Table 1.
Sequence of primers.
Figure 1.
Immunostaining for TLR2, PECAM-1, podoplanin, and VE-cadherin.
Mouse and human leukocyte cell line J774A.1 and HL60 were immunostained with both anti-PECAM-1 (CD31) and anti-TLR2, and nuclei were stained by DAPI. In the mouse heart section, lymphatic vessels (arrowheads) were immunostained by anti-podoplanin (PDPN) and a blood vessel (arrows) was stained by anti-VE-cadherin (VE-cad). In the human kidney section with type II diabetic nephropathy, there are glomerular endothelial cells immunostained with both anti-PECAM-1 (CD31) and anti-TLR2 (arrows), and cells that are only immunostained with anti-TLR2 (arrowheads). Bar: 20 µm.
Figure 2.
Distribution of TLR2-positive cells in diabetic mouse kidneys.
The immunostained sections were re-stained by HE staining. The HE staining shows that renal tubules are expanded in the cortex of the kidneys of STZ-induced type I diabetic ICR mice (ICR-STZ) and of HF-induced type II diabetic KK/Ta mice (KK/Ta-HF). In the non-diabetic ICR mouse kidney section, all of the glomeruli were immunostained with the antibody for the podocyte marker, podoplanin (PDPN, arrows, staining red), while there were no cells reacting with anti-TLR2. In the ICR-STZ and KK/Ta-HF mouse kidney sections, there are areas immunostained by anti-TLR2 (arrowheads, staining green) in all of the podoplanin-positive glomeruli. In the KK/Ta mouse kidney sections, podoplanin-negative proximal tubules which are more strongly stained with eosin than the distal tubules are also immunostained by anti-TLR2 (white arrowheads, staining green). In the ICR-STZ and KK/Ta-HF mouse kidney sections, distal tubules, collecting tubules, and blood vessels outside glomeruli are not stained. Bar: 100 µm.
Figure 3.
Localization of TLR2 in the glomeruli of STZ-induced type I diabetic ICR mouse kidneys.
The HE staining showed that glomeruli (Gl) of the diabetic mice are subject to sclerosis. In the laser-scanning confocal microscopy, the region reacting with anti-podoplanin (PDPN, arrowhead) does not coincide with the region reacting with anti-TLR2 (arrows) in the merged image while the regions reacting with VE-cadherin and anti-PECAM-1 (arrowheads) coincide with the regions reacting with anti-TLR2 (arrows) in the merged images (rightmost column). Bar: 20 µm.
Figure 4.
Localization of TLR2 in the glomeruli of HF-induced type II diabetic KK/Ta mouse kidneys.
The HE staining showed that glomeruli (Gl) of the KK/Ta-HF mice are subject to sclerosis. In the laser-scanning confocal microscopy, epithelial cells of proximal tubules (PT) did not react with anti-podoplanin (PDPN) but reacted with anti-TLR2 (arrows) at the inside. The regions reacting with VE-cadherin and anti-PECAM-1 (arrowheads) coincided with the regions reacting with anti-TLR2 (arrows) in the merged images. Bar: 20 µm.
Figure 5.
In situ hybridization for TLR2 mRNA of the diabetic mouse kidneys.
DAB signals for the genes hybridized with the probe for TLR2 mRNA (closed arrowheads) are present weakly at the glomeruli (Gl) of non-diabetic ICR and KK/Ta mice, and strongly at the glomeruli of STZ-induced type I diabetic ICR mice (ICR-STZ) and HF-induced type II diabetic KK/Ta mice (KK/Ta-HF). DAB signals are also positive at the proximal tubules (PT) of ICR-STZ and KK/Ta-HF. There are leukocytes with DAB signals on the internal peroxidase (open arrowheads) in the blood vessels (BV) of the non-diabetic ICR mouse section (top left). Bar: 20 µm.
Figure 6.
Quantitative analysis for TLR2 gene expression in the diabetic mouse kidneys and the effects of Porphyromonas gingivalis LPS on diabetic nephropathy.
(A) Tissue PCR for TLR2 mRNA of the mouse renal cortex. The RT-PCR and real-time PCR analysis show that the mRNA amounts were larger in the STZ-induced type I diabetic ICR mice (STZ) than in the non-diabetic ICR mice (ICR). *Significantly different (p<0.05) (B) Survival curve of the STZ-induced type I diabetic ICR mice with repeated intraperitoneal administrations of Porphyromonas gingivalis LPS. All of the 6 diabetic mice with LPS (red curve) died within the survival period of all of the diabetic mice without LPS and the non-diabetic mice with LPS (blue curve). (C) Urinalysis for STZ-induced type I diabetic ICR mice with LPS at the 8th week of survival. Urine reagent strips show sugar, protein, and bleeding in the mouse urine of non-diabetic ICR mice (ICR), non-diabetic ICR mice with LPS (LPS), STZ-induced type I diabetic ICR mice without LPS (STZ), and the diabetic mice with LPS (STZ+LPS). Extensive amounts of urinary sugar was observed in the diabetic mice and the diabetic mice with LPS, and the urinary protein level was higher in the diabetic mice with LPS than in the diabetic mice without LPS.
Figure 7.
Progress of collagen accumulation in the kidneys of diabetic mice repeatedly administered Porphyromonas gingivalis LPS.
(A) Immunohistochemistry for the expression of type I collagen and podoplanin (PDPN, red) on glomeruli by laser-scanning confocal microscopy. The intensity of the immunostaining for type I collagen is stronger in the glomeruli of STZ-induced type I diabetic ICR mice with the repeated intraperitoneal administrations of Porphyromonas gingivalis LPS (STZ+LPS) than in the diabetic mice without LPS (STZ) and in the non-diabetic ICR mice (ICR). Bar, 20 µm. (B) Quantitative analysis for type I collagen positive areas in the glomeruli. Areas immunostained by anti-type I collagen and anti-podoplanin in ten different glomeruli of laser-scanning microscopic images were measured by ImageJ. The relative volume of type I collagen accumulation in the glomeruli was expressed by a mean ratio: area of type I collagen in a glomerulus/area of a glomerulus within podoplanin-positive podocytes. There were statistically significant differences in the relative accumulations of type I collagen in the glomeruli of non-diabetic ICR mice (ICR) and STZ-induced type I diabetic ICR mice (STZ), and between the diabetic mice and the diabetic mice with LPS (STZ+LPS). Data are expressed as means+SD. *Significantly different (p<0.01).
Figure 8.
Cytokine induction by Porphyromonas gingivalis LPS in the diabetic mouse kidneys.
(A) Immunohistochemistry for the expression of cytokines and podoplanin (PDPN) in glomeruli by laser-scanning confocal microscopy. The IL-6, TNF-α, and TGF-β were detected in the glomeruli of STZ-induced type I diabetic ICR mice with the repeated intraperitoneal administration of Porphyromonas gingivalis LPS (STZ+LPS) whereas the no cytokines were detected in the kidneys of the diabetic mice without LPS (STZ). Bar: 20 µm. (B) Tissue RT-PCR for cytokine mRNAs in mouse kidneys. The amplicons of IL-6, TNF-α, and TGF-β mRNAs were detected from both the renal cortex of STZ-induced type I diabetic ICR mice with the repeated intraperitoneal administrations of Porphyromonas gingivalis LPS (STZ+LPS) and cultured mouse macrophage J774.1 with LPS, whereas they were not detected from the diabetic mice without LPS (STZ). (C) Tissue real-time PCR for cytokine mRNAs in mouse kidneys. The gene expression amounts of IL-6, TNF-α, and TGF-β were statistically significantly larger in the kidney tissue of STZ-induced type I diabetic ICR mice with the repeated intraperitoneal administrations of Porphyromonas gingivalis LPS (STZ+LPS) than in the diabetic mice without LPS (STZ) and in the non-diabetic ICR mice with LPS (LPS). Data are expressed as means+SD. *Significantly different (p<0.01).