Slide 1

Thank you Doctor Murer. The title of my talk is too long for my short talk so I’d like to focus on the two very complicated clinical issues.

Slide 2

Hypophosphatemia after kidney transplantation and ectopic calcification.

Slide 3

But before going to this main problem I’d like just to review the role of FGF23 in CKD very briefly.

Slide 4

As you know, this is FGF23

Slide 5

And this was originally discovered as a phosphaturic factor which is responsible for these diseases with hypophosphatemia and low 1, 25 D levels and rickets and osteomalacia but now we know that this molecule has a very physiological role for phosphate balance.

Slide 6

So with normal kidney function FGF23 secreted in response to the loaded phosphorous will excrete the phosphorous product from the kidney without affecting parathyroid function. But as kidney failure progresses, despite very high concentrations of FGF23 the kidney is unable to excrete – phosphorous, so hypophosphatemia develops and together with low 1, 25 D levels the secretion of PTH will be activated resulting in severe hypoparathyroidism. In dialysis patients without any residual function very high, extremely high levels of FGF23 have been reported. So please remember this very special condition for the discussion at the end of my talk.

Slide 7

Now, let’s go on to the first programmed role of FGF23 in hypophosphatemia after kidney transplantation.

Slide 8

As shown by the Harvard group in this slide it is well known that serum phosphate levels decrease rapidly after kidney transplantation leading to a sustained hypophosphatemia in many cases.

Slide 9

Urinary phosphate excretion is enhanced especially directly after kidney transplantation. Thus, it has been considered that urinary loss is the main cause of hypophosphatemia after kidney transplantation.

Slide 10

In a prospective study of living donor kidney recipients by my colleagues in Tokyo, a decrease of serum phosphate level was already eminent in the first week after transplantation. But it was maintained for about 6 months and recovery of the normal renal handling of phosphorous will take about 6 months as shown in this slide.

Slide 11

This slide shows another set of data after kidney transplantation from cadaver donors. Acute decrease is absent in this case but hypophosphatemia was maintained for a couple of months.

Slide 12

So far several mechanisms have been suggested for hypophosphatemia after renal transplantation.
Persistent hyperparathyroidism is one of the major candidates. PTH levels do decrease, but slowly and may remain high in some cases. Messa and associates reported in 1998 that persistent hyperparathyroidism is associated with the severity of pre-existing hyperparathyroidism and characterized by an autonomous pattern of PTH secretion suggesting the development of nodular hyperplasia in the remaining parathyroid.

Slide 13

This is the time course of serum phosphate in the lower panel after renal transplantation. As you can see, this thick line means patients with persistent hyperparathyroidism and the thinner ones show that these are patients with controllable hyperthyroidism. As you can see, the degree of hypophosphatemia was severe in patients with persistent hyperparathyroidism after 6 months from kidney transplantation. But the initial decrease within 3 months are comparable between these two groups. So we should think of another factor for this initial decrease of serum phosphorous.

Slide 14

Other candidates for this are phosphatonins such as FGF23 whose serum level increases before kidney transplantation. But before the variation of FGF23 pre- and after kidney transplantation one should be careful about the selection of assay.

Slide 15

There have been 2 available assays for serum FGF23. Initial assay, c-terminal assay as shown on the right panel detects not only the full length molecules but also c-terminal fragments that accumulate in patients with kidney dysfunction. By contrast, intact FGF23 assay shown on the left panel, only detects full-length human FGF-23 using two kinds of monoclonal antibodies.

Slide 16

As we reported earlier, serum concentrations detected by intact FGF-23 kits are extremely high and with a wide variation among them.

Slide 17

Then what were the changes of FGF23 levels after kidney transplantation?

This figure by a Harvard group shows the time course of FGF23 detected by c-terminal assay. FGF23 level decreases, but rather slowly after kidney transplantation.

Slide 18

By contrast, in our patients, a more rapid decrease of FGF23 level within three weeks was demonstrated by intact FGF23 assay.

Slide 19

In another set of patients by a Japanese group, serum intact FGF23 levels decreased progressively and reached normal level after 6 months after kidney transplantation. Thus, Serum FGF23 level decreases after successful kidney transplantation, but it takes several months to reach the normal range.

Slide 20

Then, which is more important for the development and maintenance of hypophosphatemia after kidney transplantation? PTH or FGF23?

Slide 21

In the study by Bhan and associates which used c-terminal assay, FGF23 levels, but not PTH levels were independently associated with hypophosphatemia. Furthermore, they showed as in this slide, that persistently high levels of FGF23 were more likely to develop severe hypophosphatemia as shown in this slide.

Slide 22

Another recent study by Evenepoel and associates, showed that the enhanced urinary phosphate excretion was evident at 3 months after kidney transplantation in the presence of high FGF23, either with low or high PTH.

Slide 23

These data favour a more important role of FGF23 rather than PTH in the development of hypophosphatemia after kidney transplantation.

Slide 24

Such analyses have been also performed by my colleagues in Japan. According to their data, PTH and FGF23 levels were the major determinants of serum phosphate levels at two months after transplantation, but PTH is the only major factor at six months.
So now this study is still underway so a longer observation will elucidate the role of these factors more clearly in the future.

Slide 25

So, in summary, an increased level of FGF23 contributes much to the development of hypophosphatemia in the very early phases after kidney transplantation.
But since the half-life of FGF23 should be very short, this may imply that increased production of FGF23 in the bone persists for several months even after transplantation and a persistently high PTH level may be more important in the later phase.

Slide 26

This abnormality is dependent on the speed of regression of parathyroid hyperplasia. The effect of drugs used in transplantation should be considered. However, severe hypophosphatemia is rare in transplantation of other organs, thus the contribution of these factors seems to be minor.

Slide 27

Now, let’s go on to more complicated issues, the role of FGF23 in the development of ectopic calcification.
In FGF23 null mice, a high phosphate level and high 1, 25D level with bone abnormalities develops, such as altered skeletal mineralization and low bone turnover.
In addition, remarkable soft tissue calcification was noted, suggesting there is some role of FGF23 in ectopic calcification.
Then, which abnormality is more important for the deranged bone and the mineral metabolism in FGF23 null mice? Hyperphosphatemia or high levels of 1, 25D?
Sitara and associates showed that genetic ablation of vitamin D activation pathway reversed the abnormalities in FGF23 null mice.
By contrast, Stubbs and associates have recently shown that a phosphate deficient diet prevented vascular calcification in FGF23 null nice, but a vitamin D deficient diet did not.

Slide 28

As can be seen here so this is a more important role of hypophosphatemia but however, this issue still remains controversial.

Slide 29

Such a role of FGF23 has been further supported by the analysis of gene mutations in patients with familial tumoral calcinosis.

Slide 30

In this type of mutation reported by Larsson and associates, the serum level of c-terminal FGF23 is very high, while the level of intact FGF23 is very low, suggesting the increased intracellular proteolysis of FGF23 due to the mutation.

Slide 31

The same mechanism has also been reported in the gene mutation of an enzyme which is involved in the glycosylation of FGF23 leading to augmented processing of FGF23. Also with this mutation severe to more calcinosis develops.

Slide 32

Next example of impaired FGF23 action is associated with disrupted klotho gene.
Klotho knockout mice showed aging syndrome including vascular and kidney calcification. These abnormalities are very similar to those of FGF23 knockout mice.

Slide 33

And as will be discussed by Professor Kuro-o in the plenary lecture in detail, klotho is a membrane bound protein that forms a complex with FGF23 and the FGF receptor. In addition, a soluble type of klotho protein has also been discovered.

Slide 34

In this recent study, recent report by Ichikawa and associates, a missense mutation of klotho gene caused several severe tumoral calcinosis in humans despite a very high level of FGF23.

Slide 35

This mutant klotho protein did not have the ability to form a complex with FGF23 and FGF receptor and impaired the signalling of FGF23.

Slide 36

Thus, the klotho gene mutation resulted in the end-organ resistance to FGF23, leading to tumoral calcinosis.

Slide 37

I have shown several mechanisms of impaired FGF23 action so far, as shown in this table and all of these results in ectopic calcification.

Slide 38

Then, does FGF23 have any role in vascular calcification in dialysis patients?
As shown before, these patients have a very high level of FGF23. Of course, dialysis patients already have several factors that favour calcification, such as diabetes and inflammation and can be further modified by medical therapy.

Slide 39

This is one example of such studies in dialysis patients. Inaba and associates have shown that log FGF23 was negatively associated with hand artery calcification, but strangely not with aorta calcification. So, the real world of dialysis patients may be much more complex than this.

Slide 40

First of all, already a very high level of FGF23 further increases by the use of active vitamin D sterols as shown here.

Slide 41

But recently, Silver and associates clearly showed that FGF23 directly suppresses PTH production in the parathyroid.

Slide 42

But however, as shown by Koh, at least in the remnant kidney of CKD patients, production of membrane type and soluble type of klotho was suppressed

Slide 43

So taken altogether, in dialysis patients with severe hyperparathyroidism, high levels of serum phosphate and high PTH stimulates the production of FGF23 in the bone.
In addition, as we have shown in dialysis patients, 1,25D, including exogenous vitamin D analogues, further increases the FGF-23 levels. On the other hand, production of membrane bound klotho and soluble klotho is decreased. So, it is not clear whether this very high level of FGF23 exerts its effects on the target organs including parathyroid in dialysis patients.

Slide 44

In summary, impaired activity of FGF23 by several mechanisms associated with ectopic calcification including blood vessels and low klotho expression may cause organ resistance to FGF23.

Slide 45

Thus, despite a very high serum concentration of FGF23, actual activity of FGF23 may not be high in CKD5d patients and many factors, including the use of active vitamin D, can further modify the development of vascular calcification.

My final question is whether the role of FGF23 on vascular calcification is direct or indirect? Maybe many of you agree with an indirect effect because FGF23 only acts on the cells equipped both with FGF receptor and klotho, such as cells in the kidney, parathyroid and choroid plexus so no one is sure whether the cells in the blood vessels have these two molecules for the response of FGF23.

Slide 46

Thus, it may be possible that impaired activity of FGF23 may not directly cause ectopic calcification but through evoking abnormal mineral balance, such as hyperphosphatemia and altered bone mineralization. So, pathogenesis of ectopic calcification in CKD is very complex including the mineral imbalance and high or low bone turnover so we still have lots to do.
Thank you for your attention.

Slide 47

Chairman: Thank you very much, we have time for one or two brief questions.

Question: So, I have one quick question. FGF23 suppresses PTH secretion and production and on the other hand, in CKD patients the FGF23 level is very high but still they develop secondary hyperparathyroidism. Is there a difference in short-term or long-term effects? What do you think about this discrepancy?

Dr. Fukagawa: This is a very complicated problem. At first we thought that a high FGF23 may stimulate PTH secretion but the data has clearly shown that it suppresses PTH. So two possibilities so the parathyroid is too active to be suppressed even by very high levels of FGF23 or the action of FGF23 is impaired in CKD.