Some of us overdo things with shakes and powders, some with 2 pound steaks. Others love sweets too much and don’t eat much protein. Like all the diet factors in stone and bone disease, protein intake is complex. Certainly, we all need protein in our diet but how much? Experts debate the best course, and patients wonder what to do.
Abraham van Beijeren was, by the way, little recognized in his day but now considered a major painter of ‘luxuries’ like this standing roast. I chose it, as opposed to others more brilliant, because it looks modern – I have seen something like it on my own dining room table.
In preparing this article I have made considerable use of the analyses performed by professor Tanis Fenton. She graciously read and edited the article up to the details of renal physiology, and the work much benefitted from her expertise which I gratefully acknowledge here. All errors are entirely mine, however, should you find any.
How Much Protein Do We Need?
The National Research Council (US) Recommended Dietary Allowances Tenth Edition (1989) suggests (Table 6.4) 0.8 gm/kg of dietary protein for adult men and women.
A subsequent WHO meta analysis of mostly the same underlying data supplemented by more recent studies comes to much the same conclusions, but in perhaps a more nuanced manner. A more recent analytical critique of the whole matter is not remarkably far off in estimates for adults, though pregnancy and childhood seem controversial.
This summary graph from the critique gives a sense of how the protein requirements are set. The median requirement is where about half of all studied subjects were in neutral nitrogen balance – their body protein mass would be stable, a very important matter. The safe population intake is set higher. The safe individual value is high enough enough that 97.5% of the individuals in a population would be in balance: almost all people would not lose protein mass consuming this amount of protein – for example muscle. The ‘Safe population intake’ is set higher. Although the safe individual intake is correct, within a population individual requirements vary, so the recommended level needs to be increased so that 97.5% of the individuals in a population offered that recommendation will be in balance.
That number from the WHO meta-analysis, the safe population intake, is about 1.05 gm/kg body weight/day.
Given the safe individual intake is 0.83 and the upper bound of the intake range is 1.05 gm/kg/d no one should choose a protein intake below 0.83 gm/kg/d and very few need more than 1.05 gm/kg/d unless challenged with unusually high demands for physical work. This means that in helping patients choose a protein intake for stone prevention we work in that range 0.83 – 1.05 mg/kg/d.
The Story in Brief
Because commercial vendors provide a measure, albeit indirect, of protein intake in every 24 hour urine – using the amount of urine urea to calculate the protein catabolic rate (PCR) in gm protein/kg body weight/d – patients know their intake and can control it by diet. This measure is applicable to healthy people who are neither gaining or losing protein mass.
Like sugar, protein loads raise urine calcium, and urine calcium is a major risk factor for stone formation. Low protein intake may reduce urine calcium but is bad for overall health. Whether or not high protein intake raise urine calcium at the expense of bone integrity is fiercely debated right now. Low protein intake is not good for bone.
So with respect to protein intake stone treatment steers between too high and too low.
I could just tell remind you that the high and low markers for normal people – not otherwise compelled to high or low protein diets – are between 0.8 and 1 gm/kg/d and be done, but that is not my way. I believe understanding is the key to long term treatment and encourage patients to read about protein. Physicians already know everything I write about, but many way enjoy a ride through a familiar and charming countryside.
Protein Imposes an Acid Load
For several generations we have known that the sulfur containing amino acids cystine and methionine produce an acid load and that rising diet protein acid loads correlate with increased urine calcium excretion. Giving acid loads experimentally increases urine calcium excretion.
Some believe acid loads promote bone fractures by mobilizing bone mineral stores and that alkali treatments prevent this form of bone loss. Others believe that the protein increases urine calcium by increasing intestinal calcium absorption and does not adversely affect bone,
Fenton and her colleagues performed what I think is a rigorous ‘meta-analysis’ of studies available up to 2006 concerning effects of acid load on the urine calcium excretion. The acid loads were varied by variation of diet protein, by giving alkali such as potassium citrate and by giving acid loads like ammonium chloride – a purely experimental strategy. No matter how acid load was varied urine calcium varied linearly.
The points on this graph are from Table 2 of her paper. I have redrawn her figure to suit my taste. My data set is available for others.
The negative changes in net acid load are from alkali loading such as potassium citrate. The changes from, as an example, 50 mEq of alkali – the -50 position on the horizontal axis – corresponds to a fall of about 50 mg/d of urine calcium.
In my article on prevention of calcium stones, we found the effect of potassium citrate corrected for urine sodium was in fact -92 mg/d, which is very close to the results from this large analysis (see page 8 of the statistical analysis linked from the article).
Acid Loads Apart From Protein Raise Urine Calcium
I would be remiss to leave matters as if Fenton’s meta-analysis were a sufficient guide to this vast literature. Her approach emphasizes the quality of the human trials. Another review more emphasizes the underlying technical problems of assessing net acid base balance. In this work acid base balance was altered by NH4Cl (closed triangles), methionine (open triangles), egg white (*), beef (closed circles), soy protein (open circles), deprived of KHCO3 (+), given KHCO3 (closed squares), or given NaHCO3 by replacing some of the dietary NaCl and maintaining Na intake constant (open squares).
Despite the differing formalisms and even scientific instincts of the investigators who reviewed the topic (the one a group of skilled analysts, the other a group of expert acid base physiologists) the overall result is amazingly uniform. For example at about -50 net acid excretion, there would be about 50 mg less urine calcium and at about 200 mEq of extra acid about 300 mg more urine calcium in both studies alike.
Whereas the Fenton points easily fit a linear regression, the larger range of the Lemann review shows the response is not linear but has a curving character. If you look closely at the Fenton points there is indeed a slight sag around 0 meaning that perhaps a curving regression might have a higher multiple R2.
The point of showing all this is obvious: However you review the papers, acid loads increase and alkali loads reduce urine calcium, meaning this is a vigorous phenomenon, not some houseplant that cannot stand up to the weather. It has been found in many laboratories over many decades, in humans – shown in these two figures – and animals alike.
Does Protein Itself Raise Urine Calcium?
By this one must mean does a change in diet protein raise urine calcium more than would a corresponding increase in net acid load from some other source. Alternatively, it could mean what happens if one gives a protein load with enough alkali to offset the acid from the protein.
Four studies appear to fit the needs for data in that they are trials of protein loading in a rather pure form, using foods and with considerable care for total nutrition. Of these, one used alkali to offset the protein acid load. The points taken from these additional reports are here.
The Fenton data are in a faded blue, for visual contrast. The protein load studies are in red. The pentagon and diamond are protein load +KCl and the same protein load + K Citrate in a single trial.
In these trials, protein intake was varied over a two fold range, mostly from about 1 to about 2 gm/kg/day.
More or less the data fall on the Fenton plot.
In the one special trial it is obvious that the K Citrate (diamond) lowered the change in NAE without affecting the change in urine calcium. So it is possible to dissociate a protein effect from its acid base effect within the controlled environment of a trial. Given the modest quantitative changes in NAE it is possible that natural variability of urine calcium excretion might have permitted apparent stability despite the lack of a change in NAE in one point and a significant increase of NAE in the other, but the statistical testing is based on the observed variability and gave a low probability from chance alone.
Essentially the trial of diamonds and pentagons tells us that protein itself has a renal effect on calcium handling.
Does Diet Acid Load Damage Bone?
I am not so unwise – being an expert in stone disease not bone disease – to enter into this debate on my own. All I can do is present recent reviews by the real experts who do not seem to have convinced one another.
Meta analyses up to 2011
Fenton has performed what appears to be a rigorous screening of available balance studies concerning the effects of acid loads and protein intake. She culled out eight studies that met the criteria used by the Institute of Medicine in their assessment of dietary requirements for calcium and vitamin D. Her dataset is not presently available to me but I have replotted her summary data in a manner I find ideal for this site.
The abbreviated names refer to her Table 2 in the reference. The data I extracted and plotted are here.
Change in calcium balance does not vary importantly with change in protein intake (upper left panel) nor at all with change in net acid excretion (NAE) from the protein amounts and types (upper right panel). There is perhaps a slight inverse relationship between change in balance and change in urine calcium (lower left panel) and, as in less curated studies shown above, a marked direct relationship between urine calcium and NAE (lower right panel).
The only way balance (upper right panel) can be indifferent to change in NAE and yet inverse on urine calcium which is itself dependent on NAE is that changes of intestinal calcium absorption make up the difference from calcium lost in the urine. One presumes this but proof may be beyond the resolution of calcium absorption measurements.
Balance Data after 2011
At least one important research group performed what appeared to be a fine balance study in which potassium citrate was given to neutralize diet acid load and calcium balance was measured by an expert in such work. The data for urine calcium and balance are in mg/day and I simply transformed them to mmol/d for graphing purposes.
The way the new study was performed was to compare change in NAE and both urine calcium (left panel) or calcium balance (right panel) after 6 months of treatment with placebo, or with 60 or 90 mEq/d of potassium citrate. I have plotted the new study points in red over the Fenton analysis in blue and have removed linear smoothers for clarity.
In a sense all of the studies agree about urine calcium. A change in NAE gives rise to a reasonably stable and predictable change in urine calcium here as in the much larger data sets I already reviewed.
For balance (right panel), the placebo and 60 mEq potassium citrate points are not out of range of the other studies Fenton considered although with these two points – and of course their many more internal measurements – there might be a slight inverse relationship between NAE and balance. The single 90 mEq point would make a more powerful negative regression, as is obvious.
Whether or not the Fenton group would consider this study as being of the highest quality I do not know, as I am not a student of meta analysis or of the rather elaborate and – to me at least almost incomprehensible – criteria for ‘high quality’ as opposed to ‘low quality’ studies. But if this study were indeed part of the elect minority it might change opinions.
Perhaps I should say something, however, and it is this. Potassium citrate does lower urine calcium just as acid loads raise it, and so far as I can tell the agent will not worsen bone mineral balances. So in giving it I have no reservations about bone just as I cannot have definitive hopes for better bones as a result, at least right now.
Sodium Intake Affects Urine Calcium Response to Acid
In a prior article I showed the urine calcium lowering effects of potassium citrate were independent of the effects of diet sodium. But those data, and trial data like it are observations. Suppose you give people an acid load so their urine calcium goes up as a result of what you did and also varied sodium intake – a direct experiment, not observations.
I did that, and although others also may have done the same, my experiment was a good one and I like it.
Four people were studied during three control days – points to the left on the graph. They had normal urine calcium excretions (lower panel), normal serum PTH levels (middle panel) and normal serum total calcium (upper panel filled circles) and ionized calcium (open circles). That is what one expects from normal people.
I gave them ammonium chloride which is an acid load, and as you might expect by now their urine calcium should go up. But, I also lowered their sodium intake to 40 – 80 mEq daily (average about 1500 mg) and – take a look – urine calcium did not change.
Their blood became distinctly acidic – you will have to look at the paper. The calcium ion rose and the total calcium fell because the acidity tends to liberate calcium ion from binding to blood proteins and from phosphate complexes.
I then raised the diet sodium to about 200 mEq (4000 mg) and there was the urine calcium increase. Serum PTH went up, too, perhaps because the kidneys were losing calcium.
All of this experimental data emphasize the importance of diet sodium. Given our protein intake imposes a tonic acid load, low sodium diet should presumably act as it did here and lower the calcium losses acid loading may well create. In a well performed study of bone mineral balance in menopausal women, it was the combination of reduced sodium intake and increased calcium intake that brought bone mineral balance into a positive range.
Given this, I would add to my prior statement that low sodium diet is itself safe for otherwise normal people and is likely to benefit bone health, or at least do no harm to bones.
How Does Acid Base Change Alter Renal Calcium Handling?
A Fair Warning
What comes is technical.
Physicians and scientists know this material and can read it fluently. If you are not one of those, you can read what is coming and find is very interesting. I avoid jargon, and explain things reasonably well.
However response to acid or alkali loads is a fundamental property of kidneys, crucial for life, deeply embedded in the way they function and elaborated and refined by millions of years of evolutionary biology. It is, therefore, elegant in its way and hard to grasp.
Many investigators have shown that chronic acid administration raises urine calcium by reducing tubule calcium reabsorption. My data are as good as any to illustrate the point.
Here are the same four people from the graph just above. The vertical axis shows urine calcium excretion and the x axis shows the measured filtered load of calcium, the rate at which calcium is being filtered out of blood into the fluid moving down the tubules of the kidneys.
At any filtered load, urine calcium was lowest when people ate a low sodium diet without an acid load (closed circles). High sodium diet without acid raised urine calcium, as expected (filled triangles). Chronic acid load with low sodium diet lowered filtered load (open triangles are to the left of the filled triangles) but urine calcium was slightly higher.
High sodium and acid load caused very high urine calcium excretion at any filtered load – reduced tubule calcium reabsorption.
Let me put this into perspective. In other articles about treatment I have shown you much the same thing. Sodium raises urine calcium and that is why low sodium diet is a good treatment for stones. Moreover, the effect of sodium on urine calcium is far more marked in stone formers with idiopathic hypercalciuria than in normal people. What I show here is that acid loading makes normal people behave like those with IH – urine calcium becomes overly sodium dependent. The high sodium diet raises the urine calcium far more than it could without the without the acid load – the sodium load + acid load points are far above the other points.
We have already taken trips down the nephron. For those who have forgotten the road, here is a good review. The nephron tour begins at about page 3 of the link.
Likewise here is a good review of what is to come concerning how kidneys respond to acid loads. Read it later.
What I have Said Already
In the oxalate review noted just above we focused on SLC26a6, which can permit chloride and oxalate or other negatively charged species to move through the cell membrane facing the tubule fluid. In another article we considered the citrate transporter that faces the same way and reclaims filtered citrate. That transporter is regulated by acid and alkali and I presented the outlines of that regulation.
As part of the citrate article I introduced NHE3, a sodium hydrogen exchanger that moves protons into the tubule fluid, reabsorbing filtered bicarbonate, and also moves ammonium ion (NH4+) out into the lumen.
Chronic acid loading increases NHE3 and therefore proton transport and therefore the completeness of bicarbonate reabsorption. This latter means less alkali (bicarbonate) can be lost in the urine – and is instead returned to the blood to counteract the acid load. Also less is present in later parts of the nephron. This means that protons secreted into the tubule fluid downstream are not used up reabsorbing bicarbonate but can flow into the urine as acid excretion.
The article also told us that acid loads increase the conversion of glutamine, an amino acid, to α-Ketogluterate which process takes up protons thus generating alkali. The extra ammonia from the nitrogens of glutamine is moved into the tubule fluid by NHE3 and migrates downstream. Eventually much of it leaves in the urine. We say the sum of the protons titrating phosphate and the ammonia in the urine are the net acid excretion (NAE), which we spoke of in relation to protein acid loads in this article. The ammonia counts as acid lost because metabolism of the α -Ketogluterate produces new bicarbonate.
What about Sodium?
As NHE3 moves protons into the tubule fluid to titrate bicarbonate and therefore reabsorb most of what was filtered, it reabsorbs a sodium ion for each proton secreted (therefore called a sodium hydrogen exchanger). That sodium is transported out of the cells at the blood side of the cell by the ancient and universal sodium potassium active transporter (ATPase) and reenters the blood. In fact one might point out that NHE3 runs off the sodium current created by the ATPase.
NHE3 is like a revolving door with people coming in and coming out but those coming in are doing all the pushing. This is because the sodium concentration in the cell is kept very low by the ATPase so there is a driving force favoring sodium entry into the cells from the tubule fluid.
In the process of reabsorbing sodium, the proximal tubule reabsorbs bicarbonate via NHE3. This reabsorption of bicarbonate makes the concentration of bicarbonate in the tubule fluid lower than that in the blood. The proximal tubule cells are linked together by junctions that are rather water permeable, so as sodium and bicarbonate are reabsorbed water will move with its dissolved salts back into the blood. There is more to this, but perhaps I can add it later.
Effects of Acidosis on Sodium Reabsorption
This should be simple. More NHE3 from acid means more sodium reabsorption and more bicarbonate reabsorption, therefore more water reabsorption. So proximal tubule sodium reabsorption will go up. In the proximal tubule, calcium is reabsorbed almost completely passively – meaning along with sodium. Therefore with acid loading calcium reabsorption will also go up.
But that is not what happens.
Chronic metabolic acidosis in rats produced by ammonium chloride administration, reduces sodium reabsorption in both the proximal tubule and along the nephron downstream from the proximal tubule. There is no increase in filtration of sodium but rather a reduction of tubule reabsorption. This means that calcium reabsorption would necessarily be reduced. In fact, I just showed you in my own experiment how acid loads reduce calcium reabsorption.
How can it be that NHE3 can increase and yet sodium – and therefore calcium – reabsorption go down?
This problem led to an important experiment in animals.The rats were given acid loads and as expected bicarbonate reabsorption increased significantly. But, there was trouble with respect to chloride – an element we have not as yet mentioned.
Sodium reabsorption via NHE3 is limited by the fact that only 20% or less of the sodium filtered uses bicarbonate as its counter ion – negative ion to balance the positive charge on sodium. The rest – a majority – uses chloride, and I did not say how chloride is reabsorbed. It is in fact reabsorbed passively in part because of the sodium bicarbonate reabsorption; as bicarbonate is reabsorbed with sodium and water more and more of the sodium left behind is with chloride so the concentration of chloride rises compared to blood. This chemical concentration difference causes chloride to move back into the blood, but through what can it pass?
It moves through chloride exchangers – more revolving doors, and one of these is our old friend SLC26a6, the oxalate transporter.
What if that transporter is not working right?
You would find what was found in these rats. Sodium bicarbonate reabsorption was above normal when the rats were loaded with acid but chloride oxalate exchange was not functioning. This means that sodium chloride reabsorption could not proceed at a normal rate so neither could sodium chloride and water reabsorption – or calcium reabsorption.
In this bar graph, the control rat kidney cell membrane transport of chloride (vertical axis) rose markedly when oxalate was added – black bars so labeled. Membranes from the acid loaded rats lost their oxalate response. Another pathway, chloride formate exchange, was also abolished.
When sodium reabsorption is thus limited, calcium reabsorption is also limited, and therefore the stage is set to deliver more calcium downstream as without acidosis, and potentially more out into the urine.
This oxalate requiring transporter is SLC2a6, our friend from oxalate days. One must wonder if acid base changes affect renal oxalate handling.
Distal Convoluted Tubule (DCT)
I am skipping over the thin and thick ascending limbs of the loop of Henle. Though dear to me, there is a powerful effect of acid in the DCT that can greatly affect urine calcium excretion and this article can be only so long
We have not much spoken about the cells here, but I will tell you that they have a protein in them that can ferry calcium through the cell from the urine side to the blood side and a transporter to move calcium out of the cells into the blood.
Most importantly they have a gated transporter that controls entry of calcium into the cells, bearing the stately name TRPV5.
When the outsides of the cells are made alkaline, more of these transporters are moved into the cell membranes facing into the tubule fluid.
Transporters moved from just inside cell membranes into the membranes themselves show up here as the colored splotches in these images of single cells. The whole experiment is over 400 seconds. The pH of the medium bathing the cells was raised, and then lowered again. During the high pH period the transporter entered the membranes and could therefore increase calcium entry.
Chronic acid loading lowers the pH in blood slightly and lowers the pH in the fluid delivered to the DCT – because bicarbonate reabsorption in proximal tubule is more complete. This in turn will reduce membrane TRPV5 and therefore less calcium can enter the cells and move back into the blood.
The transporter can be put into a larger host cell than its native DCT cell so its function can be studied in isolation. When this is done, one can demonstrate that the pH outside the cell controls the entry of calcium.
In this experiment the transporter is anchored in the membrane, so the effects on calcium transport are related to the intrinsic carrying capacity of the transporter itself. As the pH is lowered (pHe, x axis) the calcium entry falls from 100% (at pH 9) to nearly 0. The solid line labeled ‘WT’ shows results for the normal transporter (wild type) whereas E522Q refers to a transporter genetically modified so as to alter the pH receptor at amino acid 522.
The pH range of interest is of course not from 9 to 4, but from about 8 to 4.5 which is the range over which the tubule fluid pH might vary. When alkali loads are
given, potassium citrate as an example, the tubule fluid pH can easily be as high as 7 or more because protons are added downstream from DCT in the collecting ducts. This could permit alkali loads to increase calcium reabsorption markedly without necessarily a corresponding increase in urine pH. WIth acid loads the fluid reaching the DCT would be more acid than normal because proximal tubule bicarbonate reabsorption is more complete via the increase in NHE3 that acid loads produce.
TRPV5 is regulated by many factors other than acid base change and therefore urine calcium itself is multiply regulated. Here is an excellent review that lists some of the important regulators. In other articles I will explore these additional regulators.
Synthesis – Clinical Recommendations
Protein is more or less the acid load to which most of us are exposed lifelong who eat a Western diet. All carnivores and omnivores like ourselves have adapted to this kind of acid load over vast expanses of evolutionary time, and the systems we have that are affected by acid loads and can respond to acid loads include the kidneys, no doubt the GI tract – that can secrete bicarbonate, for example – and bone. It is partly because diet protein imposes an acid load that potassium citrate is a useful medication. It neutralizes that acid load and in so doing lowers urine calcium losses and potentially raises urine citrate losses. Given in amounts above net acid load, it imposes an alkali load that can in principle lower urine calcium further.
As for diet protein, the correct amount is certainly that recommended by WHO: between 0.8 and 1 gm/kg body weight/day. Very high intakes are not unpopular, and if pursued will raise urine calcium and stone risk. For various reasons, some may wish or need to use such diets. Probably potassium alkali will reduce urine calcium to some extent by offsetting the acid load. The one experiment that employed protein loading and potassium alkali, however, showed that urine calcium rose anyway, so alkali cannot be relied upon to compensate for high protein diet.
Sodium and acid interact as if partly independent of each other, in that acid load raises urine calcium at any level of sodium excretion presumably by varying filtration and proximal tubule reabsorption via pathways separate from chloride exchange. That makes low sodium diet and alkali a powerful synergistic approach to stone prevention. Possibly low sodium diet will reduce the increase of urine calcium from high protein diet; this has not been tested.
Although protein can raise urine calcium, it is unclear if spontaneous variation of diet protein raises stone risk on average. In his male cohort studies, Curhan found at most a weak and variable association between diet protein intake and stone onset. There have been no convincing trials showing that low protein diet reduces stone production.
Whether alkali spares bone mineral loss from diet protein is a vexed issue because the range of acid load from food is modest and balance changes might be lost in the general variability of the measurement. One cannot therefore recommend chronic alkali for bone health or condemn meat eating as a sure path to bone disease. With time perhaps this important matter will become clearer. But, there is no obvious benefit to high sodium intakes and high sodium intake certainly did interfere with the achievement of bone mineral uptake in at least one well done study, so the use of reduced sodium diet – 65 to 100 mEq (1500 to 2300 mg) daily – is prudent and should be a national health measure.