The Proximal Tubule: Reabsorption and Secretion

Active TransportActive transport is the key to the reabsorptive processes in the proximal tubule. Glucose and amino acids are reabsorbed across the apical membrane of the proximal tubule by sodium-coupled secondary active transport. Na+ glucose transport is mediated by the low affinity, high capacity SGLUT2 transport protein. Glucose gets out of the cell across the basolateral membrane by facilitated transport via GLUT2.

Which transporter carries amino acid uptake depends on the type of amino acid. Neutral amino acids are carried by the B0AT1 transport protein which will carry all amino acids without charge as well as a couple of the cationic ones.

Cationic amino acid transport is via a related transporter b0,+AT. This transporter is only loosely coupled to the Na+ gradient. b0,+AT exchanges neutral amino acids (out) for cationic amino acids (in), but as the neutral amino acid uptake is Na+ dependent the Na+ gradient can drive uptake.

Uptake of anionic amino acids is by EAAT3, which is tightly but confusingly coupled to the Na+ gradient. EAAT3 cotransports 3+ plus 1 H+ plus 1 amino acid into the cell and 1 K+out.

All of the above are principly in the proximal convoluted tubule. The few transporters I mention here are the tip of the iceberg, there are loads more. I'm not even going to mention how amino acids get out of the cell . Stefan Broer has written an exhaustive and almost incomprehensible review for Physiology Reviews (2008) 88:249-286. Read it at your own risk. If it makes you feel any better, close analogues of all these transporters do very similar jobs in the intestine.

The inwardly directed Na+ gradient that energises all of this glucose and amino acid uptake is created and maintained by primary (ATP dependent) active transport of Na+ out of the cells by the Na+ / K+ ATPase. Sodium transport across the proximal tubule is unidirectional, towards the blood, mainly because the Na+ / K+ ATPase is only to be found on the basolateral membrane but also because most of the Na+ coupled transporters which let Na+ back into the cell are found on the apical membrane. Unidirectional Na+ movement across the proximal tubule is electrogenic, in other words, it carries a net charge and this will help to energise the reabsorption of anions, such as Cl-.

Moving all this salt and amino acids and glucose creates an osmotic gradient across the proximal tubule which drags water back from the urine and into the blood. The proximal tubule is brilliant at isotonic water transport and reclaims about 70% of the 180l filtered every day.

 

Bicarbonate Reabsorption

Bicarbonate reabsorptionBicarbonate (HCO3-) reabsorption is lots of fun. HCO3- and H+ in the in the 'primary urine' will be in equilibrium with carbonic acid (H2CO3). Carbonic acid will spontaneously dehydrate into CO2 and water, but this process is normally very, very slow. The ever resourceful proximal tubule cells secrete an enzyme called carbonic anhydrase on to their apical membranes. This enzyme greatly accelerates the production of CO2, which is of course cell membrane permeable, and so diffuses into the proximal tubule cells. Once inside, intracellular carbonic anhydrase sets to work and makes carbonic acid from the carbon dioxide and the water The H2CO3 dissociates into HCO3- and H+. The HCO3- is transported across the basolateral membrane and into the blood (bicarbonate reabsorption accomplished!) and the H+ are actively transported, in exchange for Na+, across the luminal membrane into the urine. These H+ are buffered by HCO3- in the urine to form H2CO3, extracellular carbonic anhydrase catalyses the formation of CO2 an... (We've hit a loop here. I'm going to stop before it gets silly). The net effect of the whole process is that bicarbonate is reabsorbed and protons cycle backwards and forwards across the luminal membrane.

HCO3- efflux across the basolateral membrane into the blood is a bit weird. This is one of the rare transport proteins that takes Na+ out of the cell, against the Na+ gradient. It can only manage this because 3 HCO3- ions are coupled to a single Na+ ion which makes the overall process highly electronegative so that it can use the -ve membrane potential as a driving force.

We can take this process one step further to show how the kidney can manufacture HCO3- if circulating HCO3- levels fall. Imagine that, instead of using CO2 which has entered from the lumen, the renal cells use CO2 from the blood. This CO2 will also be transformed into H2CO3, which will dissociate into H+ and HCO3- and the net result will be extra HCO3- heading towards the blood and extra H+ in the urine. This is one reason why urine may be acid. If this were all that happened, the urine would rapidly become very, very acid and the processes of HCO3- reabsorption and manufacture would cease. BUT, HCO3- is not the only acid buffer. Ammonia (NH3) will also buffer H+, becoming ammonium (NH4+) in the process. NH3 is membrane permeable but NH4+ is not (it is charged!), so NH4+ has nowhere to go except the bladder and ultimately the outside world. In recent years, that last sentence has been shown to be less and less accurate. NH4+ is produced and excreted, but the mechanisms of NH4+ handling appear to be significant more complex than simple solute trapping. The proximal tubule cells can manufacture NH3 and dump it in the urine to mop up protons, become NH4+, stop the urine becoming too acid and support the manufacture HCO3- on-demand. This mechanism can account for as much as 70% of new HCO3- produced by the kidney. The key step in triggering NH3 production and HCO3- manufacture is metabolic acidosis and specifically a fall in the pH of the urine. See the paper by Garibotto et al in Metabolic Brain Disorders for more detail. The bad news is that chronic acidosis is not good for the kidney......

There is also another buffer system in the urine. Both alkaline (HPO42-) and acid (H2PO4-) phosphates are filtered into the primary urine in a ratio of 4:1, as the pH falls (becomes more acid), HPO42- will buffer protons and become H2PO4-). This happens in the proximal tubule, but is more important in the distal tubule. Protons bound to phosphate buffer constitue the titratable acid in the urine.

The proximal tubule reabsorbs most (>85%) of the filtered bicarbonate, the remainder is picked up by the intercalated cells of the distal tubule and collecting duct. The process of reabsorption is pretty much the same as in the proximal tubule except that the intercalated cells have a proton pump, a primary active transporter that extrudes protons into the urine. Useful though the Na+ gradient is in energising secondary active extrusion of H+, nothing can compare to the power of an ATPase. The intercalated cells are therefore capable of supporting a much, much bigger H+ gradient than are the proximal tubule cells. The bottom line in HCO3- uptake is what to do with the H+. With the power of an ATPase to remove them from the cytoplasm, the intercalated cells can (if necessary) just dump them in the urine and allow the urine to become more and more acidic.

If you do any job well, your reward is usually another job. The kidney is so good at reabsorbing and manufacturing HCO3- that it also gets to regulate the pH of the whole body, a responsibility it shares with the lungs. The dominant buffer system in the body is a bicarbonate buffer. pH depends therefore on the relative concentrations of carbonic acid and bicarbonate in the blood. The kidney controls bicarbonate concentration and the lungs control carbonic acid concentration

Reabsorption and Secretion

filtration absorption secretionEverything in solution in the plasma gets filtered into the primary urine. Unless actively reabsorbed, everything that is filtered will ultimately be excreted. Bearing in mind that the filtration fraction is 20%, this means that at most 20% of any substance in the blood will be excreted in a single pass through the kidney. The blood repeatedly circulates back to the rest of the body and then back to the kidney again. A volume equivalent to the entire blood supply of the body passes through the kidney every 5-6 minutes. Some simple arithmetic will show that it will take about 10 passes through the kidney to remove 90% of a substance from the blood by filtration alone and about 21 passes to remove 99%. Roughly speaking then it will take at least an hour to remove 90% of any substance from the bloodstream.

organic anionThis is too long for some substances. The kidney is so keen to get rid of some classes of compound that, in addition to filtering them, it actively secretes them. The proximal tubule is the site of active secretion of potentially toxic substances. In order to actively secrete these nasties the proximal tubule has two broad spectrum secondary active transporters, one for cations and the other for anions. Whereas most active transporters are highly substrate specific, these transporters will take any old rubbish. The organic cation transporter will accept any (small, monovalent) positively charged organic molecule and the organic anion transporter will accept any (small, monovalent) negatively charged organic molecule. (organic anions are sometimes called organic acids and organic cations, organic bases). In both the anion and the cation transporter the active step is getting the substance from the blood into the cells. Once inside the cell it is allowed to exit into the urine by passive (facilitated) transport down its gradient. The organic cation transporter is energised by the membrane potential (Cell negative interior attracts cations). The organic anion transporter runs by anion exchange. Dicarboxylic acids are accumulated inside the proximal tubule cell by metabolism and by a sodium coupled cotransport process, these then leave the cell down their electrochemical gradient in exchange for whatever anion happens to be bound to the organic anion transporter. In effect, the organic anion exchanger is loosely coupled to the sodium gradient. In any case, both transport processes are highly effective. Para amino hippuric acid (PAH) is a test substance that is not harmful in itself, but is nevertheless a good substrate for the organic anion transporter. Ninety percent of PAH is removed from the blood into the urine on a single pass through the kidney. Other organic anions that are good substrates for the transporter include DDT (probably just as well) and antibiotics (which can be a nuisance).

If glucose is a good example of a substance reabsorbed by the proximal tubule and PAH a good example of something highly secreted, then inulin is a good example of a substance that is neither secreted nor reabsorbed. Note this is not a misspelling of insulin, inulin and insulin have nothing in common except confusingly similar names. Inulin is one of those potentially dangerous inert substances, too big to cross cell membranes, ignored by transporters if it wasn't filtered into the urine it could build up in the plasma and play havoc with osmolarity.

test substancesThe three test substances glucose, PAH and inulin may be used as examples to demonstrate the effects of some of the different transport processes found in the proximal tubule. If the urine were sampled at different sites along the proximal tubule from the glomerulus to the loop of Henle, then the glucose concentration would be seen to diminish the further along the tubule you went. Under normal circumstances, all the filtered glucose is reabsorbed by the proximal tubule. On the other hand, the inulin concentration would increase. It might double by the end of the proximal tubule. The reason for the increase is not that more inulin is entering the proximal tubule (it can't) but rather that water is being removed and the inulin is staying behind. The PAH concentration goes through the roof. It may be concentrated 10 fold or more by the end of the proximal tubule, both by the action of water reabsorption and as a result of active PAH secretion.

test substancesA graph showing the relationship between plasma and urine concentration would therefore look very different for all three substances. The urine concentration of inulin would rise as a simple linear function of plasma concentration. The urine concentration of PAH would appear to be a linear function of plasma concentration but with a much higher slope than that of inulin (It will in fact be a combination of a linear function and one best described with a rectangular hyperbola). There will be no glucose in the urine and so the line showing urine glucose concentration will bump along the X axis at zero.
All of the above is true for low concentrations of glucose and PAH and completely WRONG for higher concentrations. test substances

At plasma concentrations above 10 mmol/l, glucose starts to appear in the urine. At concentrations above 15 mmol/l, there is a linear relationship between plasma glucose concentration and urine glucose concentration. At plasma concentrations above 0.6 mmol/l the rate of PAH excretion levels off. At concentrations in excess of 0.6 mmol/l there is a linear relationship between plasma PAH concentration and urine PAH concentration. The relationship between plasma inulin concentration and urine inulin concentration is the same throughout. In fact, at high concentration, the slope of the graph of plasma vs. urine concentrations for all three substances looks the same (the three lines are parallel). It is the same and for good reason. Glucose is reabsorbed by active transport and PAH is secreted by active transport. Active transport is an enzyme dependent process. (transporters are enzymes). All enzyme-mediated processes are saturable. Once all the active sites on the enzyme are occupied, it doesn't matter how much substrate is present the reaction (in this case transport across a cell membrane) won't run any faster. Once the glucose transporters are saturated no additional glucose will be reabsorbed and it will therefore it appear in the urine. The maximum capacity of an active transport system is called the Tm, which stands for Transfer maximum. Plasma glucose concentration is normally around 4-6 mmol/l, far below the transfer maximum. In diabetes mellitus insulin deficiency prevents normal uptake and utilisation of glucose so that it accumulates in the blood to concentrations in excess of 10-15 mmol/l and so appears in the urine.

A positive test for glucose in the urine is a diagnostic test for diabetes mellitus. Whilst glucose in the urine is definitely diagnostic for diabetes, this isn't the best way to diagnose diabetes. Much better is to catch the disease long before glucose handling has become so poor that glucose appears in the urine. Glucose is pretty easy to measure in the blood and diabetes is defined by the WHO as fasting plasma glucose in excess of 7 mM. An additional diagnostic tool for diabetes is the HbA1C blood test. In brief, glucose sticks to haemoglobin, to make a glycosylated haemoglobin called HbA1C. HbA1C levels reflect the average plasma glucose concentration for the previous month or three. In addition, a fasting plasma glucose between 5.5 and 7 mM is defined as "prediabetes" and like many diseases, the earlier that diabetes is identified the better (managing prediabetes is better still) because the long-term damage caused by diabetes mostly results from poor glucose homeostasis. Diabetes cannot be cured (yet) but it can be managed, and good management will mitigate the consequences of hyperglycaemia.

Incidentally, HCO3- reabsorption has an effective Tm of about 30mM, by no coincidence at all, this is close to the normal HCO3- concentration of 24nM. So, in the unusual circumstance that HCO3- is too high, the kidney does nothing and the excess is excreted.

Two of our three test substances, inulin and PAH may be used to determine some important aspects of renal function by measuring their clearance.

Clearance

Clearance is an imaginary value that may be defined as:

The volume of blood cleared of any substance by passage through the kidney in unit time.

Clearly there can be no such thing. The blood that emerges from the kidney will contain less of a substance (assuming that it is not all reabsorbed) but all the blood will contain less, there won't be part of the blood that contains none of the substance and the rest of the blood with the concentration you started with. But we can imagine that this is possible, and even calculate the volume of blood that would be 'cleared'. So, if 20% of a substance contained in plasma is excreted by a single pass through the kidney, the reality is that all the blood emerging from the kidney will contain 80% of what we started with. But we could imagine that 20% of the blood now contains none of the substance (the arithmetic comes out the same) That 20% would represent the clearance value for our substance.

Why bother?

Well suppose you had a substance, such as inulin, that is only filtered, not secreted, not reabsorbed and asked the question how much of the blood plasma will be cleared of the substance? The answer is the same amount of blood plasma that is filtered. If you could measure the clearance of inulin you would then know the Glomerular Filtration Rate (GFR). Clearance is easy to calculate. The clearance of any substance is given by the urine concentration divided by the plasma concentration multiplied by the volume of unit produced in unit time (usually minutes)

Clearancex = [X]u / [X]p . Vu

The clearance of inulin, which is equivalent to the GFR, is about 125 ml/min. Each minute 125 ml of plasma are filtered into the urine.

What about PAH clearance? PAH is almost completely removed from the blood in a single pass through the kidney. Suppose for a moment that it is all removed and then ask the question how much plasma will be cleared of PAH? The answer is all of it. Measure PAH clearance and you will know how much plasma passes through he kidney in unit time. This is known as the Renal Plasma Flow (RPF). Using the equation for clearance above you will get a value of 650 ml/min for PAH clearance. PAH clearance is sometimes termed the ERPF (Effective Renal Plasma Flow), I suppose because it is effectively the same as the RPF. ERPF can be converted to RPF by knowing that 90% rather than 100% of the PAH is removed by the kidney. The fraction of the substance removed by the kidney is termed the Extraction Ratio (ER). The ER for PAH is 0.9. Divide ERPF by ER and you get RPF.

RPF = ERPF / ER = 650/0.9 = 722 ml/min.

For completeness, Renal blood flow (RBF) takes into account the fraction of the blood occupied by cells (haematocrit), therefore:

RBF= RPF / (1-haematocrit) = 722 / (1-0.5) = 1444 ml/min

Both GFR and RPF/RBF are indicators of renal health. In fact, acute renal failure may be defined as any process that causes a rapid decine in GFR. In practice, an approximate measure of GFR may be obtained by measuring creatinine clearance. Creatinine is the end product of muscle metabolism and therefore normally present in the circulation. So far as the kidney is concerned, it is nearly as inert as inulin. RPF declines as a function of old age and also because of many chronic renal and systemic conditions.