Microcirculation. 2013 Jul;20(5):425-33. doi: 10.1111/micc.12043.

In vivo quantitative visualization analysis of the effect of C-peptide on glomerular hyperfiltration in diabetic rats by using multiphoton microscopy.

Nakamoto H, Kajiya F.

Department of Medical Engineering and Systems Cardiology, Kawasaki Medical School, Kurashiki, Okayama, Japan. hershey@me.kawasaki-m.ac.jp

 

Abstract

OBJECTIVE: The purpose of this study was to visualize glomerular hyperfiltration in rats at the early stage of diabetes under in vivo conditions and to quantitatively examine the effect of C-peptide on filtration.

METHODS: Type 1 diabetes was induced by streptozotocin (50 mg/kg) in Wistar rats. The rats were divided into four groups: control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide. C-peptide was continuously infused (50 pmol/kg/min). Filtration was visualized by a bolus shot of various sizes of dextrans (3 k to 70 k Da) conjugated with Texas Red and observed with a multiphoton microscope under inhaled anesthesia. Relative sieving coefficients were used to quantify filtration.

RESULTS: Almost all smaller particles (3–10 k Da) were filtered both in control and diabetic rats. Filtration of larger particles (40–70 k Da) was seen in normal rats but was more apparent in diabetic rats, where it was progressive according to the duration of diabetes. C-peptide administration restored the leakage of larger particles to the level seen for the control.

CONCLUSIONS: We visualized and analyzed hyperfiltration and confirmed that C-peptide has a nephroprotective effect. Furthermore, we found that the leakage of larger particles increased as the duration of diabetes increased.

PMID: 24003455

 

Supplement:

We have been working on in-vivo visualisation of renal microcirculation so far. What can be measured with our pencil type intravital video-microscope is limited to vascular diameters, blood flow velocities and so on. For an example, we were able to examine the glomerular effect of adenosine using dilazep under in-vivo conditions in a rat measuring glomerular arteriolar diameter changes (1). We have adopted a novel method, a multi-photon laser microscopy so that we can investigate on of the renal functions, glomerular filtration, further at the dimension of the molecules.

Previously, we reported that early diabetic rats are in a state of hyperfiltration. This is because creatinine clearance was increased in early diabetic rats. Supportively, afferent arteriolar diameters, efferent arteriolar diameters, renal blood flows and glomeular blood flow veclocities were increased in diabetic rats (2). We then visualised such hyperfiltration by multiphoton microscopy after administration of Texas Red conjugated dextrans of various molecular sizes. We quantitatively measured the intensities of the filtrate and compared them between control and early diabetic rats introducing the concept of “relative sieving coefficient”. We found there was a leakage of larger molecules of 40k and 70k Dalton dextrans from the glomeulri of early diabetic rats. The glomerular filtration function is already disturbed even in early diabetic rats.

C-peptide, a byproduct of insulin secretion was once considered biologically inactive. Since as early as the 1980s, studies have provided direct evidence that C-peptide is a biologically active endogenous peptide hormone. Administration of C-peptide to insulin-dependent diabetic patients or diabetic animals has been shown to decrease glomerular hyperfiltration, diminish the leakage of albumin or fluorescein across the blood-retina barrier, increase glucose uptake in skeletal muscle and to improve autonomic nerve and microvascular disease. All of these findings appear to be beneficial. Previoulsy, we reported another beneficial effect of C-peptide in an isolated perfused rat heart in Langendorff mode (3). During the early stages of type 1 diabetes, coronary flow was increased, and C-peptide in the presence of insulin synergistically normalized the excessive flow and NO production induced by C-peptide to the control level of normal rats. In the present study, we confirmed that C-peptide administration to the diabetic rats restored the glomerular leakage of larger particles to the level seen for the control. This finding gives us the perspective that the use of C-peptide for type 1 diabetes patients in combination therapy with insulin or oral drugs during the early stage of diabetes may prevent the progress of diabetic nephropathy.

Hiroshi Nakamoto-1Figure 1 Visualisation of glomerular microcirculation with our intravital videomicroscope in a rat. spatial resolution: 0.8 mm.

Hiroshi Nakamoto-2Figure 2. Visualisation of glomerular filtration. Denotation of colors: Green, FITC-conjugated dextrans (500 k Da) to mark plasma or the intravascular area; blue, Hoechst 33342 to stain cellular nuclei; red, Texas Red-conjugated dextrans that were to be filtered at the glomerulus. (a) A glomerular image obtained just before the dye solution (in 0.5 mL of dextran conjugated with Texas Red) was administered, at 10 seconds after the beginning of the recording. (b) Image taken four seconds after dye solution administration or 14 seconds after the beginning of the recording, showing the particles being filtered out of the glomerular capillaries into the Bowman space.

Hiroshi Nakamoto-3

ACKNOWLEDGMENTS

This work was supported by grant aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (B 20300163 and Challenging Exploratory Research 23650278) and was also supported by grant aid from Kawasaki Medical School (19-319Y, 20-3060, 21-314).

 

References:

1)      Nakamoto H, Ogasawara Y, Kajiya F. Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo. Hypertens Res 31: 315–324, 2008.

2)      Yamamoto T, Tomura Y, Tanaka H, Kajiya F. In vivo visualization of characteristics of renal microcirculation in hypertensive and diabetic rats. Am J Physiol Renal Physiol. 2001 Sep;281(3):F571-7.

3)      Nakamoto H, Sakane N, Kimura K, Yoshida T, Mochizuki S, Ogasawara Y, Kajiya F. Synergistic effects of C-peptide and insulin on coronary flow in early diabetic rats. Metabolism 53: 335–339, 2004.

Contact:

Hiroshi Nakamoto, MD, PhD

Associate Professor at

Department of Medical Engineering and Systems Cardiology

Kawasaki Medical School

577 Matsushima, Kurashiki, Okayama, Japan

postal code: 701-0192

hershey@me.kawasaki-m.ac.jp

 

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