CME Slides Forum - R. Kumar

A joint Congress by ERA-EDTA and ISN

INTESTINAL PHOSPHATE ABSORPTION: CROSS-TALK WITH THE KIDNEY

Rajiv Kumar, Rochester, USA

Chair: Natale Gaspare De Santo, Naples, Italy

Carsten Wagner, Zurich, Switzerland

kumar

Dr R. Kumar
Division of Nephrology and Hypertension
Department of Medicine, Mayo Clinic
Rochester, MN, USA

Good morning, I’d like to thank the organisers for the opportunity to speak. I’ll be talking today about intestinal phosphate absorption and crosstalk between the intestine and the kidney and try and develop some new concepts I think with respect to how phosphate is regulated.

The important point that I’d like to emphasise is that in the short-term immediately after a meal the regulation of phosphate by the kidney occurs via feedforward mechanisms and the concept is illustrated on this slide taken from the work of Lee and others and is relevant in this case to potassium.

Classically feedback control involves an increase in renal excretion in response to increases in plasma concentrations of a given solute. The increase in renal excretion then brings the concentration of that solute back to normal.
However, in feedforward control the intake of a solute in our particular case phosphate, is sensed in the gastrointestinal tract by sensors which then cause the release of a factor that in anticipation of the increase in plasma phosphate before that actually causes an increase in the renal excretion of phosphate so that plasma solute concentrations do not change in very large amounts at all.

So in my talk I’ll give you a brief overview of phosphate homeostasis, the mechanisms by which phosphate is thought to be regulated. I’ll present data supporting the presence of an enteric renal phosphate regulating access and I’ll discuss the components of this particular access.

This familiar slide shows the relationships between the intestine and the kidney with respect to phosphate homeostasis. The important point here is that in states of phosphate balance the amount of phosphate being reabsorbed and excreted by the kidney parallels or is about equivalent to that that is absorbed in the intestine. There is a large phosphate pool from which phosphate moves into bone and soft tissue and this is regulated by a variety of hormones.

Now, in terms of the current models that are relevant to phosphate homeostasis. The positive balance of phosphorous is regulated in large part by the vitamin D endocrine system whereas negative phosphate balance occurs in response to changes in PTH. But it’s clear that there are a number of other regulators that are either short-term regulators or more long-term regulators that play a role in phosphate homeostasis. I think what I’d like to do is in this talk, talk a little bit about short-term regulators and in particular about a term that I call the enteric phosphatonins which are similar to the other phosphatonins that you’re familiar with.

So in this slide the homeostasis of phosphate is summarised. Changes in serum phosphate cause reciprocal changes in serum calcium concentrations, changes in PTH which in turn alter renal phosphate excretion.
There is also a change in the synthesis of 1, 25 (OH) 2D3 that is modulated either positively of negatively by a number of growth factors including FGF, fibroblast growth factor and sFRP-4 which modulate the formation of 1, 25 (OH) 2D3 and the absorption of phosphorous in the intestine.

The take home message is that short-term adaptations in terms of phosphate intake occur as a result of this enteric renal access in which there is a phosphate sensor that is present within various parts of the gastrointestinal tract that rapidly modulates fractional excretion of phosphate in the kidney. Whereas more long-term alterations occur and are mediated by PTH or the classical phosphatonins as shown here.

The concept of phosphate sensing and changes that occur in response to alterations in phosphate concentrations in the extracellular milieu is depicted on this slide. In unicellular organisms like bacteria and in yeast changes in phosphate concentrations are sensed by a protein which in turn causes a variety of changes through a signalling cascade in the expression of genes that will cause either a retention or a rejection of phosphate.
However, in multicellular organisms the situation is somewhat more complex. There certainly appears to be regulation at the level of the organ that sees the phosphate but there are also changes that result in the elaboration of a factor or factors that alter the excretion of phosphate in more distant organs such as the kidney.

So some clues to this actually came about as the result of work that we did many, many years ago now almost 20 or 25 years ago in association with Hunter Heath where we put young adults on a phosphate containing diet and we found that these individuals adapted very rapidly to the change in phosphate and had very robust increases in the fractional excretion of phosphate when placed on a high phosphate diet.

This occurred both in women as well as in men placed on these diets. Surprisingly however, parathyroid hormone concentrations changed very slightly and certainly within the normal range.

Even if one carried out more extensive almost hourly PTH measurements, there really did not appear to be a great change in PTH. 1, 25 (OH) 2D3 concentrations instead of being suppressed as you would expect actually went up a little bit perhaps because these individuals were on a somewhat low calcium diet. But certainly the hormones that are normally thought to play a role in phosphate homeostasis were not changed in humans placed on a high phosphate diets.

Now, another clue came from a series of elegant experiments done by Nishita and colleagues who fed normal humans volunteers increasing amounts of phosphorous and then looked at changes in PTH and FGF23 and also the fractional excretion of phosphate.

What they found was that there was a dose-dependent increase in phosphate excretion in the urine as one increased the amount of phosphate in the diet but there were hardly any changes in FGF23 in fact, they went in the opposite direction that you would predict and there were modest changes in PTH that were associated with an increase in the fractional excretion in phosphate. The point being there must be something else controlling phosphate under these circumstances.

So, we did a series of experiments in which we infused phosphate into the duodenum of rats and found that both in intact rats as well as in parathyroidectomised rats there was a very rapid increase in the fractional excretion of phosphate following the installation of phosphate into the duodenum.

When one infused saline there was no change. In the case of parathyroidectomised animals there was an equally robust change that started from a lower fractional excretion of phosphate that you would expect in parathyroidectomised animals.

There was no difference in the phosphate excursions in fact they did not change in the early time points following the infusion of phosphate into the duodenum and they were similar in both phosphate and saline infused animals.

So this suggests that the filtered load of phosphate does not regulate the fractional excretion of phosphate following the infusion of phosphate into the intestine. PTH doesn’t change as you would expect from the parathyroidectomy experiments and neither does FGF23 either in the intact rats or in the parathyroidectomised rats.

s-FRP-4 which is another phosphatonin does not change following the infusion either.

So what is it that mediates these changes in the fractional excretion of phosphate? Well, a candidate for this are the renal nerves but surprisingly we found that following unilateral denervation there was still a robust increase in the fractional excretion of phosphate following the infusion of phosphate into the duodenum thereby, showing that renal nerves were not responsible for this process.

The process is activated by relatively low concentrations of phosphate going all the way from 0.1 molar up to 2 molars. You can see there is a dose-dependent increase in the fractional excretion of phosphate in the kidney. Surprisingly, PFA which is a competitive inhibitor of phosphate transport causes just a very modest change in the fractional excretion of phosphate suggesting that transcellular phosphate transport per se is not required for the activation of this particular response.

When you take homogenates of the intestine, grind them up and infuse them into animals one finds that there’s an increase in the excretion of phosphate in the kidney and this is mediated by proteins that are in the large molecular weight fraction greater than about 50.000 daltons although there is some modest activity with low molecular weight fraction as well.

If you carry out parabiosis experiments looking for the presence of the circulating factor, one sees that there is in the animal that receives phosphate in the pair an increase in the fractional excretion of phosphate but in the contralateral animal there is also an increase in the fractional excretion of phosphate suggesting that there is a circulating factor that is moving from the animal that receives phosphate into the contralateral animal in the pair.

This is supported by experiments done by Landsman and others showing that animals adapted on a high phosphate diet had a circulating factor that inhibited phosphate transport in OK cells.

There appears to be a mechanism in the intestine for that particular organ to sense phosphate and this is work done by Martin and others showing that uremic animals receiving either high or low phosphate diets were able to regulate PTH very rapidly following the infusion of these diets

So this data, as well as our data, suggests that there is a mechanism by which the intestine is capable of sensing phosphate.

So in conclusion the data suggests that short-term adaptations to dietary phosphate loads involve novel processes that include sensing and the release of phosphaturic substances from the intestine that alter renal phosphate absorption.

The concept that I’d like to leave you with is that changes in dietary phosphate change phosphate uptake in enterocytes by local mechanisms but also release a factor that alters renal phosphate reabsorption, thus preventing large excursions in serum or extracellular phosphate concentrations following a high phosphate meal.

This is depicted here and demonstrates that short-term post meal adaptations to phosphate are mediated by factors that are liberated by the intestine after phosphate concentrations are sensed by enterocytes.
We don’t know what the phosphate sensor is, we do not know where the phosphate sensor is located nor do we know the cells that elaborate or the nature of the cells that elaborate this phosphaturic factor. They could be enterocytes or they could enterochromatin cells or some other cells within the intestine. We do know that these cells are present in the duodenum and in the jejunum and are not present in the stomach or in more distal portions of the intestine. I think future work should and perhaps will focus on determining the nature of this sensor and also the nature of the factors that signal from the intestine to the kidney. Thank you.

Chairman: Thank you Rajiv for this wonderful talk. The paper is open for discussion. There is a microphone and I would ask everybody to identify themselves with name and affiliation.

Question: Silver, Jerusalem. Rajiv thank you very much for a beautiful talk. When you get your mucosal extract and you put it intravenously into recipient rats is the phosphorous content, actually all electrolyte content of that mucosa is there more actual phosphorous in it compared to control rats?

Dr. Kumar: Right well we do know that serum phosphate concentrations following the infusion don’t change. Now you know, the question I think you’re getting at is could it be due to an infusion of a phosphate load? In a sense that’s a difficult question to answer because once phosphaturia is induced, the phosphate levels if they stay constant could well be staying constant because there’s phosphaturia but what one can do and what we have done is extensively dialyse the homogenate to try and keep phosphate concentrations low and we’ve actually measured phosphate concentrations prior to the infusion and they’re very low. Also the nature of the factor or the behaviour of the factor on size exclusion columns is such that it’s unlikely to be a small solute because it elutes at a very high molecular weight.

Question: -- Atlanta Georgia it was a very nice talk. I’m curious what happens in renal failure. Have you done a parabiosis experiment where you have a uremic animal connected to a non-uremic? What happens to this system in renal failure’

Dr. Kumar: Well I think that’s a very interesting question and I don’t have any answers to that. We don’t know whether this is blunted or it’s upregulated and what happens with different levels of renal failure because it’s conceivable that you might have a more robust response in early renal failure and then it might just disappear towards you know more severe renal failures. So I don’t have any data to tell you one way or the other which way this would go.

Question: Maybe I can ask you what is your interpretation of the phosphonoformic acid experiment? Does it indicate that there is a primary sensor that is sitting on the luminal membrane the duodenum or jejunum?

Dr. Kumar: We did the phosphonoformic acid experiments thinking that phosphonoformic acid would mimic phosphate. We said it should do the same thing but it didn’t. So what my interpretation of the data is that the sensing is occurring somewhere else other than in the transporting apparatus, a sodium phosphate transporter or other transporters that play a role in the transcellular movement of phosphate. So it could well be that this could be on the enterochromaffin cells which then elaborate the factor and have an effect. But I am not quite sure how to interpret the phosphonoformic acid experiments other than what I’ve just suggested. Do you have any clues as to what it’s telling you?

Dr. Kumar: Well, it’s a high molecular weight substance, so we don’t know if it’s a protein because protease experiments have not given us clean answers.