Quantitative Morphology in Kidney Research
February 13-14, 2012 Conference Videos

Panel Discussion
Takamune Takahashi, Vanderbilt University
Norbert Gretz, Universitat Heidelberg, Germany

Video Transcript

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TAKAMUNE TAKAHASHI: Good afternoon. I am Takamune Takahashi at Vanderbilt University and we are also developing the imaging technique

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which assesses glomerular numbers. Actually, our strategy is a bit different from Kevin’s. Kevin’s strategy labels the glomerular capillary

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walls but our technique labels the glomerular mesangium. As you know, this panel shows the flow of the macromolecular proteins in a

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glomerulus and as you know, the smaller proteins pass through the glomerular capillary walls and go to the urine. However, macromolecular

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proteins go into the mesangial area and eventually move to the lymphatic vessels and this flow is called the mesangial channel. So, we

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thought the macromolecular protein, which is non-toxic and which stays in the glomerular mesangium for a while, but it is rapidly cleared

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from the saturation and this protein could be used for labeling the glomerulus. So, we examined the macromolecular protein and we got a good result

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with the Dextran 500, and this panel shows our FITC-Dextran 500 injection experiment in mice, single intravenous injection, 10 mg per kg, and as

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you can see in this way, Dextran 500 deposited and stayed in the glomerular mesangium up to 24 hours after injection while, in the Dextran 500 it is

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rapidly cleared and eliminated from the plasma within 6 hours, and the plasma half-life is 102 minutes. Dextran 500 is known to be uptaken by

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the liver and the spleen and it is also degradated by the enzyme dextranase, and fundamentally all glomerulus, while labeled by the FITC-Dextran

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500 and this property may make it possible to label the glomerulus without the high background. So, we conjugated Technetium 99 to the Dextran

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500 and did the SPECT imaging. This is a lateral orbital injection and it’s three hours after injection, and as you can see in this slide, Dextran 500

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strongly labels a kidney cortex and the autoradiogram data suggested that Dextran 500 is deposited in a [---] glomerulus. As you can see

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in this slide, there is some of the cortical tubular background but there is no signal that appears in the medulla. We also observed some urinary and

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fecal excretions here and this protocol still has some tubular background, so we found that optimized the protocol, deducing that dosage and

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the change in the time point from the three hours to the six hours, so this is six hours after injection. Then we imaged the wild-type mouse

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kidney and the Os mice kidney. As you know, the Os mice is a low nephron number mice and in this mice the glomerular number is decreased by 50%

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and as you can see in this slide, Os mice kidney shows a much weaker signal as compared with a wild-type mouse kidney, almost half of the wild-

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type mice kidney. So, these data suggest that this technique may be used for counting that glomerulus. The merit of this technique is that this

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is a SPECT-based imaging technique, so this technique is highly sensitive and quantitative, also less motion effect and a shorter scan time. This

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technique utilizes a glomerular mesangium, so even if the glomerular volume is increased, maybe mesangial volume is not increased that

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much. This means changes in glomerular volume may not affect the results very much, but this also means this technique may not be the [---] for

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assessing the glomerular size. Also, this technique labels a mesangium, so even if the glomerular filtration barrier is impaired, this

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technique still may work. An additional property of SPECT is low resolution, so we cannot detect the individual glomeruli but we think that even with

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MRI it may be very difficult to detect the individual glomeruli in the [---] subject because there is less periphery motion and also MRI needs a long time

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scan to get the high resolution imaging data. This technique would be less sensitive to the mesangial changes and function and there is

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some tubular background at the early time point because a high molecular weight Dextran 500 is known to be degradated by the enzyme, the

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dextranase, and this dextran degradation makes a small dextran fragment, 10 kilodalton on the 25 kilodalton and these small fragments pass

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through the glomerulus and are absorbed by the proximal tubules or maybe segregated from the proximal tubules. So, this causes some tubular

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background. So, we really need to check how the dextran kinetics and the dextran degradation are different in acute individuals and how this

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difference affects imaging data. This is a potential problem, but anyway, we are going to further evaluate this technique and there are many things

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that we need to assess, including the biodistribution of the imaging probe, also sensitivity of the imaging of this technique, the

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single injection varies as much per injection and also correlation between the histological assessment and imaging assessment. For the

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sensitivity we think that gDNA heterozygous mice in which the glomerular number is decreased by the 25% will be very helpful. Anyway, we will

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see how this technique works, and we also think that there is another way to label the glomerular mesangium. This is a PNAS paper published by

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the Caltech Chemical Engineering Group last year and this group generated gold-based nanoparticles with different sizes and they found

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that nanoparticles of the 75 nanometer diameter, that a single IV injection nicely targets a glomerular mesangium with 100% efficiency and

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there is are no tubular depositions. So, we think this strategy, this approach, could also be useful for labeling that mesangium. Our study, as I said,

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is at the very preliminary stage and this is still just the one possibility, but I hope that this gives you some idea. Thank you for your attention.

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KEVIN BENNETT: That’s great, Takamune. So, how does the Dextran actually get cleared from the mesangium over time? Do you know? How

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does it get cleared out of the mesangium? TAKAMUNE TAKAHASHI: Actually, the precise

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mechanism of the clearance of the macromolecular in the mesangium we don’t know, but probably the proteinase degradates that

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macromolecular protein and then this goes to the lymphatic vessels.

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KEVIN BENNETT: And that was over what time frame, did you say? Over what time frame does it get cleared out of the mesangium? How long?

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TAKAMUNE TAKAHASHI: How long? I don’t know. Probably one…but macromolecular protein stays in the glomerular mesangium for two or

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three days and the mesangium uptake…I did a lot of the study, actually, in Japan and the mesangial uptake is very stable in a normal glomerular and

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even in an injured glomeruli, but the clearance is very different in a normal glomeruli and a diseased glomeruli. So I guess the proteinase, the

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metalloproteinase or many proteinases are involved in the process of the clearance.

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JOHN BASGEN: Very nice. My understanding is,

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though, that dextran, when it’s synthesized, you tend to get it as a range of molecular weights. So, could you tell us something about how you got it

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to be a 500 kilodalton range for dextran and exactly what is the range that you’re actually injecting?

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TAKAMUNE TAKAHASHI: We just bought the Dextran 500 from the company, so yeah, you are right. The Dextran 500 probably includes a range

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of that dextran, which, this is just a crude…just bought it from the industry. Yeah.

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PAUL KIMMEL: I’m not sure I understood; I may have misunderstood. You said that this technique would not be sensitive to glomerular filtration? I

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would suspect…
TAKAMUNE TAKAHASHI: Enlargement, because

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this technique assesses the mesangial volume, so even if the glomerulus becomes bigger, maybe mesangial area is not…

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PAUL KIMMEL: No, no. That was another bullet that you had but then you had “not sensitive to glomerular filtration” and I would suspect that if

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there were decreased glomerular filtration, well, that’s not what it said.

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FEMALE: [inaudible comment] PAUL KIMMEL: Okay. I mean, I don’t want to go

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back and look at the slide, but, thank you.
KEVIN LEMLEY: Nice talk. So, this was rat?

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KEVIN LEMLEY: So, they don’t have the same

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problem rat does, the large dextrans causing an anaphylactic reaction in the mouse? It doesn’t have a problem with that?

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TAKAMUNE TAKAHASHI: We never tried the rat.
MICHAEL MAUER: I’m curious about what you’re

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finding with the dextrans. What you’re saying, in the injured glomerulus, the uptake by the mesangium is the same as the normal glomerulus

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but the loss from the mesangium is different.
TAKAMUNE TAKAHASHI: We never tested.

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That’s a good question, yeah.
MICHAEL MAUER: When we used a

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macromolecule of aggregated gamma globulin, which is also taken up by the mesangium, in the proteinuric animals we found the opposite: that

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the uptake by the mesangium was greatly increased, but the rate of fall-off was the same as non-proteinuric animals. So, do you think that

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this particular macromolecule that you’re using, the Dextran, is handled differently from proteins or immune complexes? Is it handled in a different

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way by the mesangium?
TAKAMUNE TAKAHASHI: Actually, we don’t

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know the mechanism of the dextran deposition in the mesangium, so we don’t know, yeah. It probably depends on the protein and the dextran

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is a polysaccharide, so maybe some special mechanisms are involved in the dextran deposition in the mesangium because nobody

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examined the mechanism of the dextran deposition in the mesangium, so we don’t have an answer. Sorry.

Date Last Updated: 10/5/2012

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