American Society of Nephrology Renal Week
Philadelphia, November, 2002
Pathophysiology of the Stone Diseases
Moderator: Dr. John Asplin, who is an old colleague and friend of mine, as well as Dr. Coe. He is currently the Medical Director at Litholink Corporation. He is going to be talking about the Pathophysiology of Stone Formation. John.
Dr. John Asplin
I would like to thank Dr. Slatopolsky and Dr. Sprague for inviting me to speak on the Pathophysiology of Stone Disease.
In this talk I am going to be covering mainly uric acid and calcium stones, since those are the most common stone types found in the industrialized countries.
Physical Chemistry of Stone Formation
First, I want to go over some physical chemistry of stones to point out a few important things about crystal formation. First off, supersaturation, which is here on the y-axis, is a necessary chemical driving force for crystal formation. If you donít have supersaturation, then you are not going to get kidney stones. Saturation, which is here marked by the #1, is the point at which crystals in solution are in equilibrium with the salt of that crystal in solution. Crystals neither grow nor dissolve. In the under-saturated urine, crystals will dissolve. Urine is almost never under-saturated for calcium oxalate, but it is frequently under-saturated for uric acid. Supersaturation, which is in this zone, has enough chemical driving force such that crystals will grow. However, in order to form new crystals, you need a high enough supersaturation to pass what is called the upper limit of metastability. You need enough driving force to numerically add a form. So this is a form of nucleation. Without a supersaturation high enough to exceed the ULM, the upper limit of metastability, you will never get kidney stones because this is the physical property required for crystallization.
Many factors come into play when looking at kidney stone formation, but I am going to focus mainly on supersaturation because that appears to be the most important.
Here I am also obligated to mention that water intake and urine flow rates are important. Gary has made this point already. When we look at kidney stone risk factors, we shouldnít just look at excretion rates. We also need to look at concentration. And, of course, concentrations are basically the excretion rate versus the urine flow rate. So I am only going to mention water here. If you have high urine flow rates, you will reduce supersaturation. That is true for all stone types, and it is an effective therapy for stones. I am not going to make anything more out of water intake, other than low water intakes will obviously raise supersaturation and increase stone risk.
The other point I want to make about supersaturation, because supersaturation is tremendously affected by concentrations, is that the supersaturation reported by a laboratory or measured by a laboratory has significant clinical relevance to kidney stone disease. We have shown that supersaturations we measure in 24-hour urine chemistries correlate very well with the stone types that the patients actually have. So there must be some significant relationship between what we can measure in 24-hour urine chemistries and what is actually going on with the patient.
Uric Acid Stones
First off, I am going to start off with uric acid stones because they are pretty clean. They are pretty much due to high urine supersaturation with uric acid. We will come back to inhibitors and promoters when we get to calcium stones. Uric acid stones. This is a figure made by Fred Coe and I am going to borrow it. I want to make a point about uric acid stones. Uric acid supersaturation is dependent on three things: uric acid excretion rate, and urine flow rate, which theoretically determine uric acid concentration, and urine pH. And urine pH is by far the big winner. On the x-axis is total uric acid concentration in a solution.
On the y-axis is the undissociated or the protonated form of uric acid. This is what forms stones. It is the protonated form, which is insoluble or relatively insoluble. This crosshatch bar here at around 100 mg/liter on the y-axis is the solubility of undissociated uric acid in urine. These isobars are basically the solubilities, if you will, or the concentrations of undissociated and total uric acid at different urine pHs. So letís look at a couple of urine pHs. At a urine pH of 5, you can keep almost no uric acid in solution, about 200 mg/liter at most. Above that you are in a supersaturation and crystallization is quite frequent. If urine pH is 6.5, you can put 1,200 mg of uric acid in urine and it is not going to crystallize. This is basically a six-fold change by changing pH from 5 to 6.5. You can never get that kind of change in urine volumes or uric acid excretion rates.
Uric acid stone formation is a pH-mediated phenomenon. It is not really a uric acid excretion problem. I also want to point out that at this low rate, a pH of 5, normal individuals, people with uric acid excretions that are normal and urine flow rates that are normal, will be highly supersaturated with uric acid when urine pH is very low.
What do the absolute urine chemistries look like in uric acid stone formers? This kind of result has been shown by many people; Charlie Pak at Dallas published this recently. You can see uric acid excretion in uric acid stone formers in a control population. The control population happens to be a mix of calcium stone formers and normals. Fifty-six age matched people in each group. Actually the uric acid stone formers have a lower uric acid excretion. It is not the uric acid excretion rate that causes uric acid stones. And, in fact, hyperuricosuria is not a common feature in uric acid stones. The difference? The pH is off by a full unit. They have a much more acidic urine, much higher levels of undissociated uric acid, which is crystallizing and leading to uric acid stone formation. Again, it is this difference that is the problem in uric acid stone formers.
Why do they have this low urine pH? What is the problem? This has been investigated intermittently over the years. I am going to show data from Charles Pakís group in Dallas. They published this in KI last year. Other people have gotten similar results. They looked at a group of normal subjects, uric acid stone formers, mixed stone formers, mixed calcium oxalate/uric acid stone formers, and pure calcium oxalate stone formers and studied their acid excretion. I am going to focus mainly on these two groups, but I want to make a couple of points. One is that the ammonia excretion rate per 24 hours was the same in the controls and the uric acid subjects. However, the net acid excretion was much higher in the uric acid subjects. So the percent of the daily acid excretion by the kidney of ammonia was much lower in the uric acid stone formers than in the controls. Here it is about 50 percent; here it is about 75 percent. So they end up excreting a larger portion of their daily acid load as titratable acid, which therefore means that they have to excrete acid in a much lower urine pH. The controls and the mixed stone formers have intermediate values. Mixed uric acid and calcium oxalate and calcium oxalate stone formers look a lot like the controls.
Urine Chemistry with Acid Loading
In the second part of that study, they actually did acid loading studies in these patients. They gave them 50 mmol of ammonium chloride, oral load, and looked at the urine chemistries over the next four hours. All the patients dropped their urine pH from their starting point given the acid load. Of course the uric acid stone formers started with a lower urine pH and finished with a lower urine pH than the rest of the group. The controls excreted their acid load as ammonia. They increased their ammonium excretion. The change in ammonium per creatinine increased tremendously. Uric acid stone formers canít increase their ammonium excretion. They are excreting their acid load as titratable acid, even furthering lowering their urine pH. Calcium oxalate stone formers look like controls. The mixed uric acid - calcium oxalate stone formers are right in between.
To summarize the story in uric acid stone formers, it is about pH. They have a defect in ammonium excretion, they canít excrete their acid load as ammonium as well as other people can, and they have a persistently low urine pH and stone formation. There was also a study this year in KI by Kamel and others in Toronto which showed a similar result. They studied 14 uric acid stone formers; 12 of them had ammonium defects as a cause of their low urine pH.
Treatment of these folks? Very straight forward. If you raise the urine pH, their stones invariably will go away. These are the most gratifying patients to see in clinic; I always love it because I canít think of anyone who has ever really failed therapy. There is not much I can guarantee in other stone diseases, but there I always feel good.
Crystallization Inhibition in Calcium Stone Forming Men
Here we move on to calcium stones. And I want to come back to inhibitors and promoters of stone formation. So first thing I want to point out is that crystal inhibition in calcium stone formers has been studied for many years. The clinical relevance of it, however, has started to come into focus over the last few years. And these are studies that I did with Fred Coe at the University of Chicago.
They did two separate studies studying crystal inhibition in male calcium oxalate stone formers and on the next slide you will see female calcium oxalate stone formers. They are separated because there are clear gender differences in crystal inhibition capacity.
We studied all forms of crystal inhibition, crystal growth inhibition, aggregation inhibition, upper limit metastabilities and supersaturations to get an estimate of nucleation. There were 17 male calcium oxalate stone formers versus 17 age matched controls. The age matching also important; we found that crystal inhibition varies with age and you need to take that into consideration in your studies.
What did we find? Well, we found no difference between aggregation inhibition in the male stone formers and normals. But we found significant number of crystal inhibition defects otherwise. These are quantile plots. The closed circles, white dots, if you will, are the stone formers. The open circles here are the normals. In crystal growth, higher numbers are bad. That means less inhibition. In a quantile plot if the groups are the same the points will overlap. When you see one group shifted away it represents a significant difference.
These stone formers had a significantly elevated, or significantly worse, crystal growth inhibition than the normals. It was p= 0.003. So as a group they have poor growth inhibition. Our estimates of the nucleation inhibition for calcium phosphate and calcium oxalate are done by looking at the difference between the upper limit of metastability of a urine sample versus a supersaturation. As those two numbers come close together, you are getting closer and closer to spontaneous nucleation. A good urine will have great separations, so higher numbers, between the ULM and the supersaturation. You can see that for calcium phosphate there is significant downshift which is a bad direction for the stone formers. Again, this is a significant difference, p= .002.
For calcium oxalate there was not a statistically significant difference as there is a lot of overlap here at the high end. At the low end there appears to be a subset of people who have inhibition defects.
So, what can we say about this other than there are a lot of inhibition defects in male calcium oxalate stone formers? These stone formers were not chosen for any particular metabolic subtype. All it required were to be recurrent stone formers. So there clearly is a role that inhibitors play in stone disease.
Crystallization Inhibition of Calcium Stone Forming Women
For women, same kind of study. Seventeen age-matched women. Calcium oxalate crystal growth. As opposed to men, there is no calcium oxalate crystal growth inhibition. Points overlap completely. For calcium phosphate nucleation, women again have a poor calcium phosphate nucleation inhibition just like the men. Calcium oxalate story? Again, just like the men. There is not a statistically significant difference but there appears to be a small subset who do have a calcium oxalate inhibition defect.
Now, you may wonder, well if these are calcium oxalate stone formers, what is the story with the calcium phosphate? Why is that abnormal in both the men and women? Well if you think about it, most calcium oxalate stones have a small nidus of calcium phosphate in the middle of it. And it may well be that the nucleation of calcium phosphate is an important step which allows, then, overgrowth of calcium oxalate on that calcium phosphate nidus. They do not have direct proof of that, but it is important to recognize that calcium phosphate is often at the nucleus of a calcium oxalate stone.
So, now that we know a lot, we and others have shown these crystal inhibition defects in calcium oxalate stone formers, what is the clinical role in this? Well, first of all there are a lot of inhibitors that can be described. In fact this list can keep getting longer and longer; I havenít bothered to update the slide. There are all these protein crystal inhibitors, glycosaminoglycans, and there are even these low molecular weight inhibitors such as citrate and pyrophosphate.
One other problem we have in putting this crystal inhibition story into a clinical realm is that there are so many inhibitors we do not know which ones are most important. It would be nice to be able to say, ďuropontin is the one that is importantĒ and we measure it or we look for defects in it or mutations in uropontin, but we canít say that. The system seems so redundant with so many inhibitors all working together that trying to pick out which one is important, or do they all become abnormal at once, is still well beyond us.
So, in the day-to-day clinical practice of kidney stone work at this point, there is not a role for inhibitor measurements or inhibitors as therapy. But hopefully as we progress in research, this kind of work will play a part in day-to-day management of kidney stone patients.
Causes of Calcium Nephrolithiasis
Now to move ahead with calcium stone disease, I want to point out there are basically four major causes of calcium nephrolithiasis. Again, I am excluding low fluid intake and low urine flow rates as a risk factor. These were already mentioned by Gary Curhan. Hypercalciuria, hyperoxaluria, hypocitraturia, and hyperuricosuria. I am going to focus on hypercalciuria and just on idiopathic hypercalciuria. I am going to ignore hyperparathyroidism.
What is hypercalciuria? Idiopathic hypercalciuria is hypercalciuria with normocalcemia in the absence of other mineral disorders known to cause hypercalciuria. So if you have hyperparathyroidism, sarcoidosis, et cetera, you generally do not fall into the idiopathic group. I agree with Gary that the cut point for hypercalciuria is somewhat arbitrary and again I agree with him that calcium and all other urine chemistries must be considered as continuous variables without true cut points.
Generally most studies have found that about 50% of calcium stone formers have whatever that study uses as its definition, hypercalciuria. It is a familial trait. If you look at family members of patients with idiopathic hypercalciuria, about 50% of them will also have hypercalciuria. It is tremendously influenced by dietary factors, as Gary has suggested. And then finally I also agree with Gary that hypercalciuria is a systemic disorder. It is not just about over-absorption or renal disease or whatever. It involves many organs, and probably in most people it involves all three of these.
Idiopathic Hypercalciuria and Calcium Balance
This is my favorite slide; I cannot take credit for it. This is something that Fred Coe and Murray Favus put together a number of years ago. It has been in the stone chapter in Brenner and Rector for years. But I think it is the most important slide about idiopathic hypercalciuria, but for some reason people ignore it.
What Fred did is he went and into the literature and pulled out all the calcium balance studies in IH. On the x-axis is net calcium absorption. This is not calcium intake; it is absorption. On the y-axis, urine calcium excretion. Remember, these are balance studies. Here is the mean of the group of normal, non-IH patients. And the two bars are rounded at the 95% confidence intervals. The dashed line is the line of identity. This is about balance studies. If you are to the left or above the line of identity, you are in negative calcium balance. If you are to the right of it, you are in positive calcium balance.
I want to make one major point. At around a calcium absorption of 400mg/day if you are below that and you are an idiopathic hypercalciuric subject, you are in negative calcium balance. Now think about that. Because the low calcium diet that has been used for years for treatment of hypercalciuria is about 400mg/day. And no one absorbs all the calcium in their diet. So when you put a patient with hypercalciuria on a low calcium diet, invariably they will go into negative calcium balance. This means that the vast majority of patients, these are not really selected patients, these are just people with high urine calcium, no subtype in particular; what this means is that the majority of people put on a low calcium diet will go with a negative calcium balance. And it canít be that some significant portion of people just have hyperabsorption as a cause of their hypercalciuria. If you only had hyperabsorption of calcium from the gut, you would not ever go into negative calcium balance.
Bone Mineral Density in Kidney Stone Formers
All right, a little bit more about systemic nature of the disease. This is work from Charles Pak about bone mineral density in kidney stone formers. There have been a lot of studies looking at bone mineral density in kidney stone formers and most of them show low bone mineral density in patients who have idiopathic hypercalciuria. I choose this study because Charles Pak is the one who initiated the categorization of IH into absorptive and then fasting in an attempt to better understand this pathophysiology and its treatment.
With this study from 1992, he looked at normocalciuric stone formers versus two groups of idiopathic hypercalciurics: those with so-called absorptive and those with fasting hypercalciuria. You will note that the radial bone mineral density in all groups is the same. The vertebral bone mineral density in both fasting and absorptive hypercalciuric group is statistically significantly lower. Itís an important finding. Even in so-called absorptive. They canít be just pure absorptive hypercalciuria. If they were, they would not have low bone mineral density. It involves more. They have some sort of bone resorption problem.
And in fact, in follow up of this, Pakís group has been doing genetic studies and have found a link to chromosome 1 which suggests that these people, these so-called absorptive hypercalciurics, have a high risk of low bone mineral density if they have mutations in a gene on chromosome 1. So again I want to stress the systemic nature of this disorder. It involves kidney, bone, and intestines.
Vitamin D Levels
So what mechanisms could be the cause of hypercalciuria? Well one attractive theory has been that this abnormality is in the vitamin D axis. Vitamin D or calcitriol as you know, increases intestinal calcium absorption and would certainly explain the invariable intestinal hyperabsorption of calcium found in idiopathic hypercalciuria. Work from Jack Lemmon has shown that if you take normal subjects, put them on a little bit of calcitriol and then restrict their dietary calcium, they go into negative calcium balance through bone absorption.
So if you look at 1-25 D levels in normal subjects and patients with idiopathic hypercalciuria, you will see a trend to higher calcitriol levels in the IH patients. Now, there is a lot of overlap here. It certainly canít explain all the hypercalciuria in all the subjects. But generally the trend again points to an abnormality in the vitamin D system.
For subjects with normal calcitriol levels, and all these studies do have some patients with normal calcitriol levels, another attractive hypothesis has been that there might be an abnormality in the vitamin D receptor. David Bushinsky has shown that in a genetic hypercalciuric rat, vitamin D receptor is present in higher amounts in bone, kidney, and intestine. This might explain the intestinal hyperabsorption and bone absorption in subjects with normal calcitriol levels.
Mechanisms for Idiopathic Hypercalciuria
However, there are many possible mechanisms for idiopathic hypercalciuria and I put down some candidate genes and in fact there are many more genes than this that people have looked at. And so far, there has been no dominant gene found to be abnormal. At the top of the list is the sodium phosphate co-transporter that was recently reported in The New England Journal of Medicine. They found two patients with mutations in this transporter that seemed to make a functionally abnormal co-transporter. However, they only found this mutation in two out of twenty subjects and all the subjects were hypophosphatemic. It would only make up a small portion of total number of subjects with idiopathic hypercalciuria.
Chloride channel defects have been found in Dentís Disease and Bartterís Disease to cause hypercalciuria, so they are not mistaken for IH. Vitamin D receptor is an attractive hypothesis, as is 1-alpha hydroxylase. Initial study of this was negative. The vitamin D receptor - there have been mixed results so far, some showing polymorphisms in the gene; others not finding anything. But there are many possible genes and probably many different pathophysiologies that can to lead to idiopathic hypercalciuria.
Dietary Effects: Sodium
As I mentioned before, in addition to the genetic and physiologic basis for hypercalciuria, there is some strong interplay with diet. And I want to highlight both sodium and protein. Here we have a group of studies in which investigators looked at the effect of dietary sodium, which is reflected here by urine sodium, on urine calcium excretion. The studies are done by fixing calcium intake, protein intake, and basically just varying sodium intake in diet. And you can see that of the high sodium diets, urine calcium is higher. And when they switch patients to low sodium, all the different studies use different amounts of sodium in their studies - and different amounts of calcium, actually. But for any individual study, there is a decrement in calcium excretion.
Now, in these white bars down here, these are all normal subjects, not hypercalciuric subjects. And they all had just modest decreases. The two yellow lines up here were both hypercalciuric subjects. And you can see they actually had a significant drop in the urine calcium. So, sodium plays an important role in calcium excretion and the higher calcium certainly will promote higher supersaturation and stone formation.
Dietary Effects: Protein
Similar results can be seen with dietary protein intake. These are group studies; these are all in normal subjects. None of these are in idiopathic hypercalciuria. They are basically young, healthy men studied on low protein diets and also high protein diets with fixed calcium and sodium intake. And you can see there is actually a dramatic effect by putting a patient on a low protein diet and then comparing it to a high protein diet, with many of these so-called normal subjects becoming frankly hypercalciuric.
Now, you may look at this and say, ďNow, these are ridiculously high amounts of protein intake.Ē But I would suggest to you that if you look at enough stone formers, you will find that many of them have ridiculously high amounts of protein intake and you will find many stone formers actually live out in this range of protein intake per day.
Causes of Hyperoxaluria
So I move on from hypercalciuria on to hyperoxaluria. There are many causes of hyperoxaluria: dietary, the enteric which was hinted at earlier, and then the genetic basis for hyperoxaluria. I am going to focus only on the dietary. These two cause severe hyperoxaluria, with oxalates generally over 100mg/day. The dietary is mild to modest hyperoxaluria, but certainly much more common than the severe hyperoxaluria.
Dietary and Urinary Oxalate Excretion
For years it has been thought and been said that oxalate in the urine was 90% endogenous and 10% diet, and that had been in textbooks for years and years. And that has been challenged by me and I will tell you anecdotally from clinical experience, I have never believed that only 10% was from diet, and this work done by Rodd Holmes I think is quite important work.
He studied 12 normal subjects. The 12 subjects were put on three levels of oxalate intake. He used a synthetic oxalate-free diet, and then two so-called solid food diets which are normal diets in which the oxalate was actually measured; this one being a 50 mg/day oxalate diet and this one being a 250 mg/day oxalate diet. That was basically their range, but they are both within the range that an average person would eat. On the oxalate-free diet, urine oxalate excretion was completely due to endogenous production and was about 10 mg/g creatinine. Nice low oxalate excretion.
As they increased oxalate content in the diet, they got almost a doubling of the urine oxalate by the time they got onto the high oxalate diet, showing that dietary intake of oxalate contributes importantly to urine oxalate excretion. But what they havenít been able to show so far is whether you can affect changes in the population not in a clinical study. But certainly I believe that this data supports strongly that dietary oxalate plays a big role in the urine oxalate excretion.
Now as mentioned before by Gary and Fred, it is not only the content of oxalate in the diet that is important, it is also the bioavailability of the oxalate. The cation content of the diet, mainly calcium and magnesium, greatly influence the availability of oxalate absorbed from the gut. and oxalate and magnesium oxalate complexes are absorbed as is free oxalate from the gut.
Effect of Low Calcium Diet on Urine Oxalate
And so, I put together a compilation of studies looking at the change in urinary oxalate and urinary calcium when changing diet. Each of these studies included a group of patients studied on a so-called normal calcium diet and then studied again on a low calcium diet. The y-axis is changing urine excretion of either calcium or oxalate as a percent. And the baseline is presumed to be the normal calcium diet and then the result is some change after the low calcium diet.
As you can see, as expected, calcium falls after you lower dietary calcium, urine calcium will fall. That is absolutely true. In these first four studies, they all found a significant increase in urine oxalate when they lowered the calcium. So you can see when they lower dietary calcium.
This study at the end which is again from Charles Pakís group showing significant decrease in calcium, but no change in oxalate.
So, there are mixed results here. But I do want to highlight mainly the two studies by Holmes and Marshall. The N's here stand for normal subjects, SF for stone formers, HA and NA stand for hyper-absorbers or calcium and normal absorbers of calcium. But I do want to highlight the Holmes and Marshall studies because those are the best performed. They put the patients on a fixed diet and the only thing they varied was the calcium content. The diet was invariable as far as oxalate goes and they did see a difference.
Messa and Galosy, however, just recommended the patients avoid high oxalate foods. There was not strict control of the oxalate content, which gives more variability to the results.
Now, what is the clinical impact of this? It is unclear to me. And I strongly believe that this is a real phenomenon, that low calcium diets do cause hyperoxaluria but I do not have a good feel, because of this kind of mixed data over on this side, for what the effect is in real clinical practice.
Now in addition to the oxalate in the diet, I want to remind you that oxalate is a metabolic end product. And as such, it has many precursors. And one thing that we often ignore about dietary issues is that what happens if you eat a lot of oxalate precursors? And I want to point out that many of these precursors are amino acids.
Urinary Oxalate Excretion
Here is a study that was in KI a year ago that looked at this phenomenon. There is a group from Switzerland that did a study in which they compared normal non-stone formers with two groups of stone formers. Eight kidney stone formers who had normal oxalate excretion and twelve kidney stone formers who had what they called mild metabolic hyperoxaluria. All that meant was that on a random diet they had mildly high oxalate excretion rates. And they put these subjects on fixed diets with fixed calcium and oxalate content and then changed their protein intake from 80g a day to 190g a day. And you could see the controls, the non-hyperoxaluric controls, did not change their oxalate excretion. The non-hyperoxaluric kidney stone formers did not change their oxalate excretion. Those who had hyperoxaluria going into the study increased their oxalate excretion significantly in response to a protein load. That protein load doesnít come with any oxalate.
Now, I do want to point out that there is tremendous individual variation. You look at a subject like this who more than doubles his oxalate and there are some people who actually go down. As a group they definitely went up. What I am more impressed by myself is by the large individual variation and I suspect that there are people who are just prone to hyperoxaluria, basically from protein loading, and perhaps this happens with other precursors as well as such as glycolate and vitamin C. But there is a lot of individual variation and the importance of this gets lost in large group studies.
One last point that I want to make about oxalate, and this is an area that has gotten a lot of play in the kidney stone research world, is the role of oxalate degrading-bacteria. Oxalobacter formigines is the most well-studied of the oxalate-degrading bacteria. It is part of the normal colonic flora; it is usually acquired in the first year of life. It uses oxalate as a carbon source so it requires oxalate for growth. And it has been questioned what is the role of this bacteria in stone formation? Basically, does the loss of O. formigines lead to hyperoxaluria? Only about 70% of adults remain colonized throughout their lives. And it has been shown that recurrent stone formers have lower colonization rates with O. formigines.
One of the problems with all these studies, however, is that they have never gotten down to the cause and effect. It is certainly true that recurrent stone formers have lower colonization rates. It is also likely that recurrent stone formers have a lot of urologic procedures, receive a lot of antibiotics and it is not clear if the loss of O. formigines is the cause of stone disease rather than the effect of having stones. There are no prospective studies right now that have been done yet on the colonization and stone formation risk, or even hyperoxaluria from O. formigines.
However, even though I am personally not clear on the role of its pathophysiology relating to stones, there is some interesting therapeutic potential. Oxalobacter formigines has been used as a probiotic, meaning it has been given daily to rats receiving oxalate in their diet. And it has been shown to reduce oxalate excretion, so that is a promising start for these bacteria.
And there was a study in Kidney International, I think in the last year, of six stone formers with hyperoxaluria who were treated with lactobacillus, another probiotic. The lactobacillus used actually could degrade oxalate as well. And they showed a reduction a reduction in oxalate excretion of about 40%. So there might be some therapeutic potential even if there is not a role in pathophysiology.
Citrate and Kidney Stones
We will move on to citrate in kidney stones. Unlike calcium and oxalate, which are excreted in excessive amounts, citrate is protective, and hypocitraturia is a cause of kidney stones. I put the normal range up here because I wanted to make a point that Gary did that there is not a pure cut point. People with 324 mg of citrate are not worse off than people with 325. It is a graded variable. In our lab, we find that women have higher citrate levels than men. The other reason I put this number up is that they will see some labs who report a normal range as low as 140 mg/day. I canít tell you how many patients I have had referred to me in clinic with no metabolic abnormalities who have gross hypocitraturia that the laboratory is reporting in the wrong normal range.
Low urine citrates can be idiopathic. They can be due to metabolic acidosis or a variety of causes. We often see it in distal renal tubular acidosis. When you see a stone former with the combination of very low urine citrates and a high urine pH, certainly consider distal RTA.
And I also want to make a point that hypokalemia causes hypocitraturia. This is important in all those patients who get put on thiazide diuretics for their hypercalciuria. On thiazides if patients develop hypokalemia, they may well give off low urine citrates and basically as you treat one stone risk you are causing another.
I also want to make an anecdotal comment that I have seen patients who have potassiums fall but stay in the normal range where citrates fall. They start off with a potassium of 4.8 and it falls to 3.8. That might be, in that patient, enough to cause the citrate to drop.
Effects of Citrate on Calcium Oxalate Supersaturation(SS)
How does citrate work? A couple of issues. First of all, citrate complexes calcium, as I am sure you are all well aware. This is a slide that I made. On the y-axis, we have calcium oxalate supersaturation calculated from the computer program ďEquilĒ. On the x-axis, we have citrate concentration in mmol/L. These are all reasonable citrate concentrations that can easily be obtained in urine.
Basically this is just a computer experiment in which I fixed everything for supersaturation and only varied citrate. And as you can see, as citrate concentration increases, calcium oxalate supersaturation decreases. Simple as that, citrate will complex calcium, reduce ionized calcium levels in the urine, and therefore reduce the calcium oxalate driving force to crystallization.
Calcium Oxalate Aggregation Inhibition by Citrate
How else does citrate work? Citrate seems to be a direct inhibitor of crystallization. This is a study by Tiselius in which he studies calcium oxalate aggregation inhibition by citrate. This is an in vitro study done in aqueous solutions. And what he does is he takes calcium oxalate crystals, disburses them by stirring and stops the stirring and watches the crystals fall. He watches the crystals fall by measuring absorbance. As the crystals fall out of solution, the absorbance declines. Crystals that aggregate fall faster, so to prevent aggregation you have got a slower decline. And when there is no citrate present, you get a very brisk fall and absorbency for higher levels of aggregation. Again, at citrate levels that can be found in human urine you reduce the aggregation of the calcium oxalate crystals. So, citrate has not only complexation of calcium effects, it also has direct inhibition effects.
Effect of Citrate on Tamm-Horsfall Protein(THP) Excretion
It also has been shown that citrate can modify the effectiveness of macromolecular inhibitors. Here, this is a study by a group at Ochsner clinic. They looked at the effect of citrate on Tamm-Horsfall protein excretion. THP protein is an inhibitor of calcium oxalate crystal aggregation. They studied 33 recovered calcium stone formers. They got 24-hour urines before and after citrate therapy.
And here are all the important values that they measured: As was expected, urine pH increases during alkali therapy. Citrate excretion again increases during alkali therapy. The surprising result was Tamm-Horsfall protein doubled when patients were given citrate. Now, I donít know and they did not have an answer either whether THP production increased or whether the alkalinization and treatment with citrate just reduced Tamm-Horsfall protein polymerization and gel formation, basically preventing THP protein from making casts. I suspect it is the latter; that they basically kept THP protein soluble in solution. In so doing, they keep it a potent aggregation inhibitor active in the urine. And you can see aggregation, which they call TM; inhibition is greatly improved by citrate therapy. And they showed it was the THP protein by isolating the macromolecular phase of the urine from the low molecular weight solutes and showing that it was really the THP protein that was making a big difference in these urine samples. So, citrate has many effects. It is clearly an effective therapy for kidney stone formers and also a deficiency in citrate puts you at risk for kidney stones for many reasons.
Role of Uric Acid in Calcium Oxalate Stones
Now I would just like to finish up with the role of uric acid in calcium oxalate stones. I started off with uric acid stones at the beginning of the talk, but this is about uric acid causing calcium oxalate stones. About 20-35% of stone formers have hyperuricosuria. It promotes calcium oxalate crystallization by a salting out effect. The normal range I put up here is what you see in studies. Why do I put this up here? Because Gary is right, these cut points are somewhat arbitrary and a lot of normal people would be above this because these are the cut points used in studies. And if you want to get results like the studies get, you have to look at those cut points they use.
Effects of a low Purine Diet
Why do we have hyperuricosuria? Well, it is basically a mix from the routine calcium oxalate hyperuricosuric stone former; it is a mix of metabolic and dietary issues. This is work Fred did 30 years ago in which he studied ten hyperuricosuric calcium oxalate stone formers and compared them to five normal subjects. They are all male; they are the same age range, socioeconomic status, and weight. So they are reasonably well matched.
And what you see is that, for the normal population and the hyperuricosuric population, when you put them on a fixed, low-purine diet, hyperuricosuric patients still had a high uric acid excretion. This is due to a subset of four of those ten who actually had above normal purine excretion; they probably have some kind of mild ill-defined metabolic defect that leads to high uric acid excretion rates.
To me, the more impressive part of the study has always been the second part, which is when the patients were sent home. Have them take a diet history and measure their uric acid excretion on a normal outpatient diet. And you see that both groups increased their uric acid excretion and their purine intake when they are left to their own devices. The stone formers, however, greatly increased their purine intake. I'd like to point out that the calorie intake of both these groups was exactly the same. What is important is that these people are eating meat all day. They give themselves an incredible purine level and greatly increase in their uric acid excretion. So the hyperuricosuria seen in these kidney stone formers is not only somewhat metabolic, it is tremendously dietary. And when you see a patient like this, this is what you really need to think about because with dietary loading with purine it is almost invariably protein. As I pointed out protein causes hypercalciuria, hyperuricosuria, and a lower urine pH and also a lower urine citrate. About everything that can go wrong in a kidney stone former can be protein mediated.
So in summary, what I have tried to cover today is the importance of supersaturation in kidney stone formation and the treatment of all these diseases, which are based on treating supersaturation. And trying to affect inhibitors when you can will be reviewed by Fred. So, I am going to stay away from treatment and let him do that. Thank you.
1. Pak CY, Sakhaee K, Peterson RD, Poindexter JR, Frawley WH.. Biochemical profile of idiopathic uric acid nephrolithiasis.Pak CK et al. Kidney Int 2001 Aug;60(2):757-61.
2. Sakhaee K, Adams-Huet B, Moe OW, Pak CY. Pathophysiologic basis for normouricosuric uric acid nephrolithiasis. Sakhaee et al. Kidney Int 2002 Sep;62(3):971-9.
3. Pietschmann F, Breslau NA, Pak CY. Reduced vertebral bone density in hypercalciuric nephrolithiasis. Sakhaee et al. Kidney Int 2002 Sep;62(3):971-9.
4. Holmes RP, Goodman HO, Assimos DG. Contribution of dietary oxalate to urinary oxalate excretion. Pietschmann et al. J Bone Miner Res 1992 Dec;7(12):1383-8.
5. Nguyen QV, Kalin A, Drouve U, Casez JP, Jaeger P. Sensitivity to meat protein intake and hyperoxaluria in idiopathic calcium stone formers. Holmes et al. Kidney Int 2001 Jan;59(1):270-6.
6. Tiselius HG, Fornander AM, Nilsson MA. The effects of citrate and urine on calcium oxalate crystal aggregation. Nguyen et al. Kidney Int 2001 Jun;59(6):2273-81.
7. Fuselier HA, Ward DM, Lindberg JS, Allen JM, Husserl FE, Marcucci PA, Cole FE, Turnipseed J, Alam . Urinary Tamm-Horsfall protein increased after potassium citrate therapy in calcium stone formers. Fuselier et al. Urology 1995 Jun;45(6):942-6.
8. Coe FL, Kavalach AG. Hypercalciuria and hyperuricosuria in patients with calcium nephrolithiasis. Coe et al. N Engl J Med 1974 Dec 19;291(25):1344-50.