Dr. Robert Narins
This morning we move from the monovalent ions to the divalents. Dr. Sharon Moe is at Indiana University. As of July she'll be an Associate Professor of Medicine. She's going to start off this morning with "Calcium and Phosphorus -- Key Physiologic Principles". One commercial note: The Genzyme Corporation has sponsored a portion of the Divalent Ion Metabolism section with an educational grant, and I told them that I would mention that. Sharon.

Dr. Sharon Moe

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Introductory remarks
The first hour is going to be on calcium and phosphorus physiology. I think this is one area that is often missed in a lot of fellowship programs. It is often considered an area of endocrinology. And so what I am going to try to do is focus on the physiology as it relates to taking care of patients with renal problems. Then at the end, I am going to take that physiology and put it in the context of dialysis patients and what we might be thinking a little bit differently.

We have had a lot of changes in our philosophy of calcium homeostasis in our dialysis patients in the last really three to four years. Some of that will be expanded on in the workshop in the morning, and some of it will be expanded on in the renal osteodystrophy discussion tomorrow, as well.

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Overview
What we are going to discuss this morning in the first hour is calcium and phosphorus physiology. I am going to go through the concepts of balance and measurement; the different regulators of this, which simply is parathyroid hormone and vitamin D; and then the target organs-- intestine, kidney, and bone. And as I said, I will try to bring it all together for you by going over the same physiology in the absence of kidneys.

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Calcium stores
The vast majority of calcium, 99 percent of the total body calcium, is stored in bone. Only 1 percent of that calcium is outside of bone, and of that only .1 percent is in the extracellular space. Yet that is what we measure when we try to decide what to do with patients and their calcium physiology. When you do measure that, you are measuring generally a total calcium level; but of that, 50 percent is in the ionized component, and that is what is physiologically relevant. Another 10 percent is complexed to either bicarbonate. citrate or other anions; and then about 40 percent is bound to albumin.

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Calcium intake, excretion, and obligatory loss
The average American diet takes in about 900 mg/day, which is below the recommended dietary allowance. Of this, the majority, two- thirds, is excreted in stool. There is an obligatory secretion of calcium so that if you take in no calcium, you go into negative balance because you secrete about 200 mg/day from the intestine. In a normal steady state situation, this fluxes back and forth to maintain the extracellular concentration. They kidney only puts out about a third or even a quarter of the total dietary calcium intake. Remember that when you are in balance, what goes in must come out. In most situations, people are in balance.

As I said, we do a very poor job in this country getting enough calcium, and it has created an epidemic of osteoporosis. In fact, recent studies have shown that of kids under the age of 20, only 30 percent of kids are getting in the amount of calcium that they need to be getting in. So we are probably only going to see a worsening of that epidemic.

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Calcium content of foods
Foods that are high in calcium include basically diary products, and that is about it unless you are really into canned sardines as a healthy snack.

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Phosphorus stores
Phosphorus--about 85 percent of the total body phosphorus is in bone. The rest of it is intracellular. But again, only 1 percent of the total body phosphorus is in the extracellular space--what we measure and how we determine what to do with patients.

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Phosphorus in the serum
Fortunately, though, when we measure serum phosphorus, we are measuring the free or the inorganic component so that you don't have to do any calculations.

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Intake and excretion of phosphorus
In contrast to the low calcium intake, most of us, particularly in the state of Indiana, do very well with the dietary phosphorus intake because it is generally in dairy products and all meats. And of this, the kidney excretes at least two-thirds of the daily intake of phosphorus. That is why it is such a problem in renal failure.

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Phosphorus content of foods
Foods that contain high phosphorus, not only the dairy products but all meats, fish included, cereals, brans, wheat-based, and nuts. Pretty much everything. Vegetables--beans and some greens and that are in the medium range. So, again, most foods contain phosphorus. I would venture to say that probably all of you had a high phosphorus diet last night, not to mention high sodium. The important thing is that to dietary restrict this is very, very difficult. I know I would have a very hard time with it.

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Secretion and metabolism of parathyroid hormone
The parathyroid gland produces one of the two regulator hormones of calcium and phosphorus homeostasis. The parathyroid hormone is secreted as a pre-pro PTH. It is then converted into a pro- parathyroid hormone, and then converted into... or cleaved before it is even secreted into the intact PTH, which is an 84-amino acid, and then some extra carboxyl fragments and amino fragments.

In this day and age there is absolutely no reason to really ever measure an N-terminal PTH, a mid-region or a C-terminal PTH. Really what you need to measure is just the intact assay. The intact assay is a double immunoradiometric assay, in which case one antibody binds to this part, one antibody binds to this part, giving you the intact molecule. But it is a little bit of a cheat because it really only is amino acids 7 to 84 in the current intact assays. There is a new assay, which I will talk about tomorrow, that truly measures the 1 to 84, which is going to just make our lives even more complicated than they are.

The half-life of PTH is very short, on the order of minutes. The N- terminal component is excreted by the kidneys. Otherwise the PTH is primarily hepatically metabolized.

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Regulation of PTH secretion
Now the PTH is stored in secretory granules such that any minute changes in the calcium are sensed at the calcium sensor and PTH is then released within seconds of changes in ionized calcium. In addition, regulation occurs at the level of transcription, and this is by primarily by calcitriol, 1,25 dihydroxy vitamin D3--calcium also controls this, and then also in the form of replication, in which case again the same regulators. Phosphorus also regulates probably at both these levels, but most likely post-translationally.

PTH is maximally suppressed with respect to calcium. So most of us sitting in this room have a fairly maximally suppressed PTH. The PTH and calcium relationship is such that, when your calcium drops the PTH goes up. The mechanism includes, again, changes in PTH transcription, inhibition of exocytosis, modulation of intracellular degradation, and activation of various signaling cascades via this newly described, newly cloned calcium sensor.

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Source: Brown EM. J Clin Endocrinol Metab. 1983 Mar;56(3):572-81.

Calcium - PTH sensitivity curve
This is the curve that traditionally describes the relationship between calcium and PTH. As your ionized calcium drops, your PTH secretion increases. This curve is sigmoidal in shape and it was mathematically modeled by Ed Brown back in 1983. This mathematical modeling has led to at least 100 different papers, if not more, in the literature looking at various aspects of things that affect this curve.

Some of the parameters that have been assessed, particularly in renal failure patients, are the minimum PTH, which theoretically is the size of the gland and the basal secretion; the maximum PTH, which is the ability to change that; the slope of the curve; and then a concept called the set point, which is marked off here as C. And that is generally described as the level of calcium at which you get 50 percent maximal inhibition of PTH. It seems like a lot of work over a simple curve, but this is how we have been trying to understand the pathophysiology of this disease.

Now something comes to mind here when you look at the shape of this curve, two points. One is that at normal calciums, you are pretty much maximally suppressed with your PTH. It is only when you drop your calcium that you get the rapid release in parathyroid hormone. And for years this rapid release had been hypothesized to be some sort of a calcium sensor. In the actual mid-90s, it was cloned and sequenced by Ed Brown and Steve Hebert and has lead already to some drugs which are in Phase II trials to control or suppress PTH release.

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Source: Brown EM et al, N Engl J Med. 1995 Jul 27;333(4):234-40. Review. No abstract available.

Calcium-sensing receptor: Structure
This is the shape of the calcium sensing receptor. It is a 7- spanning membrane protein G-coupled receptor, similar to a lot of them. Many different mutations, marked here with the little x's, have been identified already in several kindreds with diseases such as familial hypercalcemic hypocalciuria, which we will talk about, as well as hypoparathyroidism.

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Calcium-sensing receptor: Characteristics
This receptor has been localized in parathyroid glands predominantly; in the kidney, predominantly in the thick ascending limb; in brain--we still don't know what it does there; thyroid C cells, where it helps turn on calcitonin; osteoclasts; maybe osteoblasts, but it might be a different sensor; and placenta, where it is very helpful in controlling neonatal calcium levels, fetal calcium levels.

Inactivating mutations are found to result in familiar hypocalciuric hypercalcemia. What that means is that the curve is shifted to the right in that disease, such that those patients walk around with higher levels of calcium and a suppressed PTH.

Then there is also a neonatal severe hyperparathyroidism, which is also now known to be associated with defects in this receptor. A lot of diseases that have been identified we now have a molecular basis for.

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Calcium-sensing receptor regulation and dysregulation
Autoantibodies have been found in one variant of hypoparathyroidism. The activating mutation has been identified in patients who used to be called autosomal dominant hypocalcemia. As was mentioned, it can be inhibited with calcimimetics. So these are compounds that bind to the calcium-sensing receptor and fool the gland into thinking that the calcium levels are high, such that PTH is shut off. It activates the IP₃ / DAG cell signaling, and it is at least in part regulated by calcitriol, and most impressively by recent data it is highly regulated by serum phosphorus.

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Vitamin D effects on PTH
How does vitamin D or calcitriol inhibit PTH secretion? Well, there are high-affinity receptors, VDRs or vitamin D receptors, on the parathyroid cells. Calcitriol, in contrast to calcium, is more important in terms of suppressing PTH. So calcium is important in stimulating PTH release; calcitriol is more potent in suppressing PTH release. I am sure you can already imagine how that relates to your renal failure patients.

The mechanism is binding on the vitamin D response element on the PTH gene. In addition, it changes cytosolic calcium concentration and also increases its own receptor abundance.

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Phosphorus effects on PTH
As I mentioned, phosphorus also has been found recently, really in the last ten years, to be a direct stimulant of PTH synthesis. This is a major issue for our dialysis patients. We have always known that it indirectly stimulates PTH secretion by lowering ionized calcium and inhibiting the 1-alpha-hydroxylase enzyme, thereby inhibiting the conversion of (25 to 1,25-D). And it used to be thought that these were the only mechanisms that were involved. But then there were some in vivo studies and in vitro studies that suggested direct effects. This is kind of hard to work out in a patient because it is hard to separate out all of these systems. The mechanism is still controversial, but clearly both animal and human data now show a direct effect (of phosphorus in PTH secretion).

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Source: Naveh-Many T et al, Endocrinology. 1989 Jul;125(1):275-80.

Calcium, phosphorus, and cacitriol effects on PTH
What is more important: Calcium, phosphorus, or calcitriol? What really gives you the most important regulation of PTH? This is in rats, but it is a nice representative slide. Here is the serum calcium. Here is the PTH mRNA in the hatched boxes. This is a control basal levels in a rat. So the basal level of calcium is around 11.5; and here is basal PTH.

When you give the animal calcium, your calcium goes up, but your PTH doesn't drop any further because you are maximally suppressed in normal baseline. You give the animal phosphorus, PTH shoots up. But in part the calcium drops as well, in part because your phosphorus elevation changes your ionized component of calcium. So it is kind of hard to separate out which is the main effect here.

When you give the animal calcitriol, the calcium is only partially increased, but you maximally inhibit PTH secretion. So it is much more potent than if you gave somebody calcium. This is true even when you treat your dialysis patients, as well. Now if you give both phosphorus and calcitriol, the increase in the phosphorus blunts the ability of calcitriol to suppress PTH--again, very physiologically relevant.

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Source: Almaden Y et al, J Am Soc Nephrol. 1998 Oct;9(10):1845-52.

Calcium and phosphorus effects on human parathyroids in vitro
It is nice to know that phosphorus has an effect in rats, but we really like to see data in humans. And as I said, this is quite difficult because you can't really separate out and do parathyroidectomies on purpose in humans. The IRBs don't really like that. But in 1998, Almaden took parathyroid tissue from patients who had the PTH gland removed. The incubated them in Petri dishes. When the put the tissue with high phosphorus and normal calcium, they increased PTH secretion. When you put the tissue in normal phosphorus but high calcium, you decreased PTH secretion. But when you had high phosphorus and high calcium, you increased PTH secretion. Phosphorus won; more important than calcium in terms of stimulating PTH. Hyperphosphatemia alters Ca-PTH relationship
This just demonstrates this in a graphical form. As you increase your calcium levels, the change in the PTH. This is normal, and this is in patients with hyperparathyroidism. So what happens in the presence of hyperphosphatemia, you blunt the ability of an increase in calcium to lower PTH. So that means in your dialysis patient who walks around with a phosphorus of 7 or 8, increasing his calcium or her calcium to 10, 11, 12, isn't going to do anything in terms of suppressing PTH.

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Source: de Francisco AL et al, Kidney Int. 1998 Dec;54(6):2140-5.

Just to show you that even further, this is another study where they took patients on dialysis, during a run they increased the calcium bath and they measured the ionized calcium, which changes within minutes on dialysis. So the open circles are the normal shape of that sigmoidal curve that I showed you. Then they did the same thing again while they were simultaneously infusing intravenous phosphorus during the dialysis run. Again, I am sure my IRB wouldn't let me do this. But it is a nice study. What it showed is that you shift that curve to the right. So that phosphorus really affects the ability of the PTH gland to be affected by calcium.

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Source: de Francisco AL et al, Kidney Int. 1998 Dec;54(6):2140-5.

This just again shows you directly the minimal PTH, which is kind of the basal secretion of parathyroid gland or the ability to maximally suppress that gland. It changes in the presence of phosphorus. With high phosphorus it goes up in every patient.

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Source: Brown AJ et al, Kidney Int. 1999 Apr;55(4):1284-92.

High phosphorus down-regulates Ca-sensing receptor abundance
Now Dr. Slatopolsky did some wonderful work looking at this in rats and tying this together because now we have evidence that phosphorus directly affects the shape of that PTH/calcium curve. For years there has been a statement made that dialysis patients have an altered set point; and therefore, you need to give really, really high calcium because the curve is shifted to the right in order to shut off PTH. But it has been highly controversial. There are lots of papers where you don't see that; lots of papers where you do. I would say in the vast majority you don't see it. Many of the differences can be explained by how you do that mathematical computation.

Finally, we may have a reason for this. These are parathyroid glands from rats, treated with a normal diet; uremic rats with a high-phosphorus diet; uremic rats with a low-phosphorus diet; and I can't read that... these are non- uremic animals. And what you see is the staining, the brown speckling, is the staining of the calcium-sensing receptor. So when you take these rats and you give them high-phosphorus diets, you make big glands with very little calcium sensor. So it looks like the mechanism by which this hyperphosphatemia affects the glands' ability to respond to changes in calcium is by essentially down regulating this calcium- sensing receptor. What that means in your dialysis patient is if you have a high phosphorus, again no matter how you push the calcium, if you don't have a sensor to respond to that, you are not going to accomplish anything.

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Source: Brown AJ et al, Kidney Int. 1999 Apr;55(4):1284-92.

This is just the quantitative mRNA for the same thing, showing that it is a transcriptional regulation. Does everybody understand that concept? Just to make sure. That is something that is very important when you are taking care of your patients... to understand that.

Summary of phosphorus effects on PTH regulation
Again, just to reiterate--basal PTH secretion is suppressed in normal situations. So as you give... if you lower calcium, your PTH goes up. If you give vitamin D, it is a much more potent suppressor of parathyroid hormone than is raising the calcium further. And even if you do raise the calcium further or you give vitamin D in the presence of phosphorus, you blunt the ability of both of those to accomplish anything. So lesson #1 in dialysis patients is: Until you control the phosphorus, you are basically going to be helpless in terms of controlling hyperparathyroidism. And we will talk more about that tomorrow.

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Parathyroid hormone receptor
The parathyroid hormone receptor is again also a member of the G protein link 7-membrane spanning receptor family. It is down regulated in kidney and osteoblasts that are exposed to PTH. It is activated by just those first eight amino acids of the parathyroid gland. It stimulates the intracellular signaling by both cycling AMP and IP3 pathways. There is recently evidence for additional PTH receptors, but they are probably of minimal importance.

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Summary of PTH regulation
So if you put all of this stuff together in a dialysis patient, what we have is parathyroid overactivity. That is, in part, due to increased phosphorus; relative decrease in calcium, particularly before they get on dialysis; decreased calcium-sensing receptor; decreased vitamin D receptor, which is prominent in renal failure; we don't have enough calcitriol in our patients; and then acidosis; uremic toxins; all of these things contribute. There is a lot of work going on trying to understand this pathophysiology. Just to let you know, there are other abnormalities, even further abnormalities in renal failure, such as abnormal gene expression for various factors that are important.

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Parathyroid hormone-related peptide
Parathyroid hormone-related peptide is homologous to PTH, with respect to the first 13 amino acids and has similar biological activity. This is an absolutely critical role in developmental biology. If you knock out this receptor, the mice die. But in adults, we kind of blew it off as being important, but it is gaining a lot of increased importance because it appears that it is very prevalent in vascular endothelium and smooth muscle and is important for controlling vascular calcification probably, as well as other vascular issues-- hypertension, etc.

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Source: Juppner H and Schipani E. Curr Opin Nephrol Hypertens. 1996 Jul;5(4):300-6. Review.

Actions of PTH on various organs
Now again, the parathyroid glands, PTH the predominant action, is in bone, kidney, and then miscellaneous action in cartilage, liver, brain, placenta, lung, smooth muscle, other tissues. It is also very important in terms of proliferation and differentiation, as well.

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Topic Index:  Chronic Renal Failure -- Bone Disease Topic Index:  Clinical Nephrology -- Hypercalcemia