Slide 1

Professor Hernando, Professor Ritz, thank you very much. Dear friends, thank you all for being here that early on a Sunday morning. What I would like to do is for the next 45 minutes or so to give you some insights and some updates in the mechanisms of renal handling of phosphate and in the second part mostly also into physiological and pathophysiological alterations of renal phosphate handling.

Slide 2

As I told you, I will mostly describe renal and intestinal transport and in the second part physiological regulation and pathophysiological alterations. As you probably all know, phosphate homeostasis in our body is controlled by several elements. The one here is the kidney controlling renal phosphate excretion. The other one is, of course, intestine mediating intestinal uptake absorption of phosphate but also some excretion of phosphate and an important player is the bone and phosphate metabolism in the bone by formation and phosphate resorption. As I will lead to it towards the end of the talk there is now growing evidence there is a significant amount of interplay between all these three components to control and maintain the phosphate homeostasis.

Slide 3

As Doctor Hernando said, I’m a transport person and I have a very simplified view of phosphate handling and phosphate homeostasis. It’s a game of transporters and transport regulations and there are two important types of phosphate transporters, there are the type II sodium/phosphate cotransporters and the type III sodium/phosphate cotransporters. The type II sodium/phosphate cotransporters are the specialised ones and these are more the house keeping ones present practically in every cell and we have type II that’s the gene family 34 and this is the gene family 20. We have transporters, the first was the type IIa which was the renal transporter but you will see shortly there is a second transporter type IIc also a renal transporter then the intestinal isoform and then in the bone where we have the expression of type IIa and type IIb that means a renal and an intestinal isoform and they are involved in bone formation and bone resorption.
The type III transporter is a transporter mediating phosphate influx in practically every cell.

Slide 4

Kidney, in the kidney phosphate is freely filtered in the glomerular capillaries and then is reabsorbed along the proximal tubule here by these proximal tubular epithelial cells and we have the rate limiting steps in the apical membrane, the type IIa transporter as I already said and more recently this cohort the type IIc transporter. They are energised by an inverted direct sodium gradient maintained by the basolaterally located sodium potassium ATPase. You see here a staining, an immunostaining against type IIa transporter nicely in the brush border membrane of proximal tubules and to see transporters, at least in the rat and in the mouse more located in deep nephrons also nicely in the proximal tubule.

Slide 5

The type III transporter is expressed all over the kidney where we have clear evidence that it’s mostly in the basolateral membrane and it can mediate uptake from the basolateral side into the proximal tubule when cell delivery from the apical membrane is insufficient.

In the intestine we have the type IIb transporter and phosphate is reabsorbed along the small intestine and in most species it is early small intestine but as you see here, in the mouse it’s mostly in more distal portions of the small intestine in the ileum where you see here a high sodium dependent uptake of phosphate into the apical membrane or across the apical membrane into membrane vesicles prepared from them. You see here an immunostaining where you see here the high expression of the transporter in the brush border membrane. Here a nice immunostaining that’s the Western Blot and here an immunostaining at the apical surface of the villi of the intestine. But as I said, this is highly specie dependent; it’s always in the small intestine but as you see here, already the difference between mouse and rat, in the mouse it’s mostly in the ileum whereas in the rat it’s more in the upper small intestine and jejunum and in the duodenum.

Slide 6

Now, having two transporters in the proximal tubule it’s important to know which one is the key player. Unfortunately, the story is not that simple we cannot just say it’s one but this is again depending on species. In the mouse we have the feeling that we have a dominant role of the type IIa transporter, this is based on experiments in knockout animals where the type IIa transporter was deleted with a massive reduction of sodium dependent transport. But when the type IIa transporter is decreased or is eliminated, then the type IIc transporter goes up and over compensates. That means this residual activity might probably in normal mice not be present but is due to upregulation as a consequence of the knockout of IIa upregulation of IIc.

In humans and I will come to that later on in my talk, the situation seems to be different. IIa is also present and we have been looking with different laboratories around the world whether we could find evidence for a crucial role of IIa but more recently different laboratories mainly the – laboratory from Boston provided evidence via genetic defects that the type c transporter is in humans probably much more important than the type IIa transporter. I will show that later that mutations in the type IIc transporter are associated with hypophosphatemic diseases.

Slide 7

Now, what do we know about these transporters? The type II transporter has two glycosylation sites, has 8-12 transmembrane regions, has intracellular amino and carboxy terminus, has intramolecular repeats as here given by the yellow dots with crucial aminoacids for functions. These are in intracellular loop 1 or in extracellular loop 3 and the evidence we obtained for that is based and I have no time to go into this kind of experiments by sophisticated cysteine scanning and fluorescent labelling experiments but we know precisely, more or less precisely how the topology of these transporters looks like. That’s the only slide I’m going to show to provide some evidence that we know in quite some detail how these transporters function.

Slide 8

There is the transporter type IIa, the transporter type IIc. One of the transporters IIa is an electrogenic transporter. I will summarise that in the next slide because it transports 3 sodiums with one divalent phosphate but the type IIc transporter is an electroneutral transporter but we can easily switch over the type IIa transporter to a type IIc transporter just by changing 3 aminoacids here in the third transmembrane loop and by doing that we obtain then a nice electrogenic transporter. That means we change from a 2:1 to a 3:1 stoichiometry. That’s just an example of how we can assign critical functions to critical portions of the molecule.

Slide 9

This is to sum up, type IIa transporter and type IIb transporter one is the renal, one the intestinal 3:1 stoichiometry. Type IIc transporter 2:1 stoichiometry they both transport divalent phosphate, then gives an electrogenicity here or electroneutrality here of transport, whereas the type III transporter accepts monovalent phosphate and is also electrogenic but has a coupling with two sodiums.

Slide 10

Now, almost enough of this data is for the kinetics and structure of these transporters. First we have interaction with two sodium ions, then phosphate, then there is the remaining, the third sodium ion being linked and when the carrier is fully charged and we have conformational change and we have a discharge of sodium and phosphate from the internal cell surface.

Slide 11

Now, let’s talk about physiology and pathophysiology of these transporters.

Slide 12

You might have already seen this slide many times. I borrowed it or I obtained it from my dear colleague Raj Kumar and it illustrates in a nice manner how the basic or the main mechanisms operate. This is an old view, I will afterwards present a more updated view of how the physiological mechanisms are operating to control normal serum phosphate. For example, when due to dietary manoeuvres serum phosphate would be lower then serum calcium goes up, serum PTH goes down then renal phosphate excretion will go down. On the other hand, when serum phosphate goes up, serum calcium will go down, serum PTH will go up and renal phosphate excretion will go up. When you have these kind of changes, this is a question of minutes here and when you have these kind of changes for a prolonged time period, then we have the intestine coming into play and this is the mostly via renal synthesis of 1, 25 (OH) 2D3 in the intestine the 1, 25 (OH) 2D3 stimulates the intestinal reabsorption of phosphate and all these mechanisms are then contributing to keep the phosphate concentration at a constant level.

Slide 13

Now, I’m into the field of regulation of phosphate and the cellular mechanism. Parathyroid hormone is the key element in the kidney and it’s always regulated by controlling the expression here of the apical transporters. These are acutely acting mechanisms parathyroid hormone, ANF or ANP and these I will come back to that later on are the more chronic effects they are all leading to inhibition of phosphate transport or reabsorption in the kidney.

Slide 14

Now what do we know about the cellular mechanisms involved in phosphate control in the kidney? This is PTH, parathyroid hormone and we have worked over the years and that’s mainly the work of my dear colleague Nati Hernando and the transporter is removed from the brush border membrane goes to clathrin-coated vesicles, we see that by co-staining with clathrin, the transporter is green and clathrin is here red then it starts to move over, then it gets a little bit yellowish and then it goes to early endosomes, you see here it gets then here yellowish, the green disappears and finally ends up in lysosomes that means we have a nice time sequence in going from one compartment over to the next one.

Slide 15

It is via receptor-mediated endocytosis and we have labelled the receptor-mediated endocytosis pathway by insulin. Transporter is green, insulin is red and as you see here, with time they go together that means it will be yellow. When we have the fluid phase endocytosis which we can label with horseradish peroxidase, they never come together.

Slide 16

The logical step, of course, after these kind of experiments showing that this receptor-mediated endocytosis is then the use and that was done together with Thomas Willnow and Sebastian Bachmann from Berlin who used animals which have the endocytotic mechanism defect that is a megalin knockout animal and when you see here in the megalin knockout condition PTH or mimicked by cyclic AMP or mimicked by activation of kinase C is unable to re-drift the transporter. Here is the control and here is unable to re-drift the transporter after megalin deletion.

Slide 17

This is a summary of a complex cellular mechanism. PTH and I don’t whether all of you know that works through apical and basolateral receptors. The basolateral receptor mostly acts through protein kinase A, the apical receptor mostly acts via protein kinase C. They activate ERK kinase and they simulate then this endocytosis pathway leading to uptake of the transporter into lysosomes. This is a complex scheme and of course, we do not know everything but we know that there is a multiplicity of proteins involved in this traffic and we have identified by a complicated or sophisticated yeast-to-hybrid experiments where we have looked for partners of the transporter. We have seen that the transporter is not sitting alone in the membrane but is part of a huge protein complex linked via the carboxy terminus to several proteins, they are all via PDZ interaction. Among them also the sodium proton exchange regulatory factor 1 and they are important to link the transport to the cellular machinery for endocytosis.

Slide 18

An important element we have identified here in this loop there is a – motif which is absolutely required here for endocytosis. For example, the type IIc transporter which has low PTH regulation does not have this motif in the last intracellular loop.

Slide 19

This makes things very complex and very complicated. As you see here, when we give PTH the transporter is regulated but the interacting proteins are staying at the membrane. This means that the interaction I described in the previous slide has to be a dynamic one and is controlled, for example, by phosphorylation reactions. But we don’t yet have a clear clue to how this control of stability of the complex is regulated by the phosphorylation reaction.

Slide 20

The interacting proteins have an additional role. I told you before that PTH acts via apical and via basolateral receptors which have a different signalling pathway and you can activate by a PTH analogue 1-34 you can activate both pathways and by a PTH 3-34 you can only activate an apical receptor which acts via kinase C protein. When we use now animals which have been deleted of the sodium protein exchange regulatory factor, then we see here that apical regulation by 3-34 PTH is completely gone.

Slide 21

And this can be explained by the fact that we have in this complex situation of the apical scaffold, we have the transporter linked to NHERF1 to the sodium protein exchange regulatory factor but we have also the PTH receptor, the apical receptor being linked here and they are coupled or they are crucial for the signal transduction of apical PTH to control the tubular transport of PTH.

Slide 22

Now, I have already told you that there is a more crucial role of IIc and this is emerging more and more for humans. Based on genetic experiments we learned that they have a phosphate urea, in mutations of type IIc and the type IIc, this is the human situation and here the mouse situation. In the mouse situation the IIc is regulated by a phosphate diet, it is not regulated by PTH because the KR motif is not present but it’s regulated, I will come to that later on, by FGF 23.

Slide 23

Here the lack of regulation type IIa I showed you before goes into lysosomes and you can see it appears in lysosomes when we isolate lysosomes from the kidney cortex after PTH treatment. Type IIc never would go into lysosomes because it is just not internalised.

Slide 24

Now these experiments with humans, the genetic experiments with humans and in this hypophosphatemic rickets disease.

Slide 25

You see here the appearance of this disease, the clinical appearance of this disease and mostly the laboratory in Boston from Harold Juppner's group but also the laboratory in Munich from Doctor Storm’s group and the lab in Indianapolis that’s Mike – group they have identified a variety of families with a variety of mutations all associated with this hypophosphatemic disease and they have all at different points of the molecule deletions which lead to misfunctions or which are --- only partial proteins. Despite one paper which showed also a similar thing in type IIa which in our laboratory we could not reproduce we have no evidence that the type IIa transporter would be involved in such kinds of pathophysiological alterations of transport in humans but the type IIc transporter is apparently a crucial target for this kind of mutations.

Slide 26

This is the slide I have shown you already the acute changes here mostly in kidney to control phosphate levels, mostly PTH. The chronic changes here. The intestine starts to come in via the synthesis of 1, 25 (OH) 2 D3 and via the control of phosphate reabsorption in the gut. We have also cloned some years ago that transporter in the gut that means we have the molecular tools to study that and the main regulatory elements are the low phosphate diet 1, 25 (OH)2 D3 in upregulation and you see here also some elements in downregulation.

Slide 27

Again, FGF 23 is appearing here on the screen. Just to illustrate that, when we feed animals a low phosphate diet, then the transport across the apical membrane is increased, the same is with vitamin D. When you look at the molecular level the expression of the type IIb transporter is increased or here at the mRNA level you see the expression of the mRNA is increased. The classical scheme would say low phosphate diet increasing 1, 25 (OH) 2 D3 then leads to an increased reabsorption of phosphate.

Slide 28

But I could afterwards speculate on how much is true of this scheme or whether we have to revise on that a little bit. We have worked a lot with different kinds of knockout animals whether this simplified scheme is true in all details.

Slide 29

Now again, a slide I obtained from my dear colleague Raj which I just modified a little bit. This is the current model which I was explaining so far in control of body phosphate. PTH the main player in the kidney, vitamin D the chronic effects, the main player via intestinal uptake.
Now, the modified model here, short-term regulation is still the same but there will be also now a paper coming out in PNS from Raj Kumar’s group that also there is some evidence for a link between the gut, an acute link between the gut and vie enteric renal nerves and via gut hormones to control or in the short-term regulation of renal phosphate handling. In long-term regulation in addition to PTH the phosphatonins play a very, very crucial role. The long-term is clear that I spoke already about the long-term regulation.

Slide 30

What about phosphatonins? There is a term phosphatonins which has been around for some time and it’s known that in tumour-induced osteomalacia there are factors around which produce hypophosphatemia, which are associated with hyperphosphaturia with all these additional symptoms here. All these changes here are corrected and you happen to find the tumour when you remove the tumour.

Slide 31

It was mostly Raj and his group which have identified then later on by differential screening methods different phosphatonins and among them is a very prominent one in the meantime is FGF 23, soluble frizzle related protein, is MEPE and other members of the FGF family.

Slide 32

What do they do? They cause phosphaturia, they inhibit renal production of the hydroxylase, they interfere with mineralization and then I will come to that also later on they are candidates for PHEX and PHEX is the gene product which is linked to XLH in X-linked hyperphosphatemia.

Slide 33

Together with Raj we could show that they inhibit sodium-dependent phosphate transport, they inhibit renal tubular phosphate reabsorption in vivo. T hat was done exclusively by – and Raj and we could contribute here by showing with the antibodies and in vesicular uptake that it is also related to the inhibition of the apical transporter.

Slide 34

It’s good to have some friends that are also good colleagues like Doctor Shimada and Doctor Shimada works a lot on FGF and Doctor Shimada has given me some of his recent slides to discuss a little bit on the role of FGF. FGF 23 as you see here, reduces the expression of type IIa, it reduces the expression of type IIc, then leads to an inhibition of the 1α hydroxylase and this leads then to a change in the lowering of the phosphate concentration in the blood and to a lowering of the vitamin D level in the blood but does not lead to altered serum calcium concentration.

Slide 35

Regulation of phosphatonins, they are regulated by 1α, 25 (OH) 2D3. They are increased when you inject or they have a higher level and they are regulated by dietary phosphate.

Slide 36

Again, a slide I just obtained a few weeks ago from Raj. He proposes an interesting model and I think it’s no longer a hypothesis I think the evidence speaks quite strongly for that. When you have a lowering of phosphate, then you have an increase in 1α, 25 (OH) 2D3. By an increase in intestinal uptake of phosphate, you get an increase in phosphatemia. This leads to a reduction of the activity here of the production of the active metabolite of 1, 25(OH) 2D3 and leads also to an increased synthesis of FGF. FGF has a negative role here on the production of the active metabolite of 1, 25(OH) 2D3.

Slide 37

This brings me back to this scheme where we can show now that a lowering of phosphate leads to an increase of 1, 25(OH)2D3, an increase of phosphate, an increase of FGF 23 and here I should now write that together with klotho makes then this loop complete, this feedback loop complete and I should also include here a direct effect on renal reabsorption of phosphate.

Slide 38

This is my very, very, very last slide where we have a new player in the field, we have FGF 23. FGF 23 is increased by lowering serum phosphate. This FGF 23 leads, as I explained to you in some detail, to the inhibition of the hydroxylase, inhibition of the renal Pi reabsorption via the effect on IIa and IIc and also reduces, I didn’t show you that, the intestinal phosphate absorption and very important for the pathophysiology the FGF 23 levels cannot only be modified by serum phosphate but there are a lot of pathological situations or genetically determined situations where the FGF 23 levels are influenced by processing of FGF 23, for example, in XLH where there is a mutation in the enzyme probably cleaving or supposedly cleaving FGF or some how involved in the inactivation of FGF. Then there is also here in the mutations directly in FGF that FGF cannot anymore be cleaved or there are the effects in the glycosylation of FGF that FGF 23 has a different stability. Most importantly in the tumour associated or the tumour-induced osteomalacia we have a high level of FGF and all leading then finally to the effects I described. That means when the – is high, then we have these phosphate wasting disorders.

Slide 39

With that I would like to conclude and these are my coworkers. My own lab we did a lot together with Brigitte Kaisling and Desa Bacic. I just have shown a few experiments with knockout animals, we have used many different knockout animals and for the second and last part of my talk I gratefully obtained some slides from my dear colleagues Raj and Doctor Shimada. Thank you very much for your attention.

Chairman: Thank you Doctor Murer for this wonderful example of a forefront in nephrology. We owe you a lot in what to use a modern buzzword is translational and we hope that you continue with your efforts to inject science into our clinical society. This brings the session to a close thank you again.