shows the development of a lumen negative transepithelial potential difference TEPD of -2mV in the early PT which then goes to zero and a lumen positive potential of +2mV develops. The -2mV early PT TEPD is caused by electrogenic Na glucose transport, the lumen +2mV potential in the later part of the PT is caused by a Cl diffusion potential as Cl diffuses through the paracellular pathway down its concentration gradient. See the slide on NaCl reabsorption in the late PT.Proximal Tubule LengthDr. Bolliger Kanas University Medical Center 1999