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. 2017 Mar;300(3):560-576.
doi: 10.1002/ar.23511. Epub 2017 Jan 11.

Renoprotection From Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

Edward C Carlson  1 , Jennifer M Chhoun  1 , Bryon D Grove  1 , Donna I Laturnus  1 , Shirong Zheng  2 , Paul N Epstein  2 , Yi Tan  2
Affiliations

Renoprotection From Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

Edward C Carlson et al. Anat Rec (Hoboken). 2017 Mar.

Abstract

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age-matched OVE diabetic mice to produce bi-transgenic OVE-JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE-JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day-old OVE-JTMT mice, blood glucose and HbA1c were indistinguishable from age-matched OVE mice. However, endothelial-specific MT overexpression in OVE-JTMT mice mitigated several DN complications including significantly increased non-fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE-JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560-576, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: diabetic mice; electron microscopy; glomerular basement membrane; glomerular cells; glomerular filtration barrier.

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Conflict of interest statement

There are no potential conflicts of interest regarding financial and personal relationships between any of the authors and other individuals that might bias their work. Also, no part of this manuscript has been submitted or is currently being considered for publication elsewhere. The ethical background for this study derives from an ethically neutral hypothesis and all data collection and interpretation have been carried out in the absence of unethical behavior. No ethical committee has reviewed the manuscript.

Figures

Figure 1
Figure 1
A. PCR genotyping of DNA samples from JTMT founder mice. Primers corresponding to the human MTII gene resulted in a 536 bp DNA band, and control primers designed for a region of the FVB background DNA resulted in a 200 bp band. DNA containing the transgene displayed two DNA bands, and the corresponding animals were given the nomenclature JTMT‐1–5. B. Glomerular MT protein expression. Glomeruli were isolated from both wild type FVB and JTMT transgenic mice. The MT protein expression was detected by Western blot as previously reported (Wang et al., 2006).
Figure 2
Figure 2
Representative light micrographs of paraffin embedded renal cortex immunohistochemically stained for MT (A, B, E, F) and PECAM‐1 (C, D.). A. FVB: Negative control, primary antibody omitted. B. FVB: Endogenous MT staining was not observed in the vasculature, but was occasionally observed in proximal tubules (PT). C. FVB: Negative control, primary antibody omitted. D. FVB Staining was observed in the cytoplasm of EC in the capillaries of the glomerulus (arrow) and in renal cortical arterioles (arrowhead). E. JTMT: Negative control, primary antibody omitted. F. JTMT: Cytoplasm of glomerular capillaries (arrow), peritubular capillaries (double arrowheads), and and other cortical microvascular structures displayed intense MT stain. Gl; glomerulus.
Figure 3
Figure 3
Representative confocal micrographs of FVB and JTMT renal cortex frozen sections immunofluorescently stained for MT and EC. A. FVB: MT expression visualized with FITC (green) immunofluorescence. There was little to no MT expression in tissue sections from the control animal. B. JTMT: MT expression visualized with FITC (green) immunofluorescence. In the transgenic mouse, strong MT staining was observed in the glomerulus (blue arrow), peritubular capillaries (orange arrow) and arterioles (yellow arrow). C. FVB: Merged tomato lectin (red) and MT (green) immunofluorescent staining. The control mouse displayed limited MT expression and it was not associated with the vasculature. D. JTMT: Merged tomato lectin (red) and MT (green) immunofluorescent staining. Due to the somewhat “leakiness” of the TIE‐2 promoter (tends to promote staining of hematopoietic cells) and its ability to bind non‐vascular structures in the kidney, there was limited co‐distribution (yellow staining) of MT with the tomato lectin. However, the MT overexpression was localized to EC in the glomerulus (blue arrow), peritubular capillaries (orange arrow), and arterioles (yellow arrow).
Figure 4
Figure 4
Body Features of 150 day‐old Mice. A. Body Weight: Although JTMT mice weighed slightly less than other genotypes, of the four genotypes utilized in the current study, no group varied more than 15% from any other. B: Combined weight of left and right kidneys. Total renal weight was significantly greater (∼86%) in OVE animals than in non‐diabetic controls, but diabetic nephromegaly was mitigated significantly by breeding with JTMT mice, reducing the average weight in OVE‐JTMT mice by ∼29%. C. Blood Sugar. Serum glucose frequently reached 600mg/dl in OVE mice, and these levels were not mitigated by overexpression of MT. D: Likewise, HbA1c rose to more than 10% in severely diabetic OVE mice, and was maintained at highest levels with or without EC targeted MT.
Figure 5
Figure 5
Representative transmission electron micrographs of the GFB in 150 day‐old FVB, JTMT, OVE and OVE‐JTMT mice. FP, podocyte foot process: GBM, glomerular basement membrane; Double arrow, non‐fenestrated capillary endothelium.
Figure 6
Figure 6
Morphometric differences in components of the GFB in 150 day old FVB, JTMT, OVE and OVE‐JTMT mice. A. Mean percent non‐fenestrated endothelium ± S.D: FVB vs. OVE, JTMT vs. OVE, and OVE‐JTMT vs. OVE all show statistically significant differences. OVE mice show greatest percentage of non‐fenestrated endothelium, while OVE‐JTMT mice exhibited significant protection against non‐fenestration. B. Mean glomerular basement membrane thickness ± S.D: FVB vs. OVE, JTMT vs. OVE, and OVE‐JTMT vs. OVE all show statistically significant differences. OVE mice show the thickest glomerular basement membrane, while OVE‐JTMT mice show complete elimination of basement membrane thickening. C. Mean podocyte foot process width ± S.D: FVB vs. OVE, JTMT vs. OVE, and OVE‐JTMT vs. OVE all show statistically significant differences. OVE mice show the widest foot processes (greatest foot process effacement) while OVE‐JTMT mice show significant protection against increased foot process width.
Figure 7
Figure 7
Mean glomerular volume in FVB, JTMT, OVE and OVE‐JTMT mice ± S.D. FVB vs. OVE, OVE‐JTMT, and OVE‐JTMT vs. OVE all show statistically significant difference. OVE mice show the greatest glomerular volume, while OVE‐JTMT mice show significant renoprotection against glomerulomegaly, and though glomerular volume is not reduced to control levels, it is mitigated significantly (∼29%).
Figure 8
Figure 8
Mean podocyte, endothelial cell, mesangial cell, and total glomerular nuclear density in FVB, JTMT, OVE and OVE‐JTMT mice ± S.D.: While mesangial cells showed no significant differences in cell density in any of the four genotypes studied, podocyte, endothelial cell and total glomerular glomerular cell density exhibited significant differences in OVE vs. FVB, and OVE‐JTMT vs. OVE comparisons. FVB mice showed the greatest density in mesangial cells and podocytes, while JTMT mice exhibited the greatest density in endothelial cells and in total glomerular density. On the contrary, OVE diabetics exhibited the least cell density in all cell types except mesangial cells. Interestingly, when the endothelial cell‐targeted MT transgene was present, both diabetic (OVE‐JTMT) and non‐diabetic (JTMT) control mice showed strong tendencies toward increased endothelial cell density.
Figure 9
Figure 9
Mean podocyte, endothelial cell, mesangial cell, and total glomerular cell numbers per glomerulus in FVB, JTMT, OVE and OVE‐JTMT mice ± S.D. Podocyte cell number was not significantly different in any of the four genotypes studied. Endothelial cell proliferation increased significantly in diabetic mice, with or without the presence of the MT transgene. Mesangial cell number (i.e., 547), was significantly greater in OVE mice than any other genotype and were sufficient to drive the total glomerular cell number in OVE mice significantly greater than in any other genotype. OVE‐JTMT mice had an intermediate total glomerular cell number, and total glomerular cells was least in FVB mice.
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