CME Slides Forum - D.W. Swinkels

A joint Congress by ERA-EDTA and ISN

HEPCIDIN - BRINGING NEW KNOWLEDGE OF MOLECULAR MECHANISMS INTO THE CLINIC

Dorine W. Swinkels, Nijmegen, Netherlands

Chair: Ivan Rychlik, Prague, Czech Republic

David van Wyck, Tucson, USA

Dr Dorine W. Swinkels
Department of Clinical Chemistry
Radboud University Nijmegen Medical Centre
Nijmegen, The Netherlands

Good evening. I’m very happy with the introduction of Sophie that really helps me to focus on hepcidin in CKD.

So my talk will be on hepcidin bringing new basic knowledge into the clinic. I will start my presentation giving you an idea on why hepcidin could be relevant in renal disease and I will give you some recent insights in iron and hepcidin in iron regulation and I will end with a few slides on recent results of hepcidin in renal disease and I will finish with some conclusions.

As you all know, EPO allowed effective treatment of anaemia in CKD. However the optimal target haemoglobin is debated and many patients are resistant to EPO. There’s also an association of higher EPO dose to get to the target of haemoglobin and mortality.

As you have heard in the previous presentation, hepcidin is a recently discovered peptide hormone. It has a role in systemic iron metabolism and so as such it may be of help in understanding anaemia in CKD. So hepcidin may be an important tool to predict iron and EPO response. It might also a tool to guide iron and EPO treatments.

What is hepcidin? I will focus on those items that are important for the rest of my presentation.
Hepcidin is primarily produced in hepatocytes. It’s produced as 84 aminoacids, a pre pro-hormone and via the pro-hormone pro-hepcidin that consists of 64 aminoacids. It’s processed in the bioactive hepcidin 25 aminoacid hormone but hepcidin has also two smaller isoforms: hepcidin 22 and hepcidin 20. These have unknown biological function although in high concentrations that is about 1000 of hepcidin in the circulation, it might be anti-microbial. It is the hepcidin isoform 25 that is involved in iron metabolism. Hepcidin is small and as Sophie pointed out, compact and it might have 4 disulfide bridges. Therefore, it’s difficult to elicit antibodies to set up immunochemical assays.
Hepcidin is thought to be filtered in the kidney and reabsorbed by the proximal tubulus. We found a fractional excretion of about 1-3%.
Hepcidin can be measured by both immunochemical methods, such as ELISA or radioimmunoassay or by mass spectrometry. There is also a commercial pro-hepcidin kit produced by DIG, I can tell you it’s not very useful.
Immunochemical methods measure in general total hepcidin, so hepcidin 25, 22 and 20. Whereas, most mass spectrometry assays can separately measure these 3 isoforms. So remember immunochemical methods measure total hepcidin in general, whereas mass spectrometry can separate the 3 isoforms and pro-hepcidin is not very useful.

I will go briefly through iron metabolism and the role of hepcidin especially in CKD. Iron is absorbed in the enterocytes, binds to transferrin and is used in the bone marrow to synthesise HEME for haemoglobin. After about 120 days, maybe a little but shorter in CKD, erythrocytes are broken down and iron is liberated from the macrophages re-entering the circulation. This is about 20-25 mg/day.
There’s about 1-2 mg losses of iron and this in physiological conditions is compensated by iron absorption. Excess of iron is stored in the liver and the liver produces hepcidin. In this production of hepcidin by the hepatocytes the three main peptides involved and Sophie has already pointed them out HFE/TfR2/HJV that when mutated give haemochromatosis.
There are 3 main regulatory pathways in hepcidin regulation. Hepcidin blocks ferroportin and by blocking ferroportin it decreases the only cellular iron exporter that blocks iron release from the intestine and from macrophages leading to low iron in the circulation.

The three main regulatory pathways regulating hepcidin, bone marrow activity, the higher the bone marrow the activity, the lower the hepcidin and iron stores and inflammation both upregulate hepcidin. So three main regulatory pathways of which bone marrow activity decreases hepcidin. Anaemia and CKD are thought to involve cytokines, elevated cytokines. These inhibit the bone marrow for producing red blood cells giving rise to anaemia.
Next to that cytokines might also inhibit the production of EPO thereby also inhibiting erythropoiesis. Next to that cytokines increase hepcidin. It’s not very clear yet but if hepcidin is also freely circulating, a decrease in GFR will lead to an increase of hepcidin. I’ll come back to that later.
Again hepcidin will decrease iron availability by blocking ferroportin thereby blocking erythropoiesis.

So, anaemia and CKD have a complex pathophysiology that is likely to involve hepcidin. So then if we focus on the role of hepcidin in CKD, we have again cytokines increasing hepcidin and blocking of ferroportin. But hepcidin might also be increased by a decrease in GFR. Administration of exogenous iron might also increase hepcidin although this has not been proven. Administration of EPO is likely to decrease hepcidin because of the increase of erythropoiesis.

So we can see that hepcidin is likely to be a spin in the web of anaemia of CKD. So therefore, the question is could hepcidin be a new tool in the anaemia of CKD?

The pros of hepcidin could be that it reflects iron availability and needs for red blood synthesis. It integrates both inflammation and iron and in erythropoietic pathways. So it might be a single parameter of measurement of iron homeostasis in these patients.
However, the drawback of hepcidin is that there are not many assays available. It started out with mass spectrometry analysis, let’s say about 4 or 5 years ago and now more assays are coming up also radioimmunoassays. So in the future we really need simple and cheap accessible assays that are standardised throughout the world. We are waiting for that and working on it also.

Here I would like to present some of our recent data of renal handling of hepcidin just to show you because it might be important to understand the role of hepcidin in CKD. Until now it was thought that hepcidin is a freely filtered by the glomerulus and then reabsorbed by the tubulus. What we did to assess tubular reabsorption of hepcidin we infused two healthy controls with gelofusine that is thought to inhibit tubular protein reabsorption and we found that the fractional excretion increased from 1-3% to 14% and 70%. So suggesting that hepcidin when it is freely filtered by the glomerulus is reabsorbed in the tubulus. Moreover, we found and this can be seen in this graph, we found that at the start of dialysis you can see that there’s a difference in hepcidin levels between arterial line and a venous line suggesting that hepcidin is filtered, is taken out by the artificial kidney. However, this was done in 11 patients. However, when we also measured hepcidin in 5 patients at the end of dialysis, we found that hepcidin was not decreased. So this suggests that hepcidin is very tightly and swiftly regulated and might be in agreement with the recent findings of hepcidin binding to alpha 2 macroglobulin.

There’s also recent data on hepcidin in CKD. Here you have to remember and it has been done by 3 studies by Ashby, by immunochemical assay, by Zartisky immunochemical assay and by us Peters by mass spectrometry. The immunochemical methods measured total hepcidin whereas mass spectrometry measures only hepcidin 25 or 20 or 22. All three methods found hepcidin to be increased both in hemodialysis patients and CKD.
We found with our mass spectrometry that all three isoforms were increased. All three reports found that total hepcidin was increased and was inversely related to glomerular filtration. But we found that hepcidin 25 increase in CKD patients was not related to GFR whereas hepcidin 20 was. All groups found and here you can see that there was no relation in our study between hepcidin 25 and GFR because the data are all scattered around.
All groups found a relation between hepcidin 25 and ferritin. As you can see from our data with hepcidin on the y axis and ferritin on the x axis there’s a good correlation between hepcidin 25 and ferritin.
Two groups found that there was no relation between hepcidin and inflammation in CKD, whereas one group found there was a relation.

Recently it was also found that EPO administration in CDK decreases hepcidin but there are no prospective studies available on the relation between bioactive hepcidin and EPO requirements in CKD. So these studies are really needed.

To conclude, the relevance of hepcidin CKD, inflammation plays a central role in CKD associated anaemia and hepcidin production reflects the sum of most processes involved in CKD associated anaemia. Increased hepcidin levels in CKD can lead to iron restricted erythropoiesis. Recent findings comprise the following. Hepcidin is increased in CKD and in hemodialysis. Hepcidin isoforms accumulate in CKD and hemodialysis and the significance of this is unknown because as far we know hepcidin isoforms 20 and 22 are not involved in iron metabolism.
A decreased GFR is a significant determinant of elevated total hepcidin and hepcidin 20 but not of hepcidin 25.
All studies consistently find that hepcidin in CKD is related to ferritin.
So what I foresee as a potential application of hepcidin in CKD is that hepcidin levels might be predictive for EPO resistance, for EPO responsiveness and need for parental iron. Moreover hepcidin measurements could be a good tool to monitor treatment of hepcidin antibodies as mentioned by Sophie.

I want to thank my collaborators in Nijmegen in the Netherlands. At the department of clinical chemistry we have developed post mass spectrometry and immunochemical assays for hepcidin and we use these assays for several disorders of iron metabolism among which is CKD and hemodialysis patients. For the studies I showed on patients with renal diseases we collaborated with Hilde Peters and Jack Wetzels. Thank you for your attention.

Chairman: Thank you Doctor Swinkels we have time for questions. Any questions from the audience?

Question: My question is the following, some information arises concerning production of hepcidin in different parts of the proximal tubule as such as distal tubule. My question is despite the fact of knowing that hepcidin is produced or is accumulated in the proximal tubule, there is some information of hepcidin in other parts of the nephron such as the distal tubule or in Henle? What do you think about this? Is it a local production or an accumulation?

Dr. Swinkels: I agree hepcidin might be produced in the kidney although there’s only few data on that and it has not been reproduced as far as I know, so I doubt it’s really there. But I completely agree with you that hepcidin especially in the urine might also not only come from GFR but also by production of the kidney itself in the tubuli. So that could be the case but there’s only sparse data about that. In fact it can also be very interesting because in high amounts hepcidin could be ant-microbial so the suggestion is that it could have an antimicrobial function in the urine of course. But this has not been confirmed.