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

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

Video Transcript

1
00:00:00,000 --> 00:00:11,066
TAKAMUNE TAKAHASHI: Good afternoon. I am Takamune Takahashi at Vanderbilt University and we are also developing the imaging technique

2
00:00:11,066 --> 00:00:21,899
which assesses glomerular numbers. Actually, our strategy is a bit different from Kevin’s. Kevin’s strategy labels the glomerular capillary

3
00:00:21,900 --> 00:00:33,600
walls but our technique labels the glomerular mesangium. As you know, this panel shows the flow of the macromolecular proteins in a

4
00:00:33,600 --> 00:00:44,566
glomerulus and as you know, the smaller proteins pass through the glomerular capillary walls and go to the urine. However, macromolecular

5
00:00:44,566 --> 00:00:56,066
proteins go into the mesangial area and eventually move to the lymphatic vessels and this flow is called the mesangial channel. So, we

6
00:00:56,066 --> 00:01:06,899
thought the macromolecular protein, which is non-toxic and which stays in the glomerular mesangium for a while, but it is rapidly cleared

7
00:01:06,900 --> 00:01:19,266
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

8
00:01:19,266 --> 00:01:34,832
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

9
00:01:34,833 --> 00:01:49,666
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

10
00:01:49,666 --> 00:02:03,266
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

11
00:02:03,266 --> 00:02:15,432
the liver and the spleen and it is also degradated by the enzyme dextranase, and fundamentally all glomerulus, while labeled by the FITC-Dextran

12
00:02:15,433 --> 00:02:31,099
500 and this property may make it possible to label the glomerulus without the high background. So, we conjugated Technetium 99 to the Dextran

13
00:02:31,100 --> 00:02:43,033
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

14
00:02:43,033 --> 00:02:56,499
strongly labels a kidney cortex and the autoradiogram data suggested that Dextran 500 is deposited in a [---] glomerulus. As you can see

15
00:02:56,500 --> 00:03:07,800
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

16
00:03:07,800 --> 00:03:20,033
fecal excretions here and this protocol still has some tubular background, so we found that optimized the protocol, deducing that dosage and

17
00:03:20,033 --> 00:03:30,999
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

18
00:03:31,000 --> 00:03:42,600
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%

19
00:03:42,600 --> 00:03:53,766
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-

20
00:03:53,766 --> 00:04:08,499
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

21
00:04:08,500 --> 00:04:20,566
is a SPECT-based imaging technique, so this technique is highly sensitive and quantitative, also less motion effect and a shorter scan time. This

22
00:04:20,566 --> 00:04:30,932
technique utilizes a glomerular mesangium, so even if the glomerular volume is increased, maybe mesangial volume is not increased that

23
00:04:30,933 --> 00:04:43,266
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

24
00:04:43,266 --> 00:04:55,199
assessing the glomerular size. Also, this technique labels a mesangium, so even if the glomerular filtration barrier is impaired, this

25
00:04:55,200 --> 00:05:08,866
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

26
00:05:08,866 --> 00:05:22,066
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

27
00:05:22,066 --> 00:05:33,232
scan to get the high resolution imaging data. This technique would be less sensitive to the mesangial changes and function and there is

28
00:05:33,233 --> 00:05:44,633
some tubular background at the early time point because a high molecular weight Dextran 500 is known to be degradated by the enzyme, the

29
00:05:44,633 --> 00:05:57,066
dextranase, and this dextran degradation makes a small dextran fragment, 10 kilodalton on the 25 kilodalton and these small fragments pass

30
00:05:57,066 --> 00:06:05,799
through the glomerulus and are absorbed by the proximal tubules or maybe segregated from the proximal tubules. So, this causes some tubular

31
00:06:05,800 --> 00:06:22,633
background. So, we really need to check how the dextran kinetics and the dextran degradation are different in acute individuals and how this

32
00:06:22,633 --> 00:06:28,866
difference affects imaging data. This is a potential problem, but anyway, we are going to further evaluate this technique and there are many things

33
00:06:28,866 --> 00:06:38,866
that we need to assess, including the biodistribution of the imaging probe, also sensitivity of the imaging of this technique, the

34
00:06:38,866 --> 00:06:48,499
single injection varies as much per injection and also correlation between the histological assessment and imaging assessment. For the

35
00:06:48,500 --> 00:07:00,400
sensitivity we think that gDNA heterozygous mice in which the glomerular number is decreased by the 25% will be very helpful. Anyway, we will

36
00:07:00,400 --> 00:07:13,833
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

37
00:07:13,833 --> 00:07:24,466
the Caltech Chemical Engineering Group last year and this group generated gold-based nanoparticles with different sizes and they found

38
00:07:24,466 --> 00:07:38,299
that nanoparticles of the 75 nanometer diameter, that a single IV injection nicely targets a glomerular mesangium with 100% efficiency and

39
00:07:38,300 --> 00:07:52,266
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,

40
00:07:52,266 --> 00:08:19,299
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.

41
00:08:19,300 --> 00:08:26,066
KEVIN BENNETT: That’s great, Takamune. So, how does the Dextran actually get cleared from the mesangium over time? Do you know? How

42
00:08:26,066 --> 00:08:34,732
does it get cleared out of the mesangium? TAKAMUNE TAKAHASHI: Actually, the precise

43
00:08:34,733 --> 00:08:43,299
mechanism of the clearance of the macromolecular in the mesangium we don’t know, but probably the proteinase degradates that

44
00:08:43,300 --> 00:08:49,033
macromolecular protein and then this goes to the lymphatic vessels.

45
00:08:49,033 --> 00:08:56,466
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?

46
00:08:56,466 --> 00:09:07,366
TAKAMUNE TAKAHASHI: How long? I don’t know. Probably one…but macromolecular protein stays in the glomerular mesangium for two or

47
00:09:07,366 --> 00:09:17,732
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

48
00:09:17,733 --> 00:09:26,466
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

49
00:09:26,466 --> 00:09:36,232
metalloproteinase or many proteinases are involved in the process of the clearance.

50
00:09:36,233 --> 00:09:41,299
KEVIN BENNETT: Thanks.
JOHN BASGEN: Very nice. My understanding is,

51
00:09:41,300 --> 00:09:51,800
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

52
00:09:51,800 --> 00:09:59,233
to be a 500 kilodalton range for dextran and exactly what is the range that you’re actually injecting?

53
00:09:59,233 --> 00:10:11,333
TAKAMUNE TAKAHASHI: We just bought the Dextran 500 from the company, so yeah, you are right. The Dextran 500 probably includes a range

54
00:10:11,333 --> 00:10:19,266
of that dextran, which, this is just a crude…just bought it from the industry. Yeah.

55
00:10:19,266 --> 00:10:28,266
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

56
00:10:28,266 --> 00:10:31,032
would suspect…
TAKAMUNE TAKAHASHI: Enlargement, because

57
00:10:31,033 --> 00:10:43,033
this technique assesses the mesangial volume, so even if the glomerulus becomes bigger, maybe mesangial area is not…

58
00:10:43,033 --> 00:10:49,633
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

59
00:10:49,633 --> 00:10:57,733
there were decreased glomerular filtration, well, that’s not what it said.

60
00:10:57,733 --> 00:11:00,133
FEMALE: [inaudible comment] PAUL KIMMEL: Okay. I mean, I don’t want to go

61
00:11:00,133 --> 00:11:07,299
back and look at the slide, but, thank you.
KEVIN LEMLEY: Nice talk. So, this was rat?

62
00:11:07,300 --> 00:11:11,766
TAKAMUNE TAKAHASHI: This is mouse.
KEVIN LEMLEY: So, they don’t have the same

63
00:11:11,766 --> 00:11:17,866
problem rat does, the large dextrans causing an anaphylactic reaction in the mouse? It doesn’t have a problem with that?

64
00:11:17,866 --> 00:11:29,832
TAKAMUNE TAKAHASHI: We never tried the rat.
MICHAEL MAUER: I’m curious about what you’re

65
00:11:29,833 --> 00:11:40,233
finding with the dextrans. What you’re saying, in the injured glomerulus, the uptake by the mesangium is the same as the normal glomerulus

66
00:11:40,233 --> 00:11:45,133
but the loss from the mesangium is different.
TAKAMUNE TAKAHASHI: We never tested.

67
00:11:45,133 --> 00:11:49,599
That’s a good question, yeah.
MICHAEL MAUER: When we used a

68
00:11:49,600 --> 00:12:02,633
macromolecule of aggregated gamma globulin, which is also taken up by the mesangium, in the proteinuric animals we found the opposite: that

69
00:12:02,633 --> 00:12:14,199
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

70
00:12:14,200 --> 00:12:29,200
this particular macromolecule that you’re using, the Dextran, is handled differently from proteins or immune complexes? Is it handled in a different

71
00:12:29,200 --> 00:12:33,500
way by the mesangium?
TAKAMUNE TAKAHASHI: Actually, we don’t

72
00:12:33,500 --> 00:12:47,266
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

73
00:12:47,266 --> 00:13:02,032
is a polysaccharide, so maybe some special mechanisms are involved in the dextran deposition in the mesangium because nobody

74
00:13:02,033 --> 00:13:10,466
examined the mechanism of the dextran deposition in the mesangium, so we don’t have an answer. Sorry.




Date Last Updated: 10/5/2012

General Inquiries may be addressed to:
Office of Communications and Public Liaison
NIDDK, NIH
Building 31, Rm 9A06
31 Center Drive, MSC 2560
Bethesda, MD 20892-2560
USA
Phone: 301.496.3583