Good morning, it seems like we were here dancing not very long ago.
So those of us who live here on planet earth are keenly aware of the ubiquity of water.
Water of course is essential for life, it's also necessary for commerce and a source of great recreational enjoyment.
But in this equilibrium water can be quite hazardous.
This tranquil artic stream which I was paddling with my son, turned into a torrent by dropping a metre in altitude.
In our bodies water is also ubiquitous, all of our tissues are mostly water.
The release of fluids such as spinal fluid, tears, sweat, saliva, the concentration of urine
are all examples of fluids were water release or water uptake is essential.
And a disorder of this causes problems such as brain edema, glaucoma, cataracts, cystic fibroses,
renal failure and other problems.
So I'll talk about the issue of water in biology but I thought I would start, since we're surrounded here by our young people,
by pointing out that as laureates we didn't start in our dotage, we started as young scientists, like yourselves.
I know it's the ravages of time but that was me when I started my scientific career, about your age, seeking adventure
by travelling through Asia. Experiencing for the first time the third world.
And looking for an opportunity to create something in the laboratory that might be useful for the wellbeing of others.
So our laboratory, not a very photogenic group at Johns Hopkins, included people from pretty unusual backgrounds.
A Spanish anarchist, an Italian playboy, a big wave surfer from Hawaii.
I'm the one with the little eye patch, I thought that was so clever at the time, it looks ridiculous.
My project in this laboratory was to identify and isolate the toxin causing travellers' diarrhoea.
A horrific problem and a problem of the natives in the developing world where 10's of thousands of small children
and infants will die every year because of diarrhoeal diseases.
So really it was this project that made me think about water and biology for the first time.
And gave me a purpose in the laboratory.
And I re-purposed my life as a physician to become a physician scientists studying the basis of disease with the hope
that we could prevent or cure horrible diseases such as traveller's diarrhoea.
So when I began my academic career in the faculty after haematology training, we were studying red blood cell proteins
and one of the unsolved, one of the many unsolved problems in red cells is the nature of the rhesus blood group antigen,
the Rh antigen being very well recognised for the importance in maternal foetal incompatibility.
Surprising that despite the clinical understanding it was not understood at a molecular level.
And we isolated it for the first time, the core Rh polypeptide and by serendip, co-purified a second polypeptide
which we thought was a photolytic fragment.
So the Rh being 32kDa in size, the second protein being slightly smaller 28k.
And we found these not only in human red cells but in red cells of other mammalian species.
So we dismissed the notion that this 28k protein would be even interesting, till we discovered
it was totally unrelated to Rh and had some features which just provoked my curiosity.
No glimmers that this would lead to anything interesting but curiosity is a powerful force in science.
And we discovered this protein while it stained very poorly with traditional methods was exceedingly abundant in red cells,
one of the foremost abundant proteins in the cells. Never before seen.
Be a little bit like paddling down the Bodensee here and coming to a metropolis of a million people that is not on the map.
It gets your attention. So we decided the best way to figure out what this new 28k protein is, is to clone it out, which we did.
The predicted sequence indicated it would be a membrane spanning protein, 6 bilayer spans.
We knew it was tetrameric, something like a channel.
But what kind of a channel and we looked at the genetics data base at this time, this is now 20 years ago.
The genetics data base was much smaller but still had a lot of entries.
And there were some homologues that were clearly recognisable from diverse species, from bovine lens, from drosophila brain,
from bacteria, from plants. But none of them had been functionally defined.
So we really were at a loss, what does this new protein do.
And a protein without a function is a protein with no future, you will not get funded to work on things
just because you found them, you have to have an important purpose.
But fortunately like many, science was an important part of my life, but I was also a member of a family.
My wife Mary and I have 4 children, here's Mary and our son Clark when he was a toddler.
Here are the 4 kids growing up. And the issue of work life balance comes into play.
And sometimes it's really the life part, the family part that drives the work.
Every year we would take the children camping, it's the vacation you can afford when you're a basic scientist.
The kids loved it, we went to all the national parks, not all of them but many of the national parks, Yosemite,
Yellow Stone, Glacier, the Great Smokies.
And after doing this the kids loved it so much we said next year children you get to pick the national park
and they immediately said: Disney World.
Well Disney World is not a national park but we compromised, we went to the Everglades and then we went to Disney World.
And at times I would be sitting quietly drinking my coffee in the morning thinking about this protein
but having a lot of fun with the kids.
And it was on a camping trip to the Everglades and Disney World we stopped in Chapel Hill North Carolina,
a wonderful place, Oliver mentioned it yesterday in his talk. And a home to many wonderful academics and friends.
And so Mary spent some time chatting with her friends, the kids played with some playmates
and I talked to my friend John Parker at the University of North Carolina and told him about this new protein
that I was stymied with. We thought it would have an important function, we didn't know what.
And this is an example I think of where the breadth of the scientific information
that our colleagues bear can help us so much, because we were stuck.
And John thought for a while and he leaned over, he was quite exhausted, he'd been up all night working in the clinic.
He said, "Peter, red cells, renal tubules, plant tissues, all highly permeable to water, have you considered
that this might be the long sought water channel, the channel that physiologists have been searching for,
for a century to explain how osmosis occurs in biological tissues?" It had never occurred to me, it was John's suggestion.
But it was a good one.
And when we returned to Baltimore I teamed up with Bill Guggino in the physiology laboratory of John's Hopkins
to test the hypothesis. So these are frog eggs, xenopus laevis oocytes.
And on the left is a control oocyte and on the right is an oocyte injected with 2 nanograms of the complementary RNA
for our new protein. So the idea is if this is a channel the RNA will encode a protein, it will be inserted into the membrane
and the test cell should now be osmotically active and that's exactly what we found.
Here in isotonic culture medium there's no difference.
When transfer to hypotonic medium the test oocyte is rapidly swelled and exploded. This caused much jubilation in the laboratory.
I think as every scientist knows, if you discover something this is the source of joy, this is what drives us.
I have to tell you the truth, this is a picture of Greg Preston, a post doc who did some of these early studies.
And I took this picture of Greg actually 3 years after our first report, he was still celebrating.
So I know we're not like other people. Our rewards are not financial.
They're many times intellectual, but they're intense and that's what we really should be striving for.
This produced a lot of more interest in our lab than we'd ever received before.
We had requests from around the country, around the planet for our plasmid.
Others had sought to identify the water channel and we had it. So we had to pick our way very carefully.
As a small lab how do you compete with the big labs. And the answer is by collaborating with intelligent, well meaning colleagues.
And this brought great, great success. We teamed first with this group to solve the structure.
On the left is Yoshinori Fujiyoshi from Kyoto University and on the right is Andres Engel from the University of Basel
who were pioneers in the technique of membrane crystallography. And together we solved the structure in 3 dimensions.
Showing that the channel as shown in the left panel has a single pore, top to bottom and on the right panel
and cross section you can see that there are key residues lining the pore.
And these key residues explain the function of the protein.
Just briefly in the top vestibule of this channel and we refer to it as the hour glass because there's an adverse symmetry,
we have water in bulk solution at the top, hydrogen bonding between these molecules causes water to become illiquid.
Water in bulk solution in the intercellular vestibule but notice a 20 angstrom span in the centre.
Where water travels in single file, meeting barriers it blocks the movement of protons, charged solutes and other larger solutes.
So it's a water selective channel allowing rapid movement of water and this has been solved by membrane dynamics simulations.
Here in Germany by Helmut Grubmüller and Bert de Groot. In the United States by Claus Shilton and his group.
So all of the investigators largely agree, this channel explains how water crosses membranes.
And really gives us some insight into the generation and re-absorption of biological fluids.
This model was prepared by David Kozono when he was a student with us.
Now genes often times exist in families and the aquaporins as we've termed them, have 13 different homologues
in the human genome but 100's of different homologues when you include the other life forms
because every life form has at least one aquaporin.
And the human repertoire contains aquaporin 1, you can see in the upper right which was the subject of our first discovery
and several others. Each expressed in different tissues providing water transport with a specific biological function.
In the bottom are some highly related channel proteins which we termed the aquaglyceroporins
which allow the movement of water post glycerol.
And I've highlighted in this human repertoire the 2 members from E-coli, AQPZ on the left
and GlipF on the lower right following water and glycerol transport in E-coli.
So you can see these are distinct from one another but highly related.
So I'll tell you a little bit about AQP1 and I think you've already figured out, part of my story is not just the science
but the facebook of science, my friends, my colleagues and I'll introduce each one briefly.
To localise the protein in kidney we teamed up with Søren Nielsen from the University of Aarhus in Denmark,
anybody here from Aarhus? Yes, good at least one. Look for Søren.
So Søren is a pioneer, he was a young man when we did these studies, he was 29 years old.
He localised with great precision the protein in the proximal nephron.
So in the model on the left you can see the individual nephron of the kidney, each kidney having a million nephrons.
At the glomerulus, at that point where filtration occurs, the primary urine passes through the proximal nephron
which is highly water permeable.
In the presence as shown here in this thin section stained with a specific antibody to AQP1,
the gold colour represents specific presence of the aquaporin in the lumen surrounding the space with a P on it.
As well as in the lateral and the basil membranes.
It's not present in the collecting duct overlaid with the C, I'll talk about that in a moment.
So this explains how water is transferred from the primary urine in the tubules to the apical surface
and out through the lateral or basal surface, going from urine to cytoplasm to interstitium.
And every day our kidneys will rapidly filter 180 litres of plasma, of course it's the same plasma again and again and again.
So this 180 litres is concentrated down to about 1 litre of urine by the re-absorption of water through aquaporins.
Now defects in the aquaporins have clinical problems and I'll just tell you briefly about some of them.
Shown in the snapshot on the left in the centre, with permission I should point out is a photograph
of a retired school teacher from the south of France, who we identified has a genetic defect
making it impossible for her to synthesis the AQP1 protein.
And we were able to identify her because the surface of AQP1 has a rare blood group antigen.
So by collaborating with the blood group investigators we identified these rare people and were able to study them.
And we brought them to John's Hopkins where these 2 other people, the tall gentleman, that's Landon King,
a lung physician at Hopkins and Melanie Bonefare a French speaking post doc, established the clinical significance of AQP1.
And in short it turns out to be very important in the final concentration of urine.
So on the panel to the right you see the urine concentration of 15 normal controls after overnight thirsting.
We go to bed at night, if we're fortunate we sleep through the night and we wake in the morning
and we've had no water for 7 or 8 hours.
And our bodies respond by concentrating the urine, so we don't release dilute urine when we're thirsted.
That's how we prevent ourselves from becoming dehydrated.
So normal individuals can concentrate their urine up to about 1000 milliosmolars,
but notice the 2 AQP1 null individuals from different families with different genetic lesions are stuck,
they can concentrate a little bit from the 280 isotonic to about 400 milliosmolar, they can concentrate that far but no farther.
If indeed these people are thirsted beyond that and we did with very careful testing,
show that they can go no further than 400 milliosmolar.
So of course if they were in a water restricted environment, in a desert, in a nursing home
where the aids are not paying attention, they would become dehydrated by releasing dilute urine in the face of dehydration.
So it's a significant phenotype and we refer to this as mild nephrogenic diabetes insipidus.
Because on basal levels free access to water, they can compensate.
Now a race ensued with the discovery of a new protein of some significance and this is often the case in biology.
A race because of the interest of other scientists and this is what really makes science exciting, things can develop so rapidly.
And by homology Fushimi and his colleagues cloned a homologue of the AQP1 protein from renal collecting duct.
And as you can see in the diagram the collecting duct is the terminal part of the kidney
where the vasopressin regulated water transport is known to occur.
And in short the AQP2 protein is shown in the upper left by immuno gold staining resides normally an intracellular vesicles.
But in response to vasopressin, a neural hormone released from the pituitary in response to thirsting,
the protein is translocated to the cell surface.
So if you look at the upper left panel, intracellular, the lower left panel with vasopressin it's now at the surface of the cell.
Explaining how water can be regulated. And this happens often in our lives.
If you should be out jogging a long distance in the afternoon in the warm sun, you'll become water deprived,
you'll concentrate your urine maximally. This occurs because of the release of vasopressin. So this is the anti-diuretic stage.
And if you were to look at your kidneys they would look like the panel on the upper left.
If on the other hand after an evening of resting, drinking some fluids, maybe a litre of beer, you're in the diuretic state,
your kidneys would look like, excuse me I misspoke, the anti-diuretic state would be at the bottom
and the diuretic state would be at the top.
In this case water re-absorption cannot occur and so we release dilute urine, forgive me for misspeaking.
Now there are inherited defects in this pathway.
And the defects in the gene encoding AQP2 lead to a profound and severe form of renal concentration defect
referred to as nephrogenic diabetes insipidus.
These children are unable to concentrate their urine and will release at least 20 litres of urine a day,
20 litres of urine a day, a litre an hour and must drink at least that volume to maintain their body fluid status,
so it's quite severe but fortunately it's quite rare. But acquired defects in the AQP2 pathway are very common.
Excess synthesis of the protein occurs in congestive heart failure where the patients retain too much fluid.
Defects in the expression are found in common states such as bed wetting.
So the small children who wet their beds, causing much distress to their parents,
they do so because they temporarily as children underexpress their AQP2, they can't concentrate their urine.
They outgrow it but nevertheless it shows the ubiquity and the importance of this protein.
Now there are other members of the family and I'll just briefly talk about these.
The brain aquaporin, AQP4 which we worked on with our friends in Oslo, Ole Petter Ottersen and his team,
is present in a very interesting distribution in the brain because our neurons in the brain
are very sensitive to osmotic disequilibrium. And they're protected by astroglial cells.
So if capillarian brain and I'm just pointing behind me, is surrounded by astroglial end-feet.
This is the site of the so-called blood brain barrier, the movement of fluid from blood to brain
goes through the astroglial end-feet and shown here immuno decoration shows the abundance of the AQP4 protein
at the blood vein barrier. And this of course turns out to be very important in clinical problems such as brain edema.
Stroke is the second leading cause of death in Western Europe and in the United States and individuals often succumb
not to the primary stroke itself but to the brain swelling that occurs thereafter.
So management of brain edema is critical for the outcome of neurosurgical procedures after stroke and after close head injuries.
The presence of this protein explains it.
And in a study undertaken by Mahmood Amiry at the University of Oslo and in collaboration with our team in Hopkins
shows that in fact normal mice are vulnerable to brain injury.
If you look at the staining of a cross section, the normal mouse brain has a significant amount of pale,
which represents demitus and infarcted tissue.
Compared to the mutant mouse, the mutant mouse having a defect in the location of the AQP4 protein.
So it's curious isn't it, somewhat paradoxical. A mutation is protective.
Why don't all mice have that mutation and the answer is the presence of this protein
is also important in brain fluid homeostasis and so the mutant mice while protected against brain injury
do sustain epileptic seizures. So we're trading things off.
Nevertheless the presence or the protection provided by inhibition of this protein gives us hope
that we could develop pharmacological inhibitors to prevent or ameliorate the consequence of brain edema.
Now I'm going to quickly step through some of the other members of the family, AQP5 from secretory glands,
these are present in sweat glands, saliva glands, pulmonary, fluid secretory glands and important in generations of these fluids.
Shown here panels from a study of wild type mouse on the left and each blue dot represents a functional sweat gland.
And in the mutant mouse on the right, this mouse was engineered by Anil Menon and his team in Cincinnati.
And the mutant mouse on the right has sweat glands but they're hypo-functional.
The mouse is vulnerable to hypothermia from this.
And of course mice are relatively resistant because they live in the cool shadows.
But humans require degeneration of sweat to maintain body temperature in hypothermic environments.
So briefly I'll talk about the aquaglyceroporins.
Closely related to the aquaporins and this is the slide I borrowed from Bing Yap from the University of California,
who aligned the pore lining residues of a water channel and these are shaded in dark and a glycerol channel.
And you notice some residues are perfectly retained, this argenine at the top.
Whereas a cysteine here is present in the water channel, replaced by a phenylalanine in the glycerol channel.
And the difference in the mass of the c-chain in the water channel are histidine,
compared to the glycine in the glycerol channel, explains how the glycerol channel can open up the pore
to permit the passage of the larger 3 carbon-polyol. So there are structural explanations for how these things work.
Now I was a physician for a number of years and never had once considered the process of glycerol transport
to be important but it turns out to be quite important in the maintenance of skin.
And this slide prepared by Johan Agren from Uppsala when he was a sabbatical worker with this,
shows the strong expression of AQP3 aquaglyceroporin 3 in the basal levels of skin in pre-natal mouse and post-natal mouse.
It did not take long for the beauty industry to get interested in this.
A few years back, a few summers back I was visited by 3 executives from the Christian Dior company.
Now I don't usually have visitors from the Christian Dior company and I was a little suspicious and in fact
they have a product that they'd like you to buy, you can get it at the duty free shops, it's really expensive,
it's hydro action skin cream, costs about €50 for a 50 gram jar.
And I should point out I have no financial ties to Christian Dior, I would love to have financial ties to Christian Dior.
But none were offered under terms I could accept them.
Nevertheless this is the beauty industry, this is not drug development and their chemists have identified
some small molecules which lead to subtle increase in the expression of aquaglyceroporin 3 in skin.
The implication is that if you use enough of this you'll look like that, I'm not sure that constitutes proof.
But let the buyer beware. Now I actually purloined this, this is the back cover of the Marie Clare Beauty magazine
which I borrowed from the business lounge in Paris.
And those of you who read French will see that it has some bold statements, profound hydration,
spectacular results and the very bottom where it says the Nobel prize I chemistry, I show this to my mother back in Minnesota,
she smiles, said Peter you're finally doing something useful.
Thank you, when I see my mother next I'll tell her you liked that.
I think she was kidding but you never know, our mothers know us better than we know ourselves.
There is some useful information though from the aquaglyceroporin 3 studies and this is
because glycerol transport into red cells is of probably minor physiological significance
but it's the pathway for glycerol imported into red cells during a pathogenesis of malaria.
And I won't go into malaria in depth this morning since I talked about it last night.
But this intercellular parasite can rapidly synthesis glycerol lipids.
Glycerol come through aquaglyceroporins and the parasite then divides, digests the haemoglobin
and in the far right you can see a single parasite can in 2 days turn into a multitude of parasites.
And this amplification of course causes the horrific malarial fevers which kills hundreds of thousands of children every year.
Inhibitors to the aquaglyceroporins may have a therapeutic role, we're not sure but we're investigating that.
And of course the mosquitoes themselves have a problem with water transport.
When the anopheles bite us they do so because the fluid, the haemoglobin from blood is needed for egg development.
But when taking in this vast volume they have to release fluid in order to fly and they do so out of the malpighian tubules
at the back end of the mosquitoes.
It's taking in blood at this end and shooting out fluid at that end and aquaporins are involved, trust me.
There are 2 others I'll mention briefly, aquaglyceroporin 7 in fat, the release of glycerol from fat in response to starvation.
The aquaglyceroporin 9 in liver, the uptake of glycerol by liver to convert glycerol back to glucose in a setting of starvation.
This is the work of Jenn Carbrey.
And it has an unforeseen and important implication because this is also the pathway for the release of arsenite
and I won't go into the chemistry of it.
Neutral Ph arsenite, arsenic trioxide is uncharged, must resemble glycerol.
And the ability to release arsenic from our bodies is conferred by aquaglyceroporin 9.
And I'll skip through that.
Plants have aquaporins, on the right is a wild type Arabidopsis, on the left is Arabidopsis engineered
to under express rootlet aquaporins. And by over-manufacturing of rootlets it can compensate.
So I'll end with this, there's a list I could share with you if you're interested of all the clinical disorders
and there'll be more where aquaporins are involved.
I would like to show a picture of the laboratory on the morning I got a call from some very nice people in Sweden
informing me that I would share the Nobel Prize in chemistry.
My wife Mary when hearing this early in the morning called my mother back in Minnesota,
awakened her to inform her that I would share the Nobel Prize in chemistry.
Mother thought for a moment and she said, "Mary, tell Peter that's very nice but don't let this go to his head."
I don't think that was sarcasm, I think she meant prizes are nice but doing something useful is more important.
But there was a lot of celebration going on, even the local liquor store got into this.
The implication that I was their best customer is a great exaggeration.
But here we are on the stage in Stockholm and I'd just like to point out for the young people here there are 2 prizes here.
The Nobel which is a nice medal and any resemblance I had to Alfred Nobel ended when I took off the moustache.
The other prize is the people with me, my family and all of us have a cheering section, our friends, our families,
the people we love and care about, who drive us forward and encourage us in our work.
we can't forget to thank them for what they've done.
So in closing let me just wish the young people well in their careers.
I hope when you reach my stage, I'm 62, and it will come sooner than you're ready,
you look back and think that the career was fun, I had a great time. And I wish you the best.
Thank you.