Prof. Michel Jadoul Cliniques universitaires St-Luc Université Catholique de Louvain Brussels, Belgium. Slide 1

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1 Phosphate and cardiovascular disease beyond CKD: is phosphate a new cholesterol? Michel Jadoul, Brussels, Belgium Chairs: Pablo Urena Torres, Saint-Ouen, France Carmine Zoccali, Reggio Calabria, Italy Prof. Michel Jadoul Cliniques universitaires St-Luc Université Catholique de Louvain Brussels, Belgium Slide 1 Thank you very much. I want to thank the scientific committee for the opportunity to discuss with you an interesting topic and a rapidly moving field. My topic, as mentioned, is phosphate and cardiovascular disease beyond or in the absence of CKD. Is phosphate a new cholesterol? Slide 2

2 The story regarding that issue started some let's say a little less than 10 years ago when there were in the same year two important papers. Here is the first one showing that there is an association between phosphate levels not only in dialysis patients and I will not mention that, it was already well known at that time but in CKD patients initially and these will be the only few slides on the CKD population and then I will move to the population without or with very little CKD. So the first paper was by Kestenbaum in JASN and admittedly that was clearly a CKD population with a median serum creatinine of 1.7 mg/dl and what they presented here is they showed that after adjustment for estimated creatinine clearance, so for the same creatinine clearance those having a somewhat higher phosphate level had a higher mortality risk with clear differences between the three groups and these are the lowest, highest and average quintiles. The important points are that the increase in mortality is apparent for phosphate levels above 3.5 mg/dl meaning in the normal range. Now, there was in the same issue of JASN an editorial by Glen Chertow and Sharon Moe nicely quoting I would say in the title said everything 'Is it a question of calcification or classification?' In other words, is the issue that those with higher phosphate levels have worse kidney function than those who have lower and in the lower range phosphate levels or is it the issue of vascular calcification along the line of what was already known at that time 8 years ago about the hypothetical or potential risk of vascular calcification and phosphate levels in dialysis patients. Slide 3

3 Now the same year, the third speaker of this session Professor Tonelli published a post-hoc analysis of the CARE study which as you'll remember, randomised patients to Pravastatin or placebo. You have here the breakdown of the 4 groups with phosphate below 2.5mg/dL, mg/dL, mg/dL and above 4mg/dL. You will note that there are differences in several respects between these four groups but importantly below well as you would expect 50% of the patients were under pravastatin average but GFR average was 68ml/min, 71ml/min, 72ml/min, slight differences between groups significant but not impressive. Serum creatinine was mg/dl and they had importantly in that study also a dipstick and dipstick was only positive in 12-14% for proteinuria. Slide 4 The results still were I would say along the same line as the previous study, an impressive increase of the risk of all-cause death, coronary death or non-fatal MI, new CHF or fatal and non-fatal MI where a dose-dependent let's relationship has a function of phosphate after adjustment for a number of other confounders. Slide 5 Then some other groups moved the topic a step further or some steps further and the group of Alan Collins at USRDS published some years later that paper entitled 'Serum phosphorous

4 levels associated with coronary atherosclerosis in young adults' and the strength of that study is that the mean age at inclusion in those over 5000 people was 25 years. In 3 US cities the phosphate level was measured in the late 80s and many of those people, not all of them, returned 15 years later. They had calcium scoring together with fortunately, an initial phosphate level available in the vast majority of them. Slide 6 Along I would say the same line but here as you have noticed these were really young guys, along the same line there was an association that becomes significant here somewhere in the range of 4 mg/dl, the calcium score above 100, the risk is increased already when phosphate is still within the normal range. Slide 7 You can imagine that those young guys, there were few patients with CKD and here are the

5 overall results from the same paper. On the top, overall population and below after exclusion of the few patients with a GFR below 60 ml/min and you will notice that the association with phosphorous level of coronary calcification. It's virtually the same picture even though there is no association with calcium or calcium phosphorous. Slide 8 Very recently another group from Brazil published another interesting study much smaller but they had importantly elective coronary angiography and a creatinine clearance above 60 ml/min. There are two outcomes or two endpoints for that study: one is an Agatston score of above 10 so meaning significant coronary calcification. The other one is a Friesinger score and for those of you who are not familiar with the coronary lesions it's a way of measuring the extent of coronary stenosis. Slide 9

6 Here is the result of the logistic regression of the risk of having an Agatston score above 10 and you will notice that phosphorous is among others with age like you would expect older age, male gender, hypertension, diabetes even though it's borderline significant but not PTH and FGF23, there is an independent association with phosphate. Slide 10

7 Here for coronary stenosis and no longer calcification, the same is true even though it's much less significant but it's still significant even though here FGF23 again is independently associated together with a number of other factors. Slide 11 Now I must confess that when reviewing the literature in that respect, it's not a consistent finding in all studies and I will show briefly two. And of those two the first one is a prospective observational study of 3381 men in their 60s and 70s without history of MI or stroke with a mean egfr 72 ml/min who were followed up for 11 years published some weeks ago, Slide 12 in fact only on the web meaning that it's hard and poorly readable because of that ATVB that's the name of the journal visible on the proofs that I downloaded I apologise for this. But here the message is that for CVD events, coronary heart disease there is no association with phosphate for stroke, no association. For cardiovascular death, there is some association but after exclusion of CKD patients and other determinants there is no longer a significant association for major CVD events similarly. In fact, if you look at the paper at what is a

8 cardiovascular event here whereas there is no association with CHD and stroke, you will find that it's mostly borderline that associates due to aortic aneurism surgery, peripheral vascular disease, cardiomyopathy or pulmonary heart disease in other words a mix of little bit of various items that may be unrelated to each other in my opinion. Slide 13 If you then look at the link with the total mortality still serum phosphate is significantly associated like alkaline phosphatase. I will not discuss that for the sake of time. Slide 14 The last slide I'll show in that respect is again a recent one on calcium phosphate and risk of cardiovascular events and all-cause mortality. Again of course, a different population subjects who had 3 weeks rehabilitation after an acute cardiovascular event, mostly males with an average age of 62 and they had creatinine calcium and phosphate measured at the end of rehabilitation before starting the follow-up. We learned from the paper that the creatinine clearance was above 60-95% and the follow-up was 8 years. Slide 15

9 Here a smaller number of items but still long follow up and you have virtually with phosphate no association so the finding has not been completely consistent. Slide 16 Last, sorry I forgot I had another one which is a recent one from AJKD from the osteoporotic fractures in men study which looked at something complementary. Slide 17

10 Firstly, they showed like the previous ones I've shown, a dose-dependent let's say relationship for all-cause mortality and annual cardiovascular mortality with phosphate levels. Especially here for cardiovascular mortality as you can see but they interestingly showed that there is no relationship between cardiovascular mortality and urine phosphorous over creatinine ratio or the fractional excretion of phosphate, which may be of interest of course, because of the hormones involved in the regulation of phosphate. Slide 18 Now, after having shown a number of observational studies, you might wonder what the mechanisms are and if there is a mechanism and it's not residual confounding and due to other factors what are the potential mechanisms and I must say that in that respect it's a complex topic. I'm not a super expert; we may discuss it in a few minutes but it's a very complex topic. I would just point to one important study, even though as you will note it's only 11 patients. It was published in JASN some 4 years ago and the title is 'Dietary phosphorous acutely impairs endothelial function'. Slide 19

11 The baseline characteristics of those 11 healthy male Japanese subjects are here, aged 24 average and they were given in random order double blind but still they had I guess strikingly different breakfasts with 400 or 1200 mg of phosphate. We are even told that it was consumed over 7-14 minutes. Slide 20 The next slide gives you the main results of that part of the paper where you will notice that when they had the 1200 mg of phosphate diet, well as you would expect phosphate increased as compared with baseline and there is a correlation between the vasodilatation, so impaired vasodilatation meaning worse endothelial function. There is a correlation between the impaired vasodilatation and the change in phosphate level. Now of course, as I mentioned, this is a nice study but in 11 subjects that has not yet been repeated to the best of my knowledge.

12 Slide 21 The authors are of course, enthusiastic about that. They have proposed a number of potential explanations that I will not discuss and I'm not a lab scientist and I will not discuss the potential pathways that may link phosphate to endothelial dysfunction and eventually cardiovascular disease. Slide 22

13 Now the topic of today was probably proposed by the organising committee, was probably suggested by that paper establishing somewhat an analogy between phosphate and cholesterol. Now if you think about that there are similarities, both are essential for life, for cell membranes, for energy metabolism and a number of other aspects. Both have increased in our typical Western dietary intake but there are genetic determinants. Both are found in excess in pathologic samples, in lesions and both correlate, associate with vascular disease within the range considered normal in the West. Eventually, last both change the phenotype of cells found in vascular lesions but we should not forget that that is true in vascular lesions only at hyperphosphatemic levels and not within the normal range. Now still there are two major differences between phosphate and cholesterol and those differences are that there is a much tighter hormonal control for phosphate than for cholesterol number one. Number two interestingly we should keep in mind that the normal range for phosphate is higher in children than in adults even though children have no cardiovascular disease because of their age among others but I mean that should at least put into perspective the hypothetical analogy between phosphate and cholesterol. Slide 23

14 If you then want to discuss the potential mechanism, I will refrain from that and perhaps finish a little bit early because there are so many. I will try to summarise what we could do for the next steps and probably at the end of the symposium after the presentation of Professor Zoccali and Professor Tonelli but I mean there are so many, as you know stimulatory factors on this slide even though if we take only those actors: phosphate, calcium, bone, FGF23, PTH, parathyroid cells, the gut, the kidney and the vitamin D system. Slide 24 So I think I would suggest as take home messages that in observational studies, I have shown you several a high normal or normal but on the high side phosphate level associated with coronary calcification. In some studies with atherosclerosis or atheroma burden with cardiovascular events and mortality. That was in the absence of CKD in several or in some of these studies or at least adjustment for CKD present in a minority did not change the results that much even though residual confounding in any observational study will remain a potential concern. Second, that the mechanisms of the association are complex. If there is causality which is not proven at all, it may be direct or indirect, you can speculate about what I've mentioned briefly and what was suggested by the investigators along the progression of the mind of the studies I've shown. I wasn't able to show all of them but I've shown several important ones, there have been others and I apologise for having been unable to show all of them. Slide 25

15 The second part of my take home messages would be that the role of the actors FGF23, PTH, the bone vascular access, calcium and others, clearly further studies are required, observational, experimental not to say interventional in humans and that's a different topic and probably we may turn back to that after the talk of professor Tonelli. So we have known for decades that cholesterol is a well-known cardiovascular risk factor. As you have seen in the last part of my talk phosphate shares some interesting characteristics with cholesterol. There are also important differences and whether intervention, especially in the population I've mentioned in the absence of CKD could reduce the risk remains to be investigated. Thank you for your attention. I was told that because there is some recording I should leave the slides so I will leave the slide, so the recording goes on.

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