CME Slides Forum - H. Murer

A joint Congress by ERA-EDTA and ISN

MOLECULAR CONTROL OF RENAL PHOSPHATE REABSORPTION

Heini Murer, Zurich, Switzerland

Chair: Natale Gaspare De Santo, Naples, Italy

Carsten Wagner, Zurich, Switzerland

murer

Prof. Heini Murer
Center for Integrative Human Physiology
University of Zurich
Zurich, Switzerland

Good morning everyone. I would also like to express my thanks to the organising committee for having invited me here to summarise very briefly the results available on the molecular handling of phosphate in the kidney.

I can be rather brief because most of it has already been said by Rajiv before and I’ll just concentrate now on the kidney. As you all know, phosphate is freely filtered at the glomerulus and then is reabsorbed along the proximal tubule

and we know in the meantime that it’s mostly the type II phosphate transporters which are involved in this process, type IIa here located in the apical membrane of the proximal tubular epithelial cells and type IIc also located in the apical membrane of the proximal tubule cells. There is another type II transporter around and this is type IIb and this type IIb transporter is involved in the intestinal absorption of phosphate.

For many years it has been known that there are other phosphate transporters available in mammals and especially it’s the type III transporters and type III transporters are ubiquitously expressed and some time ago it was to our surprise that it was found that the type III transporter is also present in the proximal tubule, you see here the apical membrane of the proximal tubule stained by one of the type III transporters, it’s the PiT-2 transporter.

Thus, we have a multiplicity of phosphate transporters and we have characterised them in great detail and here we just summarise very briefly the kinetic or the transport properties of these transporters and you see that the type II transport like NaPi - IIa and NaPi - IIb they transport with a 3:1 stoichiometry, they prefer divalent phosphate that results in a so-called electrogenic transporter and the type IIc transporter which as I just said before, also is expressed in the apical membrane of the kidney proximal tubule has a 2:1 stoichiometry, also prefers divalent phosphate but then results in an electroneutral transport and the PiTs, PiT 1 and PiT 2 they transport also with a 2:1 stoichiometry, they prefer monovalent phosphate and have then also an electrogenic property. What is important is that there are inhibitors of phosphate transport and we have just heard about phosphonoformic acid. Phosphonoformic acid is a strong inhibitor of the type II transporter but has no effect on the type III transporter. It’s known, I have no slides with me here, that the type III transporter is also expressed in the intestine that means in Rajiv’s experiment it might well be that a type III transporter might play a role and it’s a PFA resistant transporter.

Now having this multiplicity of transporters it’s, of course, important to know who is doing the major transport load and we have no clear answer to that. We just know from knockout experiments and these are the old knockout experiments done by Suzy Tenant’s group that when you knockout IIa in mice, then you lose about 70% of transport activity across the brush border membrane and you maximally upregulate then type IIc transporter that means the type IIc transporter can take part of the load but not the complete load. These are experiments done by Miyamoto’s group in Japan. When you knockout type IIc, there is no apparent phenotype available or apparent and there is no upregulation of the type IIa transporter. We are still waiting for the experiments, they are on the way of knocking out type III transporter and then we can make all kinds of combinations.

This I borrowed, as you’ve just seen, from Rajiv and you just heard that there are many regulators of phosphate transport. I can be brief, there’s a long-term regulation mostly via the vitamin D system and there are short-term regulations mostly by PTH but also the intestinal access, as we just heard before and then there are also the phosphatonins.

What is interesting and this of course, has been known already for a few years that all these regulatory events are somehow related to apical influx and this regulation is determined by withdrawal or by the amount of phosphate transporters in the brush border membranes that means they are either inserted or they are either retrieved from the membrane. As you see here many, many different factors have been studied and for all of them the amount of transporters is regulated.

It’s mostly the IIa which has been studied in great detail and these are just examples for that. When you have a low phosphate diet, you see the transporter is increased here by immunostaining as compared to high phosphate diet or when you give PTH, the transporter disappears here from the apical membrane of the proximal tubule that’s IIa.

Now what about IIc? IIc does not, at least in our hands and also by the experiments of -- seems not to be regulated at least in the same way as IIa and what you see here in this slide is the appearance of the type IIa transporter and the story of the type IIc transporter, type IIa and type IIc transporters in lysosomes, type IIa and type IIc transporters because when it is internalised, it’s then removed and travels into a lysosomal compartment and you see that the IIa transporter goes into lysosomes but the IIc transporter never appears in lysosome that means that it does not seem to be internalised in response to PTH.

We and others have in some detail characterised the regulation of type IIc and as it is summarised here, it’s not inhibited by PTH like we’ve just heard but it’s upregulated by dietary intake that means by low phosphate intake and downregulated by high phosphate intake but compared to the type IIa with a relatively slow time course, the type IIa transporter is the fast replying system and the type IIc transporter is relatively slow.
The vitamin D axis is apparently or could also play a role and we see that from experiments which we have done in vitamin D receptor knockouts experiments where the type IIc transporter is downregulated.

What about regulation of PiT? The PiT transporter, the PiT-2 transporter and this is also work just in progress and not yet completed is regulated by dietary intake that means it’s also increased by low phosphate diet and decreased by high phosphate diet but again, it’s a relatively slow response when you compare that to type IIa transporter.
It is also regulated by PTH and you see it leads to a reduction of PFA-resistant phosphate uptake in brush border membrane and in the protein content in the brush border membrane.

What do we know about the cellular molecular mechanisms in the regulation of the type IIa transporters in terms of PTH? I’m not going through the experimental details I’m just showing it or summarising it briefly here on this scheme. What we have seen it’s internalised, goes to the lysosomes. The PTH receptor is apical and basolateral. Basolateral PTH receptors have the classical signalling pathway via the adenylyl cyclase kinase A system, the apical receptor signals via the phospholipase C system and the regulatory cascade converts in MAP kinase system.

Finally leads then to internalisation of the transporter. Probably you have all seen the very recent review by Dominique Priè from Gerard Friedlander’s group and he chose that we have here the two transporters in the apical membrane. We are in some disagreement with this here as I just showed you before we have here a receptor system which mostly signals via cyclic AMP but a receptor system also in the apical membrane which mostly signals through the phospholipase C pathway.

But what is important here that you see is that we have interacting proteins and the sodium protein exchange regulatory factor 1 is involved here in this thing and I’ll come back to that in controlling the apical location of the type IIa transporter. This is summarised here. We did extensive studies on the candidates or on interacting candidates of proteins with type IIa transporter in the brush border membrane and as you see here this is a complex picture. There is an interaction via a PDZ domain in NHERF and this involves loss of the three aminoacids and you see this brings it then to a complex of multiple proteins which all link then the transporter to the cytoskeleton. We previously showed that the transporter here has a motif which is involved in endocytosis and Japanese colleagues have shown here that there is another specific protein interacting here with this internalisation motif.
This interaction with NHERF and the other proteins is important for apical location of the transporter and this was shown in knockout experiments by NHERF knockout experiments by Ed Weinman’s group when you have NHERF minus mice then the apical content of NaPi-IIa is reduced.

Now what happens in regulation when you have this complex of proteins including NaPi-IIa in the apical domain? When you give now PTH then you see that NaPi-IIa is disappearing but interacting proteins such as NHERF and PDZK1 I did not talk about PDZK1 they are stained in the apical membrane.

This means there must be a dynamic complex or the complex must be dynamically controlled by phosphorylation and it is known that there is a specific phosphorylation site in the PTZ domain, in one of the PDZ domains of NHERF

and just to give you one experimental evidence for that for this unstable complex after PTH treatment when you give PTH and you try to co-precipitate, then you see you have less co-precipitated NaPi-IIa in the brush border membrane and you see a reduction in the complex NHERF NaPi-IIa in the apical membrane.

That means this complex is falling apart when you give PTH and it has been shown and these were experiments by Ed Weinman’s group that there is a phosphorylation at one of the specific sites of NHERF.

Now most recently we had a surprise to find by another Y2Hybridscreen by different technique involving the entire protein but we know this is not a -- interaction we found another protein interacting, it’s a GABA receptor associated protein we call it GABARAP. This GABARAP is an ubiquitin-like protein and usually its function is well-described in synaptic trafficking of proteins. What we found then later on to find something in yeast with this screening procedure is one thing but do they really interact also in the kidney proximal tubule that’s the next thing to document.

What you should see here is yes we can co-immuno precipitate that means they really do interact and what we could show and unfortunately it has not shown up very clearly in this slide that the transporter is increased, we had an access to GABARAP knockout animals and we could compare in the knockout animals the expression levels of NaPi-IIa and NaPi-IIa is increased in the GABARAP knockout animals.

I had again to take out a couple of slides but here it’s just summarised. GABARAP is expressed in the renal proximal tubule proven by different techniques. We were able to show that it interacts with NaPi-IIa. The animals have a reduced phosphate excretion and have an increased NaPi-IIa abundance in the brush border membrane paralleled with an increased uptake rate and these animals have a highly increased expression of NHERF1 and it could well be that the increased expression of NaPi-IIa is then indirectly related to the increased expression of NHERF 1. From there we could speculate that GABARAP might have a function direct or indirect in the apical trafficking of NaPi-IIa. This brings me to just to two slides back to interacting proteins and interacting proteins and genetic diseases and there was a very recent paper by Collin Tubider a former post doc in our laboratory now working with Gerard Friedlander and they could show that NHERF mutations are associated with phosphaturia and they have related that to increased responsiveness of the proximal tubule PTH.

What this means here or what this says here we have very, very different factors and they are affected by mutations and they all result or they can result then in differences in transport rates of the key player at least in mice of the key player of proximal tubular transport, it’s IIa and again in the very same paper we have a very nice overview which was made by Dominique Priè

and that is the kidney International review the recent one by Gerard Friedlander and what this table should show that many, many genetic diseases associated with phosphaturia are either related to one of the factors controlling phosphate handling is FGF. You see here the levels of FGF here are changed or in more seldom cases and only very, very recently described we have also mutations at the level of the transporter and especially the transporter IIc. In human -- mutations and these mutations are associated then with the phosphate loss and the most recent paper by Dominique Priè that also mutations in one of the interacting proteins lead to a phosphate loss.

With this slide I would like to conclude and just to say here that this work has been done certainly not by me by all the colleagues back home and I would also especially like to mention my dear colleague back home and friend Carsten who has helped us a lot in doing experiments with mice and timing also the in vivo parameters on phosphate handling in mice. Thank you very much.

Chairman: Thank you Heini for this wonderful summary. The paper is open for discussion.
No questions, good it was so clear. Could you maybe come back to that point the controversy whether IIa, IIc or the PiTs are the main transporters in rodents or in humans?

Prof. Murer: We once together wrote a short comment on a paper and phosphate transport type IIa versus type IIc which is the key element? It’s difficult to say in mice and possibly in rats it seems to be clear it’s IIa but in humans I’m not so sure. Genetic experiments would tell us that IIc is much more important than IIa because it was hard to find IIa mutations and there is this paper from Gerard Friedlander’s group that when we do some experiments on what the mutations do then after transfections then we see practically no effect of these particular mutations. But we see it’s clear they are very nice transport modifying mutations.