Slide 1

Thank you very much. First I’d like to thank the organisers of the program for their kind invitation and I would like to proceed to follow this excellent presentation with a different focus in two categories. One of them is I will focus my discussion on proximal tubule cell cytotoxicity of myeloma light chains, second, our studies that investigate the pathophysiologic mechanisms which identify potential opportunities for novel interventions in treatment of myeloma nephrotoxicity which converge on this peptide which we call PACAP, pituitary adenylate cyclase activating polypeptide in myeloma kidney.

Slide 2

As was pointed out by Doctor Bradwell, multiple myeloma involves the kidneys in approximately 50% of patients. Multiple myeloma is the 6th most common tumour, the second most common hematologic malignancy. Approximately 25% will have kidney failure. Approximately 1% of ESRD patients started on dialysis has myeloma kidney. The presence of kidney toxicity is independently associated with a 2.5-fold increase in all cause mortality and as of today there is no effective treatment for kidney involvement although as you have heard in the preceding talk, hope is underway.

Slide 3

What I would like to point out is that the most common kidney disorder associated with multiple myeloma is this lesion you?re seeing which is called myeloma kidney and it is predominantly a tubulointerstitial kidney disease. Casts are present, these are formed by interaction of light chains with Tamm-Horsfall protein but there is a prominent tubule atrophy and interstial fibrosis.

Slide 4

Those of you with pathology backgrounds will easily appreciate from this trichrome stain kidney biopsy of a patient with multiple myeloma, the extensive abundant amount of fibrosis present in the interstitium and also note the absence of casts.

Slide 5

Here I would like to point out that in myeloma kidney presence of casts is not a sine qua non. The predominant event appears to be a tubulointerstitial process. They are frequent but they are not present 100% of the time.

Slide 6

As Doctor Bradwell pointed out, that free light chains in myeloma are filtered relatively unhindered in the glomerulus, they present first to the proximal tubule cells. Proximal tubule cells are the most abundant cell types in the kidney and they participate, they have a very important role in the handling of filtered light chains, they bind to megalin and cubilin, the tandem endocytic receptors which were shown in our laboratory as the receptors that mediate internalisation of light chains and they are presented into the endosomal lysosomal pathway and in the lysosome they are degraded into the aminoacids constituents where they are reabsorbed in the peritubular capillaries as a way of conserving aminoacids perhaps.

Slide 7

This is a picture from our laboratory of 2 adjacent human proximal tubule cells demonstrating vacuolar localisation of FITC conjugated light chains in these presumed endosomes around the 2 nuclei. In excessive quantities and here let me digress and point out to you that kidney involvement in multiple myeloma with rare exceptions, perhaps in hypercalcemia, is always associated with light chain overproduction. Although there’s variability among light chains the quantity is a factor and again referring to Doctor Bradwell’s studies removing these light chains benefits these patients. In vitro, when the human proximal tubule cells are exposed to higher concentrations of light chains that may be present in the proximal tubule of patients with multiple myeloma, you see severe cytotoxicity. Here at 25 umol concentration, here at 50 umol concentration.

Slide 8

At this point I'd like to telegraph to the audience that the severe disruption and formation of filopodia in these proximal tubule cells are suggestive of epithelial mesenchymal transition which may well have been contributing to the intense tubulointerstitial fibrosis seen in these patients. Electron microscopically you see intense vacuolisation in human proximal tubular cells and at least in 2 cells apoptosis as evident here by involuted nuclei.

Slide 9

Protein overloading, in this case light chain protein overloading induces stress responses in these cells and we see here 4 different kappa light chains and in two lambda light chains inducing intense IL-6 responses in human proximal tubule cells. In comparison human serum albumin had minimal or no effect and this contrasts with some of the studies reported in other laboratories but nevertheless, this is what we consistently find in our laboratories.

Slide 10

The IL-6 response due to light chains in the medium is immediate, is brisk within about 6 hours and is sustained over 48 hours. It is dose-dependent and it reaches a plateau at approximately 50 umol concentration in the medium. These light chains not only induce IL-6 but they also induce IL-8 again in comparison to human serum albumin you can see how much more potent light chain effect on the cytokines is and here is MCP-1. In cytokine arrays we're finding many other inflammatory and proinflammatory cytokines induced by light chain addition into culture medium.

Slide 11

This next slide shows also again IL-6 and TNF-a in response to light chain exposure in these human proximal tubule epithelial cells.

Slide 12

Light chain exposure in vitro is associated with activation of NF-kB.
Here's a 5 umol nuclear extract, NE and 9, 5 ug NE is shown here. 2 different lightchains you see marked presence of p50 here this is demonstrated immunocytochemically. NF-kB is dormant inactive in the cytoplasm in control cells. After exposure to light chain, they are translocated to nuclei and they bind to promoter regions in the DNA and drive the transcription of these many inflammatory and proinflammatory cytokines.

Slide 13

That NF-kB is pathophysiologically important is demonstrated by these experiments. Aspirin and PDTC, two known potent inhibitors of NF-kB abrogated these cytokine responses here IL-8 with aspirin, here IL-6, IL-8, MCP-1 and with PDTC the same abrogation of MCP-1 is demonstrated in the right hand panel.

Slide 14

Also involved in these processes are signalling events that involve the MAPKs. Phospho-ERK is stimulated here by exposure to light chains. JNK and other MAPKs were also participating in this process but what emerged as most important was p38 MAPK.

Slide 15

As shown here within 10 minutes of exposure there is intense phosphorylation of p38 MAPK.

Slide 16

This can be inhibited by its specific pharmacologic inhibitor  SB203580.

Slide 17

And inhibiting p38 MAPK prevents light chain-induced cytokines IL-6 here and MCP-1.

Slide 18

So this emerged as a possible interventional or therapeutic opportunity.

Slide 19

The importance of p38 MAPK is demonstrated here in these experiments in which these MAPK genes were silenced using short interfering RNAs. I would like to call your attention to p38 experiments. Silencing the p38 gene with siRNA eliminated light chain-induced response with IL-6 and MCP-1. p38 MAPK is also involved in epithelial mesenchymal transition induced by light chains. Here you could see disappearance of the epithelial cell marker E-cadherin when the cells are exposed to kappa light chain and an increase in the a-SMA marker for myofibroblasts in light chain exposed human proximal tubule cells. These responses are eliminated if the p38 MAPK gene is silenced.

Slide 20

Here is where we come to PACAP 38, we kind of stumbled on this because of our interaction with a scientist who was very interested in this peptide. This peptide attracted our attention because of its well known effects on p38 MAPK. But to introduce to you this peptide. This is a pleiotropic neurotrophic factor and it can be a catecholamine secretagogue, it has potent immunomodulatory effects on various immune responses by suppressing or stimulating the production of proinflammatory cytokines, a very important modulator of p38MAPK and is currently being explored as a potential therapy for a variety of autoimmune and inflammatory diseases such as rheumatoid arthritis, septic shock, asthma, Crohn's disease, pulmonary hypertension, multiple sclerosis.

Slide 21

This is the PACAP molecule, this molecule is related closely to vaso intestinal polypeptide, VIP type peptides and it shares close homology here with the VIP up to any 7 aminoacid sequence.

Slide 22

It is most abundant in the hypothalamus where it was originally identified and isolated from it?s also abundant in the testis. Not that much presence in the kidney.

Slide 23

Again an effect of PACAP on cell morphology. These are control cells we can appreciate the confluence of these proximal tubule cells, human proximal tubule cells when exposed to light chain they lose their confluence and they are distorted and their numbers are decreased. Here when light chain exposure is antagonised by PACAP, the confluence is restored and cell numbers are also restored.

Slide 24

PACAP had a very profound effect on light chain-induced cytokines. I would like to call your attention here just to this column. These are detailed experiments. PACAP 10 to the minus 9 molar concentrations almost completely eliminated the TNF response induced by light chains. Similarly with IL-6. These responses are similar also to those induced by dexamethasone but PACAP appeared to be much more potent by molar comparison than dexamethasone.

Slide 25

How are these effects mediated? There are a number of PACAP receptors and splicing variants. This is the complete PACAP receptor which has 3 intracytoplasmic loops and there?s a splicing variant resulting with a shorter variant and all of them are responsible in mediating various biological responses and we then explored which receptors were participating in these responses.

Slide 26

As you can see here that the bone marrow stromal cells, which are important in myeloma, in multiple myeloma cells and human proximal tubule epithelial cells all had the VPAC1 receptors. These are various types of receptor inhibitory experiments and VPAC1 Receptor antagonists were  the most effective in competing with PACAP and preventing protective effect with PACAP suggesting that the effects of PACAP on the kidney cells are mediated through VPAC1 receptors. Now if PACAP had a stimulatory effect on multiple myeloma cell lines it would be useless as a potential therapy.

Slide 27

In these experiments we examined the effect of PACAP on 4 different myeloma cell lines shown here is that in increasing concentrations PACAP suppressed all these 4 different myeloma cell lines, two of them secreting kappa light chains, two of them secreting lambda light chains. The interaction of myeloma cells with bone marrow stromal cells and bone cells is a form of unholy alliance if you like. This interaction stimulates the tumour growth and secretion of IL-6 in the bone stromal cells and TNF-a which renders myeloma cells also resistant to therapy.

Slide 28

We then examined the effect of PACAP on this symbiotic relation between the multiple myeloma cells and bone marrow stromal cells. As you can see, that when the multiple myeloma cells are co-cultured with bone marrow stromal cells, there is intense production of IL-6. If you treat them with PACAP, this is completely normalised.

Slide 29

Here we see varying concentrations and at 10 to the minus 9 molar concentration PACAP prevented this response. Here this is shown morphologically, myeloma cells by themselves, bone marrow stromal cells by themselves. When These cells are co-cultured there is intense proliferation ofmyeloma cells, with PACAP this is neutralised.

Slide 30

This experiment is shown here quantitatively with PACAP multiple myeloma cell proliferation is completely eliminated.

Slide 31

Again, in these experiments we explored the participation of various PACAP receptors. This detailed experiment showed for the sake of brevity both again VPAC1 receptor and PACAP's own receptor was participatory in antagonising or opposing the actions of PACAP here suggesting that both receptors participated here.

Slide 32

PACAP in bone marrow stromal cells inhibited TNF-a both at the transcription level and also at the protein level in a dose-dependent manner. PACAP also suppressed MAPKs here shown. NF-kB shown here with light chains and IL-6 shown in this column and p38 MAPK in these bone marrow stromal cells when co-cultured with multiple myeloma cells.

Slide 33

We then moved down to an in vivo experiment. This is a slide from rats infused with light chains and when rats are infused with human light chains, there is intense production of TNF-a in rat kidney and this can be opposed very effectively by simultaneous infusion of PACAP.

Slide 34

Let me again remind you epithelium mesenchymal transition in light chain exposed human proximal tubule cells is very similar to a known positive controlled TGF-a effect. EMT inducing effect and you can see this morphologically here. The time course is shown here, a loss of E-cadherin and up here of a-SMA.

Slide 35

The protective effect of PACAP is nearly identical to that of BMP-7  a known antagonist of epithelial mesenchymal transition restoring cell morphology in light chain exposed human proximal tubule cells.

Slide 36

The prevention from EMT is also evident in this experiment. Light chain exposed human proximal tubule cells shed their epithelial cell marker, soluble E-cadherin to the medium and PACAP prevents this in a manner similar to that induced by bone marrow morphogenic protein 7.

Slide 37

We had now enough courage to infuse PACAP in a patient with multiple myeloma after informed consent from this patient who had a very strong science background and obviously permission from human research committees and with a single patient IND from the US FDA. This shows the pharmakinetics of PACAP in this single patient. It achieves rapid plateau within 30 minutes and after completion of infusion there's rapid clearance from plasma.

Slide 38

In this single patient study PACAP resulted with marked diminution of lambda light chain excretion in the urine of this patient from 13 g to 751 mg. The infusion was also associated with subjective sense of wellbeing as well as reported again subjective decrease in bone pain.

Slide 39

So to summarise PACAP appears to be a promising agent to antagonise proximal tubule mediated actions of light chains, toxic effects of light chains at a transcription level, at a cell signalling level suppressing the inflammatory and the pro-inflammatory cytokines which may also be stimulated by light chain Tamm-Horsfall protein interaction in the proximal tubule and preventing fibrosis and tubulointerstitial disease which is the most common type of kidney disease in myeloma.

Slide 40

To conclude, PACAP as I demonstrated in this presentation, preserved cultured kidney cell morphology, it prevented LC-induced cytokine responses in vitro and in vivo. It had marked antiproliferative effects on myeloma cells. It inhibited IL-6 and cell growth in both myeloma cells and bone marrow stromal cell co-cultures. It prevented epithelial mesenchymal cell transition. Its mechanism of action appeared to be through neutralising NF-kB, antagonising cell signalling molecules particularly p38 MAPK. Its effects are mediated predominantly via VPAC1R. It may be beneficial in other proteinuric kidney diseases as well and it is safe to use in humans. Thank you very much.