Figure 1.
RenTg mice are hypertensive and develop chronic kidney disease progressively with the age.
Systolic blood pressure was highly increased in RenTg mice since the age of 3 months as compared to aged matched WT controls (A). Urinary excretion of albumin progressively increased with age to reach at 12 months highly elevated values in RenTg mice (B) whereas GFR significantly decreased (C). In accordance, the percentage of sclerotic glomeruli progressively increased in RenTg mice compared to WT mice (D). Representative examples of renal cortical slices after Masson’s Trichrome histology (E–I). Renal cortex of WT (E) as well as 3 month-old RenTg appeared normal (F); inflammatory lesions and renal fibrosis were evident at 5 (G), and 8 (H) months respectively in RenTg mice. Renal cortical tissue of 12 month old RenTg mice displayed several of the characteristics of hypertension-associated renal disease such as peri- vascular and glomerular inflammation, glomerulosclerosis, fibrinoid-like deposits within renal vessels, glomerular ischemia and tubular dilatation (I). Values are means ± SEM; n = 8 and 9 for wild type and RenTg mice, respectively for each time point; *P<0,05 or **P<0,01 vs WT. Magnification of the microphotographs: X200.
Figure 2.
Alterations of endothelial function markers in RenTg mice.
Immunohistochemistry on renal cortical cryosections showed a marked increase of the PECAM-1 (CD31) expression in the 3 month old RenTg mice as compared to the control tissues (A). Quantitative RT-PCR analysis showed a progressive increase of VCAM-1 in the RenTg mice (B). Note that the ET-1 mRNA expression is up-regulated at the later stages of the renal disease (C). Values are presented as fold increase as compared to the age matched WT mice and are means ± SEM; n = 8 and 9 for wild type and RenTg mice, respectively for each time point; *P<0,05 or **P<0,01 vs WT. Magnification of the microphotographs: X200.
Figure 3.
Inflammatory cell infiltration during the progression of CKD in RenTg mice.
Representative examples of renal cortical sections for macrophages (CD68) and lymphocytes (CD3) immunostaining, respectively. Note that macrophage infiltration was first detected since the age of 5 months in the renal cortex of RenTg mice (A), whereas a positive staining for lymphocytes was observed at 7 month-old RenTg mice (B). Magnification of the microphotographs: X200.
Figure 4.
Changes in the TGFβ/BMP balance and cardiac hypertrophy during the progression of CKD in RenTg mice.
Quantitative RT-PCR analysis of TGF-β1 (A), BMPR2 (B), USAG-1 (C) and Id-1 (D) in the renal cortex of wild type and RenTg mice. Note that the expression of BMPR2 has been decreased since 3 months in RenTg mice while at the expressions of TGF-β1 and USAG-1, an endogenous inhibitor of BMPs, were highly increased from 8 months in injured tissues. Significant down regulation of Id-1 mRNA expression, a BMP7 target gene is also detected after 8 months. In addition, qPCR analysis of typical markers of cardiac hypertrophy, such as BNP (E) and β-MHC/α-MHC ratio (F), as well as measurements of the heart weight (H), confirmed that RenTg mice developed cardiac hypertrophy with age. Values are presented as fold increase or decrease as compared to the age matched WT mice and are mean ± SEM; n = 8 and 9 for wild type and RenTg mice, respectively for each time point; *P<0,05 or **P<0,01 vs WT.
Figure 5.
Progression of renal interstitial and cardiac fibrosis in hypertension-induced CKD.
Representative example of Sirius red staining in WT (A) and RenTg (B) 12 month-old mice. Quantification by morphometric analysis of renal cortical paraffin embedded sections after Sirius Red staining is shown in panel C. Fibrillar collagen content was progressively increased in RenTg mice after 5 months. Similar results were observed in heart sections of WT (D) and RentTg (E) 12 month-old mice. Quantification of cardiac fibrosis is shown in panel F. Values are presented as fold increase as compared to the age matched WT mice and are means ± SEM; n = 8 and 9 for wild type and RenTg mice, respectively for each time point; *P<0,05 vs WT.
Figure 6.
Alterations of mRNA expression of podocyte and tubular markers during progression of CKD in RenTg mice.
Quantitative RT-PCR analysis of nephrin (A), KIM-1 (B) and E-cadherin (C) in the renal cortex of wild type and RenTg mice. Note that the expression of nephrin is decreased since 8 months in RenTg mice while at the same age the expression of KIM-1 was highly increased. Significant differences in E-cadherin expression were detected in the 12 month old RenTg mice. Values are presented as fold increased or decreased as compared to the age matched WT mice and are mean±SEM; n = 8 and 9 for wild type and RenTg mice, respectively for each time point; *P<0,05 or **P<0,01 vs WT.
Table 1.
List of the primers used for the Real Time-PCR of the different genes as mentioned in the results sections.