Oliver Smithies:
[00:00:00] So this is book capital T’. Starts March 10th, 2006, and runs through March of 2007. Beginning as usual by continuing what was going on with the design of a construct with conditional deletion of the bradykinin receptors. In other words we could control when the bradykinin receptors were used or not used. It turned out to be very useful idea. And here is the detail of the construct, the usual sort of diagram on page one Friday March 10th. And as follows then [00:01:00] preparing to isolate the various fragments needed in various bacteriophages. For example, trying to get on Monday, March 20th page seven, trying to get a bovine growth hormone poly(A) fragment that will be used to make a message very stable. Following Masao’s method. So it continues for many pages on straightforward work to isolate fragments [00:02:00] and join them together. Typical Tuesday, March 28th page 15, a check on these fragments and on gels. And on the following page a review of the state of the bacteriophage minis used to isolate pieces of DNA. Nothing particularly remarkable in the pages there. Typical though as usual. Thursday, April 6th, page 27, review of the growths from previous transfer. Abandon this approach. [00:03:00] There’s bovine growth hormone poly(A) again being talked about on Friday, April 7th, page 29. Thinking of different vector following page. There’s always renewed efforts Monday, April 10th, page 33. Renewed effort at getting this particular piece that was being considered there. Having to try different things when stuff didn’t work Friday April 28th, page 45, loxP by PCR. [00:04:00] No colonies with loxP oligo. Maybe better redesign for PCR amplification. So trying to shift the method of making a multiple sequence. As we see again on Monday, May 8th, page 49, continuation of loxP and linker, loxP being needed in order to make sure that one could control the structure of the gene by using Cre recombinase. Sad comment on Sunday, May 21st. [00:05:00] Back from London with Nobuyo to see Roger, with lung cancer now. Unfortunately he’d been diagnosed and later died of lung cancer though he never smoked. But he lived in London in the time when smog was very prevalent and I’m sure that must have had something to do with it. Very likely. Not sure, but very likely. But he was getting pretty sick at that time. This is in 2006. So he was 80 bordering on 81. [00:06:00]Beginning to think about direct tests of proteins in mice. Cy5-labeled mouse albumin. That’s mouse albumin which had been made fluorescent with a fluorescent side chain Cy5. Here is work now in collaboration with Byron Ballou. Byron Ballou made three levels of label of mouse albumin with Cy5 estimated as from about one to two Cy5 molecules per molecule of albumin. And this is some attempt to see through the mouse a long wavelength excitation [00:07:00] that could cause Cy5 to emit enough to visualize in a living animal. This is one of his images. It says he concentrated the lowest labeled albumin and injected it intravenously and images of the nude mouse showed intravascular fluorescence with a 670-nanometer emission of Cy5, etc. So beginning to do direct experiments with mice and fluorescent protein [00:08:00]with regard to testing my ideas on how the kidney separates small and large molecules. Also thinking about lymphatic drainage of the kidney which was a very interesting theoretical possibility. Probably relevant. So Thursday, May 25th, page 57, received from Byron Ballou Cy5 albumin and transferred it to Nobuyuki Takahashi, who was a postdoc at the time. [00:09:00] So we’re beginning to see a transfer, a change in the focus of my work towards working more and more with kidney problems. But a good or supposedly good method of measuring fluorescence with small samples. A machine called the Fluostar. And I tested it and found it very unsatisfactory. [00:10:00] Although at first I thought I was getting a good linear curve. But as we’ll see that could be improved on very greatly. Some general talk about kidney function and clearance of albumin from kidney glomerular filtrates on Wednesday, June 7th through 9th, page 61, on albumin protein overload and transepithelial. That sentence doesn’t end. But it was transepithelial passage or uptake talking about the fetal receptor for the constant region FcRn of immunoglobulins that is. [00:11:00] It’s sensitive to amino acid differences, etc. So various comments there about what might be done. Nothing terribly striking. Not stopped doing DNA work. Wednesday, June 14th, page 65. Comparisons of two candidates. Back to thinking about what is happening with clearance of albumin using an animal that is lacking the fetal receptor for constant region of the heavy chain of immunoglobulin which also [00:12:00] affects albuminuria. So tests of clearance of Cy5 albumin in wild type versus FcRn minus minus mice which we had obtained with the Fluostar, which I didn’t like very much. So on page 71 it says conclusion. Whole blood cannot be used. Some attempts to get calibration curves. And then quite a novel idea that turned out to be very useful although I don’t think I ever published it. [00:13:00] Tuesday, June 27th lower part of the page 71. A new thought. I can use the microscope auto feature to get time required to peak the image and use one over time as a measure of fluorescence. And so check this with a rough dilution series. In other words the automatic camera of the microscope counts different pixels. And when an appreciable fraction have been saturated, in other words 256 bits or 256 level, then the camera automatically stops so that it doesn’t get washed out with overexposure. But that time obviously depends on [00:14:00] the fluorescence and so this was a beginning test of that. And on page 70 it’s pretty clear that there was a pretty good relationship there. Looked like a very nice standard curve. And this was pursued on the following pages 72 and 73 and became quite clear that we had a good new method of measuring fluorescence with equipment that we had available. So it goes on making a little apparatus to hold the — a little rack to hold the tubes. Page 75 Wednesday, June 28th. More Cy5 tests. [00:15:00] Tested with flat capillaries. And there’s a little image of the type of flat capillary which makes it more accurate. And for example showing that this — that you use an image which uses the flat part of this type of capillary. Is one and a half times more sensitive than using a round capillary. I enjoyed doing that work. Some more dilute tests on Thursday, June 29th, page 77. Spent most effort on testing 1 over 1,000 dilution versus plasma, etc. And would it work. And it was looking very good. [00:16:00] So proceeded with the first tests with the new method using immunoglobulin. So got Cy5 IgG from Byron and then looked at its half-life in animals with or without the mutation that knocks out the Fc receptor. And one had a half-life of 140 hours. The other had a half-life of 18 hours. Quite nice results. Gave good data for a first try with a comment that the half t half is longer than published but not much. [00:17:00] Proceed but include quantum dots calibration standard. And then sadly on Monday, July 3rd page 81 I record that my brother Roger died. And there’s some photographs taken of him on our last visit when he was still cheerful although very obviously having difficulty. And with his favorite poem about climbing. Mine is the freedom of the tranquil hills. And a good picture of him.
And then commenting [00:18:00] below that sad comment the visit to Indianapolis with John Hagaman and Bob Bagnell and Nobuyuki to learn about two-photon fluorescence of the renal glomerulus in Bruce Molitoris’s lab to see what would happen to clearance when the aorta was blocked. And the following couple of pages, page 83 and 82, are the summary of the results obtained with Bruce which were informative though again not at this point ever published. [00:19:00] Back to the old problems of making good plasma from whole blood on Monday, July 24th, page 89. Whole blood to plasma and how to do it with a microcapillary. More analysis of the data from the testing the new idea of obtaining fluorescent measurements looking on [00:20:00] page 90 the lower right, the bottom image on the right. Shows how beautiful the linearity of the procedure was, covering a range of concentration 1 to something diluted 2,048 times. So linear. Spectacular result even with this series. So that method we used quite a bit. And various images of it on the following page 92. I thought of maybe even having that simple [00:21:00] apparatus of the sort made commercially and patented but never did follow it up. And here we are with the results. Visually now Friday, Saturday, August 1st, 2nd , page 95. The Indianapolis experiments continued from page 82. Some more comments. Some things from Kumar on Friday, August 11th page 97 and 96, towards what later was published as we’ve seen. [00:22:00] Getting images now with Cy5 albumin into the mouse. And one can see all the glomeruli being heavily or being very brightly fluorescent. And then at a high magnification one can also see the fluorescence. These were vibratome sections, examined by confocal microscopy after fixing the kidney in paraformaldehyde, with a comment that the images are nevertheless of high quality. [00:23:00]
More analysis of the Indianapolis data on Wednesday, August 23rd page 101. Here we are on page 105 Tuesday, September 5th. Maria Dudareva’s first tests with the FcRn mutant versus wild type with albumin. Couldn’t see any differences. [00:24:00] But some comment that that conclusion might be incorrect. The intensities are tricky and need to look at — better look at five minutes. Some data now by scanning electron microscopy high resolution images, page 107, [00:25:00] Tuesday, September 5th. Two mice from Feng were examined. Indistinguishable at this point. I presume that one was mutant and one was not, but not clearly stated. The comment is that subsequently after seeing that they are indistinguishable subsequently Feng indicated that these two mice were both untreated. So these are wild type mice and that’s why they were indistinguishable and had normal fenestration in the endothelium. Very nice images. [00:26:00] More of these images on the following pages and more of the fluorescent images on page 110 and 111, and some rather spectacular fluorescent images.
And one on page 111 which I used to use often to show what was happening with a later difficulty then understanding why the basement membrane always was fluorescent but the plasma didn’t appear to be present in most of the glomerular capillaries. [00:27:00] Which we later understood was a problem during fixation that almost certainly when the glomerulus was being — when the kidney was being fixed the glomerular afferent and efferent arterioles could still contract and did so but not fully close, so that as further contraction occurred plasma was ejected from the glomerular capillary but red cells remained there. So that the space was occupied by red cells rather than by plasma. So one would only see fluorescent in the basement membrane. But these are some of the experiments that led to that conclusion. [00:28:00] With a grant writing hiatus on Monday, September 25th through October 31st page 115.
More fluorescence experiments going on here picking up work. Wednesday, November 8th page 117, a 15-minute IgG experiment showing that even IgG can get through to the tubule if you wait. Some of this work I’m even at the present time here in 2015 writing up some of this work[00:29:00] which didn’t get published when Mike was here still a postdoc. Mike Altenburg that is, who worked with Maria. Some images from the Akita bradykinin null mice. Glomeruli looked normal, except for some blown-up glomeruli. [00:30:00]
Over the next several considerable number of pages trying to understand these fluorescent images. Pulse-chase experiment from 10/26/2006 was being looked at on November 15th Wednesday, November 15th, page 125. Mike gave a quick injection of less than one second and also a slow injection over two minutes and sacrificed at 15 minutes, etc. [00:31:00] Thinking about the distribution of the label in the animal in an alpha phase and a beta phase, etc. Jen Wilder, Mike Altenburg’s wife and still a very happy worker in the lab – one of the best technicians I’ve ever had. [00:32:00] On page 129, Friday November 17th she set up a dilution series as standard for the concentrations. So a standard curve with this time method.
Mixture of Cy5-labeled IgG and Cy3-labeled IgG that had been digested with trypsin was tried on Monday, December 4th page 131. And similar things. [00:33:00] I was obviously thinking about getting a grant for doing this work. And Wednesday, December 13th page 135 shows some ideas that had been discussed in writing my hypothesis paper. And this is the beginning of what turned out to be a marathon study that is still ongoing. In preparing for a kidney hypothesis grant application I realized that by using a size-graded series of gold beads I may be able to establish the validity of Ogston’s equation in the glomerular basement membrane by directly counting particles as illustrated in my 2003 paper diagram. [00:34:00] And as used by Jarad in 2006 and following Marilyn Farquhar’s 1960 ferritin method. Develop of this now. Underlined. So that’s the beginning of what’s still very much part of my work. And in fact I’m writing up that paper on this at the present time so that nine years later with some images from Jarad’s paper being shown there. Though I now realize that those are a little spurious because Jarad was taking his points with ferritin after an hour or so instead of after seconds. [00:35:00]
Some thoughts that I’m sure are not novel to me. Second less developed thought is that the countercurrent sodium chloride gradient developed by ion pumping in the tall ascending limb may be transferred to the loop of Henle as a continuing multiplate system. If the sodium chloride gradient generated more energy than the urea water gradient requires then energy would not then be needed in the thin loop but it would still be effective. Develop this later. [00:36:00] Trying to calculate how much label I would need in order to be able to see anything on page 137, Friday, December 15th. Assuming an unlabeled 26-angstrom diameter 1.4-nanometer Nanogold which could be bought. Supplied at 30-nanomolar. Then one can calculate how many particles there are. And the volume of the basement membrane in a 70-nanometer-thick electron microscope section. And whether there would be enough to see. Ferritin used. On page 136 ferritin as [00:37:00] used by Jarad at 15 mg per 30-gram mouse would be 20-micromolar in milliliters of plasmid. And would have 1.2 times 10 to the somethingth there. But difficult to read. Particles per 1.5-microliter 2 times — it looks like 1.2 times 10 to the 13th particles for microliter, 15 grams of ferritin, it’s 31.6 nanomoles. Compared to 30 nanomoles of particles, etc., etc. Beginning to — anyway the main thing is beginning to think about how many particles one needs in order to see them. [00:38:00]
Here on Friday, December 22nd page 139 some data on aspirin and enalapril because of having read publications which suggested that you can get albuminuria by having an animal treated with aspirin after ACE inhibition. Beginning to think about using aspirin to lower the GFR. This was aspirin, enalapril data were from earlier work. [00:39:00] This work stemmed from work in Guyton’s lab a long time ago in which he tried to see how GFR could be decreased and he published some work suggesting that a combination of aspirin and enalapril could get GFR down very substantially. So beginning to think about that on this page, with a mean arterial pressure falling very substantially over four hours on page 139 [00:40:00] and with proteinuria. This eventually turned out to be an effect not of aspirin but of the breakdown product of aspirin, salicylic acid, as was studied very extensively by Ryan Sanford which no doubt we’ll come to as we go through the history. Beginning to try some things anyway, Tuesday, March 6th, page 141. Grants finished with hypertension, had a good score, in the top 2.3 percentile. [00:41:00] Looking at some of Nobuyuki’s data on urinary albumin versus GFR. But here it looks as if as GFR increases albuminuria increases. The opposite way from what I was expecting. So the comment here is that there’s no doubt that urine albumin excretion per day or per minute decreases as GFR decreases in the left [00:42:00] experimental or right experimental kidney, etc. The result is not encouraging but not clearly disproven. It fits Lazzara and Deen’s prediction. They predicted exactly this effect that if you increase GFR you would increase albuminuria. Now I think that probably there is a biphasic curve. That’s to say very low GFRs will increase albuminuria and very high GFR will also increase albuminuria for two different reasons. The first being the effect on albumin concentration as GFR decreases and saturation of the tubule, [00:43:00] and the second being too rapid passage through the tubule for the albumin to be recovered. So I think probably Deen and Lazzara are right as well as possibly I’m right.
Now looking at the endothelium using the scanning electron microscope four or five days after giving the sFlt-1 virus received from Ananth Karumanchi. So Feng’s experiment showing that actually after the virus there is definite decrease in the area [00:44:00] that can be seen to have good fenestrae versus the control. But that’s rather a limited number of tests. But no question that the sFlt virus causes albuminuria. With a comment that the proteinuria is clearly induced, p less than 0.01, and increases as GFR decreases, but the control animals with a similar distribution of GFR show no proteinuria. So experiment is therefore not free from ambiguities. [00:45:00] Review of the rationale of the aspirin enalapril experiments on page 147, Tuesday, March 6th. And in checking on the Guyton autoregulation of blood pressure versus salt balance I came across autoregulation of GFR which interferes with my attempts to decrease GFR by decreasing blood pressure, etc., etc. So finding things a little bit complicated as usual. [00:46:00]
And the business of using the energy of sodium chloride to transfer other solutes is a matter of switching channels and using the energy available from sodium chloride to move other ions and nonionic substances. I’m pretty sure this is a general idea, not mine only. [00:47:00] Starting to be worrying about what the limit of how the current experiments were following nanoparticle distribution was reviewed on Wednesday, March 7th page 149. Various comments about it. Images, none of which are particularly striking, on page 148. A need for something better. [00:48:00] Back to making a GFP fusion gene connected to albumin with a comment at page 153, Thursday, March 15th. Couldn’t bear it. Will modify the endogenous mouse albumin gene to give a GFP fusion protein with GFP on the C terminus. So that the signal peptide, etc., etc. And so began to think about making transgene that were driven by albumin or attached to albumin. In other words labeling the albumin with GFP genetically rather than chemically and with a scheme [00:49:00] for making what I called albumin GFP then on page 155 and 154. And that was indeed made and tested as we’ll see. With the usual then constructs being made page 159, Wednesday, March 28th, PCR for the 3’ short albumin piece. And the book ends on page 163, Friday, March 30th, talking about the short arm PCR fragment that was needed to continue that construct. And so ends book T’.
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