| GENERAL OVERVIEW: BONE REMODELLING AND CHRONIC RENAL FAILURE |
| Giorgio Coen, Rome, Italy |
Chair: T. Drueke, Paris, France |
J. Cannata-Andia, Oviedo, Spain |
|
Prof G. Coen |
Slide 1
Prof Coen: Thank you for inviting me. I see many experts in the floor.
Slide 2
So, the bone remodelling is a process of reabsorption of pre-existing extracellular matrix by osteoclasts, which is followed by osteoblastic secretion of extracellular matrix that is then mineralised.
Slide 3
And the adult skeleton therefore is in a dynamic state, since the process goes on for the entire life and is due to a coordinated action of osteoblasts and osteoclasts, which excavate the socalled haversian canals in the cortical bone or trench-like excavations on the trabecular bone surfaces.
Slide 4
Well, you certainly know images like this. It shows an haversian canal, which has been excavated by a group of osteoclasts, which are very well recognised at the tip of the excavation, and then there are plump osteoblasts, which elaborate osteoid, which will be then mineralised.
Slide 5
As far as the trabecular bone is concerned, there is a kind of trench which is excavated on the trabecular surface and I chose this slide by Susan Ott, she’s an expert obviously, and here you can see an osteoclast which is eroding the trabecular surface making an excavated lacuna, followed by apoptosis of the osteoclast. Then, you’ll see a team of osteoblasts which are producing osteoid, which will be then mineralised, so that the entire lacuna is replaced by new bone. This is a process which goes on continuously. Obviously, in old age this replacement probably will not be complete and this is also a cause of osteopenia.
Slide 6
Well, this schematic representation shows one very important point. You’ll see here a so-called bone remodelling compartment where the processes of reabsorption and bone formation go on. This compartment is separated by a flat layer of osteoblasts, separating the marrow from this cavity.
This is probably better seen in this section of bone where also you can see a layer of lining cells separating the bone marrow from this compartment. But , let’s say, some cells of the bone marrow have to enter into the compartment, like the osteoclasts. In the marrow there are macrophages and pre-osteoclasts and finally you’ll get osteoclasts inside the compartment. This means that this is a vascular compartment, where blood with cells is arriving inside, together with cytokines ad many other important factors of the bone remodelling process.
Slide 7
That’s why also what happens in this bone remodelling compartment is very quickly signalled outside in the circulation, so that we can say that there is a continuation of this vascular compartment with the compartment of circulating blood.
Slide 8
And finally, there will be the formation of this new bone as shown in this slide. What is shown is a lamellar bone. You can see the cement line. Bone deposited is limited by the quiescence surface. All together this is a basic structural unit as seen on a polarised light microscopy. If one measures the process of histological activation, that is the time taking place between two remodelling process at the same site of the bone surface, the so-called activation process, you have to measure this amount of bone in quite a few replicas. It is a time consuming type of histodynamic parameter to measure.
Slide 9
Well, obviously there is a coordination of cellular activities among osteoblasts and osteocytes and how does this happen?
Slide 10
The osteoblasts have adhesion complexes as shown here, which link one cell to the other. two osteoblastic cells. And the complex is made of an extracellular domain which goes inside the cytoplasm and is strictly linked to the actin cytoskeleton. Therefore the cells, one cell to the other and many others are all linked together.
Slide 11
But there is more than this, because the osteoblasts communicate through gap junctions and there is an hemi-channel on one cell which with juxtaposition on a hemi-channel on the other cell, creates a gap junction, which is very important because ions go through from one cell to the other and there are also other messengers which are able to modulate the gene expression and cellular function. In conclusion, it’s a team of cells that work in combination.
Slide 12
We have to recall that PTH treatment stimulates the genesis of gap junctions and the gap junctions are necessary for the development of differentiated osteoblast phenotypes and also gap junction communication is also stimulated by mechanical strain.
Slide 13
This has been seen repeatedly in different experiments and so, as you can see, human osteoblasts-like cells respond to mechanical strain with increased bone matrix protein production, independently from hormonal regulation. After a few days of stretch you have in the culture production of osteopontin, production of osteocalcin and other proteins. So the stretching process stimulates the osteoblasts, which differentiate and produce their proteins.
Slide 14
This has been seen also in the intact animals, where lining cells, these flat cells here in the upper panel, after mechanical loading, there is a transformation into osteoblasts, which become active cuboidal osteoblasts. As you can see, there are large nuclei, abundant cytoplasm and so on and this is able synthesize and to elaborate new bone.
Slide 15
And this process has been seen to occur as a trabecular mini-modelling, which has been probably already described by Frost many, many years ago, but again reported by these Japanese authors. You see a certain amount of bone which is superimposed on the trabecular bone. This is not the remodelling process,which is se sequence of resorption and formation. It’s a modelling process which means that there is a direct activation of osteoblasts which elaborate bone. Probably this process is able to confer increased bone competence. It has been observed also in hypoparathyroidism and probably it explains why there are not so frequently fractures in patients with adynamic bone disease and hypoparathyroidism in chronic kidney disease or also in primary hypoparathyroidism.
Slide 16
Well, everybody knows the systemic hormones influencing osteoclastic activity like parathyroid hormone, 1,25 dehydroxide vitamin D and calcitonin.
Slide 17
Parathyroid hormone has an effect mediated by regulation of osteoblastic Cbfa1 expression, which also regulates RANKL ligand expression, and we will see what is the meaning of it. The effect of PHH on the osteoclast is therefore indirect. As far as 1,25-dihydroxyvitamin D, there is obviously an effect on the differentiation of osteoblastic cells but it’s also a potent stimulator of osteoclastic bone reabsorption and through the RANKL ligand system. As far as calcitonin is concerned, there are receptors on osteoclasts but their effect at least what we know experimentally, is very transient but it is also very potent.
Slide 18
Recently it has been shown that when we speak of PTH we have to think also to other species of PTH and I will only briefly refer to this problem. If we measure intact PTH, we measure 1-84PTH but we measure also the so-called 7-84 molecular species, which is about 20-30% of the intact PTH that we measure. In uraemia there is some increase of this 7-84 PTH.
Slide 19
I’m not going through the entire problem, but I would like to remind you of this important paper published by Slatopolsky, who showed that in parathyroidectomized rats the administration of 1-84PTH leads to an increase in the serum calcium. By administering 1-84PTH together with 7-84PTH there is a blunting of this hypercalcemic effect. This finding has been considered by the authors and by other authors as a direct inhibitory effect on the same receptors.
Slide 20
However, we know now that there are different receptors, PTH-1R for the 1-84PTH, for the N-terminal portion of PTH. The other receptor is for the 7-84 PTH. Many of these receptors are, for instance, on the osteocytic cells. Therefore the problem, is still entirely open.
Slide 21
I think to make a synthesis to leave open the problem with this sentence: is PTH 7-84 interfering with bone remodelling in chronic renal failure? We have not yet reached a definitive answer.
Slide 22
I think that the results that have been published so far are not satisfactory to conclude that 7-84 PTH has really, in the physiological and pathological conditions, this effect of interfering in the osteoclastic activity.
I would like to remind you also that we spoke before of 1, 25-dihydroxycholecalciferol. We have recently shown tha also 25 hydroxyvitamin D has important functions, at least in uraemia.
Slide 23
We have shown that levels of 25 OHD lower than 20 nanograms/ml are accompanied by a decrease in bone formation rate in spite of very elevated, PTH levels all throughout, which is not shown here, and very low 1, 25-dihydroxycholecalciferol. So probably there is also a direct effect of 25 hydroxydecholecalciferol, not only 1, 25-dihydroxycholecalciferol.
Slide 24
As everybody knows, there is a very good correlation between PTH and bone formation rate but you can see that there is a large scatter of data, which has been confirmed by several authors. Many have found a large scatter of data, which probably means that there are many other factors, other than PTH, which are at work.
Slide 25
I would like to remind you that the rate of remodelling is controlled primarily by local factors. Compared to cortical bone, trabecular bone has a high surface to volume surface and in contact with bone marrow and what counts is not the yellow marrow but the red marrow, which is rich in cells and is rich also in cytokines and many other growth factors.
Slide 26
A few years ago Hruska et al. published this slide which shows the importance of interleukin 1, tumor necrosis factor, PTH on this side where osteoblasts and pre-osteoblasts are located, and through a list of factors and also matrix dissolution products, which attract osteoclasts, there is activation of the pre-osteoclasts and osteoclasts.
Slide 27
Nine years after, the same authors have shown that there is another actor coming into play which is an important new actor and player. This is the RANKLigand which is produced by osteoblasts and pre-osteoblasts, which links to the RANKL receptors on pre-osteoclasts. This linkage brings to the formation of multinucleated osteoclasts.
Slide 28
So, there is an activation of preosteoclasts, but the system is probably better shown in this slide published by Gonzalez, which shows that under many stimuli there is, in the osteoblasts, the production of the RANKLigand which is destined to be linked to the RANKL receptors on the pre-osteoblastic cells which then differentiate into active osteoclasts. But there is also the production of another protein, called osteoprotegerin, which is known to be a decoy receptor, able to block the RANKLigand and inhibit the RANKL ligand effect on the pre-osteoclasts.
Slide 29
So, we can envisage the process as shown in this slide, also by Gonzalez, that the pool and activity of osteoclasts is a stimulated by the RANKL ligand and is inhibited by osteoprotegerin production. There are several factors which are in favour of osteoprotegerin production like oestrogen, or in favor of RANKLigand, like PTH and glucocorticoids.
Slide 30
Everybody knows that knock-out animals for osteoprotegerin develop osteopenia, because probably there is an excess of osteoclastic activity. The animals show very thin cortical bones, they have fractures, they have also deformities of the spine.
Slide 31
What is the role of the OPG RANKLigand system in renal osteodystrophy?
Slide 32
We know very little, but it has been observed that osteoprotegerin, circulating osteoprotegerin, increases with decreasing renal function. This has suggested the hypothesis that the increase in osteoprotegerin serum levels in renal failure may favour the development of adynamic bone disease and of bone resistance to PTH.
Slide 33
We have shown a few years ago that in adynamic bone disease really osteoprotegerin levels are not higher, lower probably in this group, at least if compared to mixed osteodystrophy.
Slide 34
In addition, we showed that osteoprotegerin correlates negatively with PTH, which is in line with what we know also experimentally and in vitro.
Slide 35
And these authors very recently have shown that with an increase in PTH in hemodialysis patients there is an increase in RANKLigand serum levels. So, in conclusion of these observations, we think that hyperparathyroidism in chronic renal failure is mediated by an excess of RANKLigand and a decrease of osteoprotegerin.
Slide 36
There are other factors anyway which can potentate the activity of RANKLigand, like tumour necrosis factor.
Slide 37
And I show you one slide which shows that 30 nanograms of RANKLigand are less effective than 3 nanograms of RANKLigand in culture together with tumour necrosis factor 1 nanogram/ml. So TNF effectively enhances osteoclastic activity.
Slide 38
Well, this is a sentence by Hruska who has published this sentence in reference to Chronic Kidney Disease: aberrant levels of agents regulating bone remodelling are released into the system resulting in the lack of normal bone formation rates, inappropriate bone absorption and defects in mineralisation constituting renal osteodystrophy.
Slide 39
This is an hypothesis but they are very interesting observations and this is a particularly related to the bone morphogenetic proteins.
Slide 40
The bone morphogenetic proteins are multifunctional regulators of cell growth, differentiation and apoptosis. They are part of the protein family of TGF beta. Bone morphogenetic proteins induce differentiation of mesenchymal cells into osteoblasts and chondroblast lineages.
Slide 41
Well, this is a slide synthesizing experiments published by Hruska very recently.They showed that CKD rats have a pattern of renal osteodystrophy with increased osteoid. By treating with calcitriol there will be a decrease in osteoblasts and the development of the adynamic bone disease pattern, but treatment of CKD with bone morphogenetic protein 7 restores osteoblastic surface and brings about the mineralisation of osteoid.
Slide 42
It has also been shown by Gonzalez et al. that in murine renal osteodystrophy, administration of BMP 7 restores plump osteoblasts on the surface of bone, while when no bone morphogenetic protein is administered there is a marked fibrosis. So we can say that the bone morphogenetic protein 7 prevents the transformation of osteoblasts in fibroblast-like cells, with production of fibrosis.
Slide 43
So, there are very important effects on the renal osteodystrophy and treatment with BMP7 in high turnover osteodystrophy prevents the development of peritrabecular fibrosis, affects osteoblastic differentiation and decreases bone resorption. According to these authors, deficiency of BMP 7, which is produced by the kidney, may be an important factor of the lesions found in renal osteodystrophy.
Slide 44
Let’s speak now of leptin, which also is an important factor, which has peripheral and central control of bone remodelling. It is known, that leptin inhibits osteoclast generation.
Slide 45
Slide 46
In this experiment, peripheral blood mononuclear cells are transformed into osteoclasts by the addition of RANKLigand. As shown in the slide, these osteoclasts are able to erode the surface of bone, as visible by scanning electron microscopy. But the addition of leptin prevents this transformation.
Slide 47
In addition, it has been shown that with increasing leptin concentration there is a decrease in osteoclasts in the slices of bone and a decrease in bone resorption.
Slide 48
We have also shown with bone biopsies in dialysis patients that there is a very good inverse correlation between serum leptin and osteoclastic surface which is in line with the other reported experiences.
Slide 49
But we also know that leptin deficient mice have increased bone density and mass, due to increased bone formation. The effect is through the Neuropeptide Y to receptors in the nucleus arcuatus and the sympathetic nervous system. Therefore, we have to think of leptin as having a dual opposing effect on bone remodelling. There is a peripheral one and there is a central one, central mediated one.
Slide 50
These experiments by Baldock have been carried out on receptor deleted rats for the Y2 receptor, which has the same meaning of making knockout, leptin knockout animals.
Slide 51
If you abolish these receptors so that there is no central effect of leptin, there is an increase of cortical area in the KO animals, and also of cortical thickness. This result is also seen in the trabecular bone, as an increase in trabecular bone, an increase of trabecular number and thickness.
Slide 52
Takeda’s study shows that the effect of leptin is through the hypothalamus and from there initiate a sympathetic nervous system arm which, through beta 2 adrenergic receptors, brings about inhibition of bone formation. If we abolish the effect of leptin, there will be an increase in bone formation.
Slide 53
Leptin has a direct stimulatory effect on bone, an indirect opposite effect via the central nervous system. In uraemia the direct effect prevails with a reduction of bone turnover and bone reabsorption. Probably this special effect in uremia is due to a decreased blood-brain barrier.
Slide 54
I would like to conclude by saying that bone turnover in chronic kidney disease is a very complex phenomenon, with many players at work, with different roles. In spite of the many advances, the relative importance of some of them is not yet defined and still requires extensive investigation.
Thank you.
Chairman: Thank you Giorgio for this very comprehensive review. Do you have a question?
Question: Thank you for this nice review about bone remodelling. You showed that vitamin 1, 25 stimulates osteoclast, what is the implication of this and what doses work in humans and implications of this in the treatment of uraemic osteodystrophy?
Prof Coen: I didn’t understand, what’s the implication of?
Question: You said 1, 25 stimulates osteoclasts more and effect on osteoblasts and implication of this in the use of vitamin 1, 25 in uraemic bone disease and on what doses dose this effect occur?
Prof Coen: Well I’m speaking of normal levels of 1, 25, not pharmacological levels so obviously vitamin D in the form of 1, 25 is the most effective, so I think we have to replace in case of deficiency, I don’t see others.
Question: And the normal level stimulates osteoclasts?
Prof Coen: You have to distinguish between acute effects of 1, 25 which are stimulating osteoclast function reabsorbing bone and more chronic effects which of course increase serum calcium, serum phosphorous and have a pro bone formation function so it is a very different effect which both exist.
Question: Do you have an explanation for the decrease in bone mineral density when you overload your patient with calcium and depress PTH? Do you have a molecular explanation for that? So calcium will kill osteoblasts?
Prof Coen: I would say if there’s a decrease in PTH levels, you have a decrease in osteoblastic activity, you have a suppression of osteoblastic Cbfa-1 and so there is less bone, which is formed. This is one thing.
Question: But all the therapeutical treatment to increase bone mineral density in all populations is a decrease in bone remodelling.
Prof Coen: That’s true anyway there is also a direct effect I think on osteoclasts because there are calcium sensitive receptors as I remember. So it’s a very complicated thing. I think t is very difficult to put together all these things.
Chairman: Thank you very much.
 |
DO YOU HAVE QUESTIONS FOR PROF COEN? |
Send them to Question forum Prof Coen. |
|
We will publish the forum in the next issues of NDT-Educational. |
