COORDINATED REGULATION OF RENAL CALCIUM TRANSPORT BY VITAMIN D AND DIETARY CALCIUM

René Bindels, Nijmegen, the Netherlands

Chair: H. Murer, Zurich, Switzerland

T.B. Drueke, Paris, France

Prof R. Bindels Department of Physiology Nijmegen Centre for Molecular Life Sciences Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands

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Slide 1

Dr Bindels: Thank you Mr Chairman. First of all, I would certainly like to thank the organisers for inviting me. It’s really a pleasure to be here. I would like to take you back to the Ben side right now and tell you something about regulation of calcium transport proteins and vitamin D and dietary calcium.

Slide 2

So as you are all aware, the extracellular calcium concentration is normally maintained within very narrow limits and this is mainly due to a concerted action of 3 organ systems, that’s the bone where the calcium is stored, that’s the intestine where we can absorb calcium from our diet and finally, in the kidney where calcium is excreted into the urine. The calcium fluxes between these compartments are certainly regulated by various hormone systems including 1,25 dihydroxyvitamin D3 and parathyroid hormone and certainly also the calcium sensing receptor plays a very important role which is present in parathyroid glands but also in other tissues including the kidney.

Slide 3

If we now re-look at the cellular level then we can appreciate that there are specialised cells within the kidney and intestine and bone where calcium is absorbed or transported actively. In the kidney that’s the distal part of the nephron, in the intestine that’s the duodenum and in the bone resolving osteoclasts. I depict now a cartoon a kind of generalised mechanism so this is in a cell, this is the luminal compartment facing the urinary compartment and this is the blood compartment.

And you see here in the luminal membrane that there are 2 transport systems, which are called epithelial calcium channels, TRPV 5 and TRPV 6. They allow the calcium ions to enter these cells. Calcium will subsequently bind to the calcium binding proteins like calbindin 28 K or 9K and finally calcium is extruded at the basal lateral membrane by sodium-calcium exchange mechanism or by an ATP-driven calcium pump.

Slide 4

But really there are epithelial calcium channels, which are the postulated gatekeepers of this process. Actually these calcium channels command 2 flavins as I just said. TRPV 5, which was originally cloned from kidney, and TRPV 6, which was originally cloned from intestine. They belong to the super family of transient receptor potential channels. This is the super family of cation channels. TRPV 5 is really mainly localised in the kidney. You see 98% of the transcript is present in the kidney, whereas TRPV6 has a more broader expression pattern being present in the prostrate, brain but also certainly in the intestine. This is an image of the cortex you can easily see the outline of reconnecting tubule and here you can appreciate immunopositive staining for TRPV5 channel, which is very close to the luminal domain where the pro urine is present in the tubular lumen. Likewise if you turn to the intestine, the TRPV 6 immunopositive staining can be easily detected along the brush-border membrane where it faces the dietary content. So, so far so good. This slide really shows you the predicted typology of these channels so these channels cross the plasma membrane 6 times and between transmembrane second 5 and 6 there’s a hydrophobic stretch which is the postulated poor area of this particular channel. There is a large amino and carboxy terminus, which resides within the cell cytosol and this contains many regulatory domains.

Slide 5

Actually to understand the physiological function of TRPV 5, which is only or mainly present in the kidney we generated TRPV 5 knockout mice by partial gene ablation of the TRPV 5 gene and this slide shows this was successful indeed. We could show the absence of the TRPV 5 protein in the kidney by the absence in this particular case the green or yellow immuno fluorescent staining and in red you see the staining for kallikrein, which is a marker protein for the distal convoluted tubule and the connecting tubule where normally this protein is expressed. And importantly these TRPV5 knockout mice display a robust calciuresis, which is absent in the heterozygous mice. This calcium wasting in the kidney is not accompanied by the disturbances in blood calcium levels, which were normal, and also the blood pH levels were normal in these TRPV 5 knockout mice. However the 1,25 dihydroxyvitamin D levels were highly increased.

Slide 6

This slide actually shows you that the excretion of sodium-potassium ions is not disturbed showing the specificity of the TRPV 5 knockout. However, these animals produce a tremendous amount of urine as shown over here, so they have this large diuresis accompanied by a drop in pH of the urine and I must say that both adaptations are highly beneficial for these animals because they should prevent the formation of kidney stones during the production of large amounts of calcium to be excreted.

Slide 7

Actually, to re-localise the defect within the kidney or within a nephron segment we performed free flow micropuncture analysis. In this way we could first of all demonstrate that the fractional calcium delivery at a late proximal puncture site was not different between both animal groups, suggesting that the calcium absorption in the proximal tubule is unaffected in addition we looked at the fractional calcium delivery at more distal puncture sites and we could indeed observe an increase in the fractional calcium delivery in the knockout animals suggesting a defect in the distal calcium reabsorption. And to really pin point the defect we used the potassium concentration of the puncture fluid as a measure of the location in the distal part of the nephron because we reasoned that in the distal convoluted tubule and in the connecting tubule there’s a progressive potassium secretion and in addition, there is water reabsorption, so the potassium concentration can nicely tell us where we are in the distal part of the nephron. So we plotted a fractional calcium delivery as a function of the potassium concentration in the punctuate and indeed we could observe an increase in the fractional calcium delivery when the potassium concentration increases suggesting that a defect is indeed localised in the distal convoluted tubule and the collecting tubule and that’s exactly the place where normally the TRPV 5 channel is expressed.

Slide 8

We also looked at the expression of the other calcium transport proteins in the kidneys of these knockout animals. We could show that by gene ablation of TRPV 5, the other calcium transport proteins including TRPV 6, calcium binding protein and sodium calcium exchanger are down regulated. I think this is a very important finding because for the first time it shows us that there is a concerted regulation of the calcium transport proteins in which the calcium influx through the epithelial calcium channel is instrumental and I will come back to this point later on in my presentation.

Slide 9

We also turned to the gut and there we performed a radioactive calcium absorption assay and we could show that there was an increased calcium absorption in the gut in these TRPV 5 knockout animals and this was accompanied by an increased expression both on the messenger RNA protein level of the calcium transport proteins present in the small intestine, that’s TRPV 6, calcium binding protein 9K and ATP different calcium ATPase.

Slide 10

So this suggested to us that to increase 1,25 levels due to the renal calcium wasting upregulated calcium transport proteins in the small intestine and this facilitates the enhanced calcium absorption in the gut and this all to compensate for the renal calcium wasting and to really demonstrate a crucial role of 1, 25 in this process, we used the vitamin D antagonist, ZK-191784. We administered this to our TRPV 5 knockout animals. Subsequently, we performed the calcium absorption assay again and we could show that when you give this vitamin D derivative, which has a modification in the chain that you can normalise the calcium absorption in the knockout animals and at the same time you can also normalise or decrease the increased expression of the calcium transport proteins. So again demonstrated a crucial role of 1,25 in this adaptation process.

Slide 11

We also turned to bone by performing micro computed tomography and we could show that in the bones of the femurs of these knockout animals there was a reduced bone density and by performing various calculations we could indeed show a reduced trabecular and cortical bone thickness in these knockout animals. However, it is not clear at present whether this is a primary defect of the TRPV 5 gene ablation or whether this is due to the increased 1, 25 levels.

Slide 12

Very recently we could demonstrate that TRPV 5 is indeed expressed in osteoclasts both in human and in mice cultures we could show this. So this is an immunohistochemical image and you can appreciate here the immunopositive staining of TRPV 5 in a domain very close to the ruffled membrane facing the bone surface. This staining was absent in osteoclasts isolated from TRPV 5 knockout animals showing nicely I think the specificity of this particular staining. We could show at various levels that the number of osteoclasts in the TRPV 5 knockouts was significantly increased, was more than doubled, however by performing a functional assay in which we isolate osteoclasts from wild type and knockout animals, I put them on the bone surface to detect reabsorption pits, we saw a normal number of reabsorption pits very osteoclast isolated from wild type animals however, there were no reabsorption pits whatsoever in cultures from osteoclasts isolated from TRPV 5 knockout mice.

Slide 13

So this tells us really the following. First of all, this is the first time that we can show that there is indeed TRPV 5 expressed in osteoclasts both in the human as well as in the mouse level and it’s present along the ruffled membrane facing the bone surface. If you now knock down or knock out TRPV 5 and this results in a large number of additional osteoclasts that these osteoclasts are non functional. At present we don’t have a clue or we cannot re-conceal this effect that we have non-functional osteoclasts and at the same time have reduced trabecular and cortical bone thickness. Certainly, there is much more research needed to explain this bone phenotype in TRPV 5 knockout animals.

Slide 14

We have performed an extensive set of studies, I will not bore you to present all the data in which we showed that vitamin D indeed has a significant positive effect on the expression of the calcium transport proteins both in the kidney and in the duodenum. We also could show that the dietary calcium content is important and can regulate the expression of calcium transport proteins depending on the vitamin D status of the animals. That is when the animals have a normal vitamin D status, a high dietary calcium content down regulates the expression of the calcium transport proteins whereas when the animals are deficient in vitamin D, 1,25 vitamin D in high dietary calcium can increase the expression of the calcium transport proteins. We also could show an upregulation by estradiol and finally, we could show recently that PTH also has a stimulatory effect on the expression pattern of all these proteins.

Slide 15

So, we were really intrigued by the fact that the proteins were always regulated concomitantly and therefore, we actually made first of all a gene profile, so we performed a microarray assay in which we identified all the genes or sub cellular genes which are regulated either by vitamin D or by calcium, dietary calcium or by both and to this end we use the 1 alpha hydroxylase knockout mice. These animals have no 1,25 vitamin D3 and therefore have a very severe hypocalcemia. We used also wild type animals as a control and we put these animals on two regimens as normal calcium diet and a high calcium diet because we realised that a high calcium diet can rescue the phenotype of these animals including normalisation of the hypocalcemia. We isolated the kidneys of these animals and the RNA performed a microarray assay and we observed that there were many, many genes significantly up or down regulated and of the interesting genes were all confirmed by real time PCR. So this is a Venn diagram showing that there are a lot of genes either separately or concomitantly regulated by vitamin D and dietary calcium. But I would like to highlight that we observed that the calcium transport proteins like the channel, the binding protein of sodium calcium exchange were always regulated in the same direction we even identified new proteins which we further characterised, which were regulated similarly and which proved to be important for the regulation of the calcium homeostasis.

Slide 16

So to further address this, to further study the mechanism we first of all looked more carefully at the expression of the various calcium transport proteins and this is just an example of 2 but we could show that all these calcium transport proteins reside within one single cell either in the kidney or in the intestine, so that’s all already a very important clue. In addition we could show that there is a striking correlation between the expression of the calcium binding protein and for instance the epithelial calcium channel and here we plotted really very diverse situations like parathyroidectomized animals or ovariectomized animals or animals lacking vitamin D or animals supplemented with PTH, with vitamin D or with estradiol and you see indeed there’s a striking correlation. On the biochemical level, we can now even demonstrate that these proteins interact, so we could do a pulldown assay in the absence of calcium and we could show a physical interaction between the epithelial calcium channel and the calcium binding protein and its physical action is disturbed by an increased calcium concentration.

Slide 17

So here we would like now to postulate really that in calcium binding proteins when there is no calcium influx through the channel the calcium binding proteins bind to this particular channel so they are in a way waiting for calcium ions to enter and the advantage of binding to the channel is that you allow sufficient buffering of calcium in close vicinity of the calcium channel mouth. So as soon as calcium ions enter they will bind to the calcium binding protein and then the calcium binding protein in the calcium complex will release from the channel, will diffuse to the opposite side of the cell and calcium ions can be extruded. So this explains already why it’s so important to have a tight correlation.

Slide 18

But we really wanted to pinpoint the molecular mechanism by which this concomitance regulation is occurring and therefore we finally turned to previously established primary culture of calcium transporting distal convoluted and connecting tubule cells. So these cells are really in tubule in vitro and you can see that they indeed transport calcium from the luminal to the base lateral compartments and you can stimulate this calcium transport by exposure to parathyroid hormone. We also could inhibit this transport by using the specific inhibitor for the epithelial calcium channel that’s rat. It’s above the control flux as well as the stimulated flux can be modified.

Slide 19

Now we look at the expression of the calcium transport proteins in these 4 different conditions. So here you see a calcium transport data and now the expression of for instance, the calcium binding protein. This is the normal expression and this is the expression in the presence of PTH and we indeed can observe that if you increase the rate of transepithelial calcium transport then you can also increase the level of calcium binding protein. Like we observed in the animals because I just showed you a nice correlation curve. However, we performed the same experiment in the continuous presence of the retaining rat which inhibits the flux of calcium through this particular channel and now you see that the expression of the calcium binding protein is decreased in both under basal conditions as well as under stimulated conditions. So these experiments really tell us that it’s the calcium flux through the TRPV 5 channel which is regulating the activity of the other calcium transport proteins. So again it shows us that the epithelial calcium channel is the gatekeeper and is instrumental for all the other calcium transport proteins, which are necessary to facilitate transepithelial calcium transport.

Slide 20

So, I actually would like to conclude now and I have shown to you today that again that the calcium concentration in the extracellular compartment is regulated by a concerted action of 3 hormone systems and we now know more and more about the molecular details, which are the molecular players which are involved in this process both on the level of the kidney where we have implicated now several transport proteins and are even more regulatory proteins which are influenced by various hormones like vitamin D, PTH and estrogens. A similar mechanism although a little bit different players holds true for the duodenum where we also have a hormonal control by estrogens and vitamin D. We are not sure yet whether PTH also has an effect on this system in the gut and now we are also beginning to understand which molecular players are involved in bone. We have the first indication that in the osteoclasts the TRPV 5 channel is expressed and we know now also from preliminary experiments that TRPV 6 is mainly expressed in the osteoblasts. We hope now in the near future that we can really make a very comprehensive model how the calcium balance is regulated and we certainly do hope that we can translate our findings to the better physiological situations, which you all encounter frequently in the clinical practise.

Slide 21

So I finally would like to certainly acknowledge all my collaborators within my lab and that’s a group of people and I would certainly like to mention J. Hoenderop who has been instrumental for most studies which I have shown to you today and in addition we are very fortunate that we have very nice and stimulating collaborators which performed and participated in many of these studies which I showed to you today. Particularly, I would like to mention in this case the group of Rotterdam who performed all the bone analyses, which I showed to you. I certainly would like to thank you for your attention.

Thank you.