| THE PROMISE OF BMP-7 THERAPY IN CHRONIC KIDNEY DISEASE |
| K. Hruska, Saint Louis, USA |
| Chair:
K. Olgaard, Copenhagen, Denmark
|
L. Guerra, Porto, Portugal
|
| |
|
Prof K. Hruska |
Slide 1
Prof Hruska: As Prof Olgaard said I began with BMP-7 in a paper in the New England Journal in 1995 when I speculated that in chronic kidney disease, the observation that when we treated what we understood was renal osteodystrophy or secondary hyperparathyroidism, the surprise observation that you did not get normal bone, led me to speculate some principals that I hope I can convince you about today and at that time we knew, one of the things that I'll be showing you shortly, the phenotype of BMP-7 deficiency and I wondered if BMP-7 was not a therapeutic principal for chronic kidney disease and that's what I hope I can convince you of today.
Slide 2
So the objectives are to discuss other therapy of chronic kidney disease as related to BMP-7. I'll discuss its roles in chronic tubulointerstitial disease or renal fibrogenesis, the final common pathway of most chronic kidney diseases prior to end-stage disease. Because of its actions in renal fibrogenesis, I posed another question and that is whether as a therapeutic principal, BMP-7 might be broadly effective in most forms of chronic kidney disease and therefore decided to analyse what BMP-7 might do in diabetic nephropathy since its our most common form of chronic kidney disease and I'll discuss these results. But as a new therapeutic I think that, perhaps that the exciting observation or the exciting aspect of having this therapeutic is that it's also effective in many of the complications caused by chronic kidney disease, specifically today I'll discuss its role in the treatment and prevention of renal osteodystrophy and associated with its actions in renal osteodystrophy, it appears to be a new therapeutic for vascular calcification and since vascular calcification is the reason that so many of our patients with end-stage kidney disease on therapy die, related to their cardiovascular disease, I think the potential that BMP-7 as a therapeutic for vascular calcification is truly exciting.
Slide 3
So what is BMP-7, it's a member of the Bone morphogenic protein subfamily of the TGF-beta super family, it is a critical morphpogen during development, human development, for the skeleton, eye and kidney.
Slide 4
And the phenotype of BMP-7 deficiency is shown here in the whole mounts of the urogenital system of foetus, a mouse foetus and the deficient mouse urogenital system is shown on the right and you can see 2 atrophic kidneys with the adrenal glands sitting on top. So severe renal hyperplasia is the phenotype of BMP-7 deficiency and the animals die in the first week of life from uraemia. During development BMP-7 is critical for induction of nephrogenesis and in the wild type mouse, you can see that this structure called the ureteric bud is inducing a mesenchymal condensation around the ureteric bud. This is the effect of BMP-7, BMP-7 is made in the ureteric bud, causes this mesenchymal condensation, this condensed mesenchyme becomes the nephron proximal to the collecting duct, so BMP-7 is critical in inducing nephron genesis.
Slide 5
Following its inductive event in the mouse, it is expressed all the time in the ureteric bud but from 10.5 to day 12.5 DPC in the condensing mesenchyme but then it disappears from the condensing mesenchyme after that but continues to be expressed in the ureteric bud. In the deficiency of BMP-7, which you can see is a marked deficiency in the number of nephrogenic developing nephrons such as the comma shaped body and the S shaped bodies, so these are developing nephrons, their number is markedly decreased so there is a marked decrease in nephron induction, the kidneys become hypoplastic and at birth they are hypoplastic, dysplastic kidneys with the cystogenesis, so this is a major renal phenotype.
Slide 6
There's also a skeletal phenotype and the skeletal phenotype has 2 parts to it: the first part is a patterning defect and this patterning defect is seen in B here where there is an extra rib and in E, and F here where there is an extra digit but BMP-7 also causes osteoblastic differentiation and mineralisation so that there's hypomineralisation of the developing skeleton, and you can see that here in this bone from the skull whole mount where you can see mineralisation so this is an alizarin red alcian blue stained whole mount of the skeleton, this is the skull, the alizarin red stain mineralised bone and the alcian blue is cartilage, so you can see in the deficient mouse there's delayed mineralisation demonstrating then that BMP-7 is a potent osteoblast differentiating factor stimulating bone mineralisation.
Slide 7
So finally, its inductive action in kidney development. BMP-7 continues to be expressed at high levels in tubule segments derived from the ureteric bud, that is the collecting duct.
Slide 8
And about 75% of the total body burden of BMP-7 in the adult derives from its production in the collecting duct where it is secreted both into the blood stream and into the urinary space, down stream of the collecting duct BMP-7 exerts the differentiation effects in the pelvic epithelium and in the urinary bladder. The physiologic function of BMP-7 in the adult is as a differentiation factor for the collecting duct, it is also a differentiation factor for the proximal tubule but remember that I said that it disappears from the mesenchyme that's becoming the tubule distal or proximal to the collecting duct, so this represents then either a hormonal or a paracrine action of BMP-7 as a differentiation factor for the proximal tubule. It surprisingly continues to be made in glomerular podocytes so it's an important factor in the glomerulars in the glomerular epithelium. It also has actions in glomerular mesangial cells but again those are either paracrine or hormonally derived. It's also a differentiation factor for vascular smooth muscle and for osteoblasts so that the source then of BMP-7 action may be autocrine, paracrine or hormonal and as I have said most of the body's burden is in the collecting duct.
Slide 9
Renal injuries decrease the expression of BMP-7.
Slide 10
And this is an immunohistochemistry study from the collecting duct following unilateral ureteral obstruction for 72 hours and then released for 1 week, so what I'm showing you then is the normal intramedullary collecting duct with uniform expression of BMP-7 in almost all its cells except for some of the intercalated cells, however there's a marked loss of epithelial cells and loss of BMP-7 expression in the intramedullary collecting duct following unilateral ureteral obstruction and recovery. When you treat the animal beginning at day 3 with BMP-7, you see surprising partial reconstitution of the epithelium and re-expression of BMP-7, so here with a low dose of BMP-7 of 10 micrograms per Kg every other day, you can see a partial recovery but a near complete recovery when you treat it with a higher dose. The positive control in these studies was treatment with our main renal therapeutic, angiotensin converting enzyme inhibition and so this is the effect of enalapril over the same period of time and in this animal model of renal fibrogenesis enalapril did not cause re-expression of BMP-7 and you can also see it was less effective in restoring the intramedullary collecting duct epithelium, so in renal fibrogenesis it was clear that BMP-7 outperformed ACE inhibition.
Slide 11
Other sites of production do not adapt for the effects of renal injury on BMP-7 production, so chronic kidney disease then becomes a state of BMP-7 deficiency. This loss of differentiation factor influence is permissive for the cellular effects of renal injury that is, for instance in diabetes where you have hyperglycaemia induced renal injury, the hyperglycaemia effect decreases BMP-7 and increases TGF-beta production but because of the absence of TGF-beta the actions of TGF because of the absence of BMP7, the actions of TGF-beta are unopposed and TGF-beta produces sclerosis and fibrosis.
Slide 12
So what happens if BMP-7 is replaced during renal injury, well the surprise is that BMP-7 is an effective agent in preventing or treating various forms of renal disease and first I'll take you through a tubulointerstitial nephritis.
Slide 13
So our model of tubulointerstitial nephritis is a fairly faithful one and I've already mentioned it and that is that of unilateral ureteral obstruction and we specifically use release of obstruction to be able to analyse recovery of renal function and recovery of glomerular filtration rate and what I'm showing you here are trichrome stains of 2 kidneys, normal controls in A and B and in C and D corticomedullary sections of kidneys that have been obstructed for 3 days, released and then analysed at day 10, so 7 days of recovery. And these animals also had the treatment with the vehicle every other day and what you can see in this stain is that cells stain red, so here are tubular epithelial cells staining red and you can see that there is a small amount of blue stain in this perivascular space here and you can see that Bowman's capsule stains blue, so collagen stains blue in this trichrome and what you can see that has happened after the unilateral ureteral obstruction and a week of recovery is that there is tremendous collagen genesis, so this is a cortical fibrosis. You can see tremendous infiltration of the interstitial space and you can see major expansion of the interstitial space. One of the characteristics of the kidney is you can see there's very little interstitial space, that the tubules abut on each other, so for instance in terms of BMP-7 action, if this was a proximal tubule, it could be abutting a cortical collecting duct and that would be one possible way that BMP's paracrine actions are mediated, well there's tremendous renal fibrogenesis and with 3 doses of BMP-7 administered at day 3, 5 and 7 you can see at 10 micrograms/kg that there was a partial resolution of this effect. You can still see a significant increase in the interstitial space but most of the collagen deposition has been alleviated. With the higher dose of BMP-7 you have restoration of almost normal renal architecture, so BMP-7 in tubulointerstitial nephritis is a potent therapeutic agent.
Slide 14
So this is obviously an example of renal fibrogenesis and renal fibrogenesis is a final common pathway by which chronic kidney diseases progress, it is activated by stimulation of the TGF-beta signal transduction pathway and it involves epithelial-to-mesenchymal transdifferentiation.
Slide 15
The cells contributing to this production of collagen are interstitial miofibroblasts. These miofibroblasts are characterised by expression of vascular smooth muscle type collagen as a marker and studies over the past several years have demonstrated that the origin of at least some of these miofibroblasts actually represent transdifferentiation of renal epithelial cells. So renal fibrogenesis involves this epithelial-to-mesenchymal transdifferentiation and BMP-7 is a potent inhibitor of this process. As I've shown you, BMP-7 is more potent than ACE inhibition in reversing this tubulointerstitial nephritis but both agents are effective inhibitors of epithelial-to-mesenchymal transdifferentiation. Well BMP-7 has been shown to be a potent therapeutic in several other forms of chronic kidney disease by other investigators besides myself. For instance in this murine model of lupus nephritis, Zeisburg et al. showed major protective effects of BMP-7 and in a very important paper in Nature of Medicine last year, the effects of BMP-7 in preventing the epithelial-to-mesenchymal transdifferentiation stimulated in Heymann's nephritis was clearly demonstrated along with beautiful molecular mechanisms.
Slide 16
Well, importantly for consideration later, BMP-7 in tubulointerstitial disease markedly decreased inflammation, it decreased the number of interstitial macrophages and decreased the activity of the macrophages that were present so that BMP-7 is a significant anti inflammatory principle in tubulointerstitial disease. This may have important systemic ramifications for the actions of BMP-7 in chronic kidney disease and I'll come to this perhaps later.
Slide 17
Well, as I've already said the question then became whether BMP-7 would have brought efficacy in chronic kidney disease across many disease entities and thus we decided to study diabetic nephropathy.
Slide 18
It's the most common form of chronic kidney disease in the United States and Europe and early in the disease, renal hypertrophy thickened basement membranes of the glomerulars and of the tubules and mesangial expansion of the glomerular or in the glomeruli are observed. Loss of glomerular infiltration late in the disease is associated with proteinuria and glomerular sclerosis.
Slide 19
Administration of streptozotocin produces a model in animals of type I diabetes mellitus and we employed long-term streptozotocin induced diabetes to develop a model of diabetic nephropathy in the rat. This model is relatively faithful to the clinical course of human type I diabetes mellitus in diabetic nephropathy. The stages and progression of the nephropathy are similar to the human disease so that there's a stage 1, which is no disease, a stage 2 which is microalbuminuria, a stage 3 which is overt nephropathy with the onset of macroproteinuria or macroalbinuria and a stage 4 is end-stage kidney disease. The therapeutic efficacy of angiotensin converting enzyme inhibition in human medicine was first demonstrated in streptozotocin induced diabetes mellitus in the rat.
Slide 20
And therefore this was the model of diabetic nephropathy that we elected and analysed of BMP-7 and this is the model that we were able to establish.
Slide 21
A single dose of streptozotocin was introduced at time 0 here and by 16 weeks you can see tremendous renal hypertrophy and in this set of studies kidney weight was then stable from 16 weeks to 32 weeks, whereas there was loss of GFR going from this massive hyperfiltration at 16 weeks to about a 30% decrease in GFR at 32 weeks. So this is the stage of overt nephropathy and the progression of chronic kidney disease in this model of streptozotocin induced diabetes mellitus and diabetic nephropathy. These are the effects of BMP-7 in this model, so here's the 16 week hyperfiltering kidney with its GFR, the normal control in our hands had a GFR of about 0.5 ml/100 g body weight and at 32 weeks of diabetic nephropathy you can see that GFR was 0.35. With ACE inhibition as a control, you can see that GFR was normal. With the highest dose of BMP-7, the GFR was above normal but was maintained normal with the intermediate doses of BMP-7 and the low dose of BMP-7. So what the process that was going on here represents from going from here to here, is glomerular sclerosis and decrease in filtration surface. So this represents partial protection against glomerular sclerosis and preservation of GFR. Also this is early renal injury and if a potent therapeutic is actually going to reverse this, so some of this decrease in GFR is actually loss of renal hypertrophy and I'll show you that.
Slide 22
This is the protein excretion in this animal model. You can see at 16 weeks you have early overt nephropathy with the rat putting out about 25 mg of protein per day but from 16 weeks to 32 weeks nephropathy became nephrotic in range and was quite variable but very heavy with many of the rats having overt nephritic syndrome and you can see that there was a dose ordering of the effects of BMP-7, so low dose, intermediate dose and high dose with the high dose of BMP-7 normalising protein excretion to the level of 13 mg per day. So there was a potent protection in terms of proteinuria induced by BMP-7 in this animal model. The positive control here of ACE inhibition performed at the level of the intermediate dose of BMP-7. You can see that BMP-7 was effective in reducing glomerular area.
Slide 23
In other studies it has been shown to decrease glomerular volume. The interstitial volume was significantly increased but the disease here is relatively mild so that there was very little tubulointerstitial disease actually being developed. In our unilateral ureteral obstruction model to put this data in context, interstitial volume normal of about 9% went to 55% so you can see that the effects of diabetes here was significant in that increased interstitial volume from 9% to 13% but this is very mild however, it was significantly reduced both by enalapril and BMP-7.
Slide 24
The major action of BMP-7 in this model is protection against glomerular sclerosis and here you can see what we've counted is the percent of glomeruli being effected with glomerular sclerosis. This is one glomerular sclerosis, one area where streptozotocin diabetic nephropathy is not faithful to the human situation. The glomerular sclerosis of streptozotocin diabetic nephropathy is an FSGS whereas as you know the glomerular sclerosis of diabetic nephropathy in the human is a pan glomerular sclerosis, a diffuse glomerular sclerosis. Yet the sclerotic kidney is the mechanism by which glomerular infiltration failure develops and so this protection against the development of glomerular sclerosis is the major therapeutic effect of BMP-7 in diabetic nephropathy. And you can see there was again dose ordering of the action of BMP-7 and glomerular sclerosis was almost completely prevented by the highest dose of BMP-7.
Slide 25
Just to show nephrologists renal biopsy, this is a normal Spreg-Douli rat kidney and for putting 2 glomeruli here purposely.
Slide 26
So that you can see the effects then of the tremendous glomerular hypertrophy that you get with diabetic nephropathy in this model with 1 glomerular being equal to 2 almost and you can see that at 32 weeks you have a significant segmental sclerosis in this segment and an early segmental lesion in this segment with the intervening segment being pretty normal.
Slide 27
The effects of BMP-7 at high dose are to pretty normalise the renal glomerulars. You can see the mesangial expansion has been prevented, the glomerular basement membranes look distinct and very nice.
Slide 28
Enalapril was not as effective in preserving glomerular morphology in this animal model, while it prevented glomerular sclerosis you can see that there's still a significant mesangial expansion in many of the glomeruli in the enalapril treated.
Slide 29
So I've shown you that BMP-7 reverses renal injury, effectively treating and slowing progression of chronic disease. You must remember that the diabetic nephropathy trial was a treatment trial in that it began at the stage of overt nephropathy but we now began to ask additional questions. My original interest in studying BMP-7 was whether it would have an impact in renal osteodystrophy and so we first were able to look at the effects of BMP-7 in osteitis fibrosa, secondary hyperparathyroidism.
Slide 30
We published this in 2002.
Slide 31
What we were able to demonstrate is that if you induced 5/6 nephrectomy in a rat or an ablative type of 5/6 nephrectomy in the mouse using coagulation of one kidney and contralateral nephrectomy, you induced a classic form of renal osteodystrophy referred to as osteitis fibrosa and when you added to this high turnover renal osteodystrophy BMP-7 therapy, you saw a tendency for there to be a decrease in osteoblast number.
Slide 32
But a significant increase in osteoblast number, so even though it was a high turnover disease, the osteitis meaning there's increased numbers of osteoblasts, BMP-7 was still effective in increasing osteoblast number here. It decreased osteoblast surface measured here by the eroded surface, so it increased bone formation and decreased bone resorption in this model of high turnover renal osteodystrophy.
Slide 33
It had no significant impact on the osteoid surface but it significantly increased osteoblast surface, bone formation rate and adjusted apposition rate.
Slide 34
So the interpretation of this has come after the studies were actually completed. We now know from studies that I'm going to show you next is that secondary hyperparathyroidism actually represents an adaptation for the A dynamic bone disorder and as such it is a maladaptation and there's an abnormal morphology and abnormal function in osteitis fibrosa of the osteoblasts and what BMP-7 does in high turnover renal osteodystrophy is reverse this and restores bone formation or osteoblast function to its normal anabolic situation, so it maintains a high turn-over state possibly stimulated by PTH in the presence of BMP-7 but it restores the osteodystrophy.
Slide 35
So I've mentioned the A dynamic bone disorder, what about it. Well, first we needed a model of the A dynamic bone disorder and this need actually led us to an important pathophysiologic advance. We postulated that if you induced chronic kidney disease but never allowed abnormalities in calcium, phosphorous, parathyroid hormone or vitamin D to happen that you would actually develop an osteodystrophy and that's what we were able to demonstrate to establish this model. The surprise, it was not a surprise, but as predicted what you ended up with was an A dynamic bone disorder, so what we did is took our mouse model of chronic kidney disease induced by coagulation of the cortex of 1 kidney and a controlateral nephrectomy and then treated the mice with proportional reduction of phosphorous intake to prevent hyperphosphatemia and a hyperparathyroidism and we also added calcitriol supplementation to prevent calcitriol deficiency, mimicking then the clinical situation which we use in chronic kidney disease that is phosphate restriction and calcitriol supplementation. Well, of course in the animal you can be effective with this approach whereas probably in humans we can't be and the outcome was that calcium, phosphorous and parathyroid hormone levels were maintained normal, calcitriol levels were slightly elevated, however bone formation rates, mineral apposition rates and osteoblast surfaces were decreased and what we produced was the A dynamic bone disorder.
Slide 36
So here's a section from the distal femoral metaphysis showing a tubercular surface that is extremely inactive in these mice. You can see a few lining cells but this is a very inactive skeletal tubercular surface. So this is the A dynamic bone disorder and the A dynamic bone disorder is caused directly by kidney disease, so what does that mean saying that in other words, is that chronic kidney disease decreases skeletal anabolism.
Slide 37
Kidney renal injury decreases skeletal anabolism, so one way of testing that statement of course was to treat with a skeletal anabolic, so this is what happens then when we treated the A dynamic bone disorder with BMP-7. These are the animals: you can see that the level of renal insufficiency that we produced was relatively mild, about a reduction in GFR estimated by blood urea nitrogen of a little more than 50%, so this is early chronic kidney disease and you can see that in the chow fed animals we induced secondary hyperparathyroidism but in the phosphate restricted animals the yellow symbols in this and in all the subsequent slides, you can see that parathyroid hormone levels were maintained normal and BMP-7 had no further effect on PTH levels.
Slide 38
Surprisingly in these animals you can see that we started out with a normal plasma phosphorous but with BMP-7 therapy the plasma phosphorous was actually decreased and this was the first clue that BMP-7 therapy in chronic kidney disease is an anti hyperphosphatemic therapeutic and the way that BMP-7 effects plasma phosphorous or serum phosphorous in chronic kidney disease is it's driving phosphorous into the skeleton and one of the things that we didn't appreciate until these studies was the potency of the skeleton as a phosphate reservoir in chronic kidney disease and that you can significantly effect the serum phosphorous by increasing phosphate exit from the blood space into the skeleton with your therapeutic and it actually turns out that this is the critical link between the skeleton and vascular calcification. Calcium was not effected, calcium phosphorous product was decreased by BMP-7 therapy. You can see what happened as expected in the chow fed animals, they became hyperphosphatemic and these are, the green is the sham operated, low phosphate treated control group and the yellow is the vehicle-treated chronic kidney disease maintaining normal phosphorous throughout the course of chronic kidney disease, so despite normal phosphatemia, normal parathyroid hormone, normal calcitriol you can see what happened to the skeleton.
Slide 39
There was a tendency for a decrease in bone volume, tubercular number, tubercular separation and osteoid volume were not changed. The only manifestation of secondary hyperparathyroidism that we saw in this mild chronic kidney disease was a significant increase in osteoid volume.
Slide 40
But you can see that in the A dynamic group there was a significant decrease in osteoblasts number, osteoblast perimeter, whereas the osteoclast was not effected so that now there is an imbalance between osteoblast activity which is markedly decreased and maintained osteoclast activity, so the A dynamic bone disorder is an osteopenic state and if it persists for a long time will lead to osteoporosis.
Slide 41
You can see that there's a marked decrease in the double labelled surface and mineralising surface that was restored from low levels to normal by BMP-7 therapy.
Slide 42
And the key is the bone formation rate in the A dynamic group was decreased and restored to normal by BMP-7 and the adjusted apposition rate which represents the number of bone modelling units being activated per unit time is markedly decreased in the A dynamic and restored to normal by BMP-7 therapy.
Slide 43
An important therapeutic for renal osteodystrophy in that it appears to be effective in both of the major forms of renal osteodystrophy. Renal osteodystrophy is an important complication of chronic kidney disease but an even more important complication of chronic kidney disease is vascular calcification I think. And then the question became, first as a toxicity study, you may not know how the bone morphogenetic protein family was discovered but it was discovered by an investigator in 1965 by the name of Marshall Urist who took demineralised bone powder and put it into a muscle and what he generated by doing this was the entire programme of endochondral bone development. And the active proteins involved in this process were the BMPs so there was concern of giving a BMP systemically as to whether or not she would actually activate heterotopic calcification and so we looked at vascular calcification as an initial toxicity study but we ended up with major therapeutic benefit.
Slide 44
What we did here was we used an animal model of vascular calcification, this is a difficult thing to come to but we were intrigued by the finding of vascular calcification in this low density lipoprotein receptor deficient mouse fed a high fat cholesterol western type diet. So LDLR deficiency is of course familial hypercholesterolemia in the human and if you take a hypercholesterolemic subject and feed him a high fat diet, you induce the metabolic syndrome, so it turns out that the LDLR deficient mouse fed this high fat diet is actually a model of the metabolic syndrome, it develops type II diabetes over time and so it's actually a very attractive model, it develops vascular calcification and what we did is we added ablative chronic kidney disease to this model of the metabolic syndrome. What we did is we did surgery to induce chronic kidney disease and instituted diets at 10-12 weeks of age, then we began treatment and we stopped in our prevention trials at 22 weeks, this there had significant calcification, so in our treatment trials we start at 22 weeks and sacrifice at 28 weeks, so we sacrifice either at 28 or 22 or 28 weeks.
Slide 45
So these are studies from our first prevention trial and these all represent sections of the aortic route. Here is an aortic valve leaflet coming off, here is one aortic valve leaflet and here is the other aortic valve leaflet, so this is aortic route and this is aortic route and you can see at the aortic route there is this atherosclerotic plaque and within this plaque this is an alizarin red stain you can see this punctate calcification, so these are LDLR deficient mice fed chow. When you feed them a high fat diet you can see that you induce an increase in the number of these calcifications and you also induce medial calcification when you add chronic kidney disease to LDLR deficiency in high fat feeding you increase the size, this is a degraded, large, medial calcification but you also increase the number of these calcifications in the neointima. You can see that this is a plaque from the section here and you can see a marked increase in the number and you can see it well here at this lower power, the effect of chronic kidney disease to increase the number of these neointimal calcifications, so that there's a dual action of chronic kidney disease in this model of the metabolic syndrome with chronic renal insufficiency, there's both an effect on atherosclerosis and there's a stimulation of arteriosclerosis but there's clearly a direct effect of chronic kidney disease to increase vascular calcification, so again it was a model for our end stage kidney disease.
Slide 46
And this is the impact of BMP-7, you can see that we have enlarged this section but basically vascular calcification has disappeared. There was heterotopic calcification of many tissues in this animal model. The lung is effected but perhaps most obviously effected is the tracheal cartilage which becomes completely calcified in this animal model and you can see that BMP-7 reverses that.
Slide 47
When we got into mechanism of action we were able to demonstrate that part of the action here is abnormal phenotype expression of cells producing this vascular calcification, so here are medial cells from wild type mice stained for osteocalcin and you can see that the propriate is negative but with LDLR deficiency the genetic influence, you begin to see dystrophic expression of osteocalcin in various tissues, here the vascular smooth muscle. And when you add high fat feeding the environmental influence there's a marked increase in osteocalcin expression. That's further intensified when we add chronic kidney disease and BMP-7 therapy restores osteocalcin expression to its normal tissue restricted site, the osteoblast of the skeleton, so one mechanism then of BMP-7 in vascular calcification is that it's protecting the phenotype of the vascular smooth muscle cell maintaining the contractile nature of the cell and inhibiting its becoming a proliferative calcifying cell.
Slide 48
Again for the trachea you can see wild type trachea, early expression of osteocalcin by the genetic influence when you feed a high fat and induce chronic kidney disease, as extremely high levels of osteocalcin expression that are eliminated by BMP-7 therapy.
Slide 49
So in summary BMP-7 is effective in preventing and treating tubulointerstitial nephritis and progressive chronic renal diseases of various types including diabetic nephropathy. Renal fibrogenesis is a final common pathway.
Slide 50
The actions of BMP-7 in chronic kidney disease involve inhibiting TGF-beta stimulated processes, BMP-7 has multiple efficacies in chronic kidney disease, the renal disease itself, renal osteodystrophy and vascular calcification. Chronic kidney disease causes a loss of skeletal anabolism and BMP-7 is a candidate for one of the principals rendered deficient by chronic kidney disease.
Slide 51
Vascular calcification in chronic kidney disease derives in part from an abnormal function and phenotypic expression of cells in the vascular media. BMP-7 normalises vascular smooth muscle cell phenotype however, this mechanism of BMP-7 action is only part of the story, it's more complex because BMP-7 also decreases the serum phosphate and serum phosphate is a direct stimulus for this phenotypic drift of the vascular smooth muscle cell, so that it turns out that there's a direct link between the skeleton and the vascular smooth muscle cell and the effects of BMP-7 on renal osteodystrophy are a big part of the mechanism by which it protects the vasculature and therefore bringing forward BMP-7 as a renal therapeutic for renal osteodystrophy should get us vascular calcification and this may be the strategy in which we go forward.
Slide 52
I'd like to acknowledge the people that did all the work, a series of renal fellows that I've had in the lab, the pleasure to have in the lab over the past several years, Richard Lund, Matt Davies, Song Wang, Qing Chen, Suresh Matthew and Ting Ting Li. My collaborators Kuber Sampath and Marc Charette.
Slide 53
I was funded by NIH and I have a disclosure obviously.
