Detailed localization of augmented angiotensinogen mRNA and protein in proximal tubule segments of diabetic kidneys in rats and humans

Abstract

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.

Keywords: angiotensin II receptor blocker; angiotensinogen; diabetic nephropathy; proximal tubule; renin-angiotensin system..

Figure 1

In situ hybridization with biotinylated probes against AGT mRNA. Images of stained kidney tissue are shown in A - H and the calculated AGT mRNA expression is shown in I. Each value was expressed as a fold-increase against the average of mRNA level of 5-week-old LETO rats, which was taken as 1. * P < 0.05 vs. each LETO, ** P < 0.01 vs. each LETO. Dotted lines indicate edge of kidney. Magnification, x100 (scale bar, 200 μm). High magnification images of E, F, G, and H are presented as insets. AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; CO: cortex.

Figure 2

Immunostaining for AGT protein. Images of kidney tissue staining images (A - H) and the calculated AGT protein expression in I. Each value was expressed as a fold-increase against the average of protein level of 5-week-old LETO rats, which was taken as 1. * P < 0.05 vs. each LETO, ** P < 0.01 vs. each LETO. Magnification, x100 (scale bar, 200 μm). High magnification images of E, F, G, and H are presented as insets. AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty.

Figure 3

AGT mRNA localization in proximal tubule segment (S) 2 in OLETF and LETO rats. The merged images of AGT mRNA (A and D) and the S2-specific marker CA IV (B and E) are shown in C (LETO rats) and F (OLETF rats). The calculated AGT mRNA expression in S2 is shown in G. Each value was expressed as a fold-increase against the average of protein level of LETO rats, which was taken as 1. Dotted lines indicate edge of kidney. Magnification, x100 (scale bar, 200 μm). AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; CA IV: carbonic anhydrase IV; CO: cortex.

Figure 4

AGT mRNA localization in proximal tubule segment (S) 3 in OLETF and LETO rats. The merged images of AGT mRNA (A, D, G, and J) and the S3-specific marker ecto-ATPase (B, E, H, and K) are shown in C (LETO rats), F (OLETF rats), I (OLETF + olmesartan), and L (OLETF + HRH). The calculated AGT mRNA expression in S3 is shown in M. Each value was expressed as a fold-increase against the average of protein level of LETO rats, which was taken, which was taken as 1. ** P < 0.01 vs. LETO + vehicle. Magnification, x100 (scale bar, 200 μm). High magnification images of C, F, I, and L are also attached. AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; ecto-ATPase: ecto-adenosinetriphosphatase; HRH: hydralazine, reserpine, and hydrochlorothiazide.

Figure 5

AGT protein localization in proximal tubule segment (S) 1 in OLETF and LETO rats. The merged images of AGT protein (A, D, G, and J) and the S1-specific marker SGLT2 (B, E, H, and K) are shown in C (LETO rats), F (OLETF rats), I (OLETF + olmesartan), and L (OLETF + HRH). The calculated AGT protein expression in S1 is shown in M. Each value was expressed as a fold-increase against the average of protein level of LETO rats, which was taken as 1. * P < 0.05 vs. LETO + vehicle. Dotted line indicates edge of kidney. Magnification, x100 (scale bar, 200 μm). High magnification images of C, F, I, and L are also attached. AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; SGLT2: sodium glucose cotransporter 2; HRH: hydralazine, reserpine, and hydrochlorothiazide; CO: cortex.

Figure 6

AGT protein localization in proximal tubule segment (S) 2 in OLETF and LETO rats. The merged images of AGT protein (A and D) and the S2-specific marker CA IV (B and E) are shown in C (LETO rats) and F (OLETF rats). The calculated AGT protein expression in S2 is shown in G. Each value was expressed as a fold-increase against the average of protein level of LETO rats, which was taken as 1. Dotted lines indicate edge of kidney. Magnification, x100 (scale bar, 200 μm). AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; CA IV: carbonic anhydrase IV; CO: cortex.

Figure 7

AGT protein localization in proximal tubule segment (S) 3 in OLETF and LETO rats. The merged images of AGT protein (A, D, G, and J) and the S3-specific marker ecto-ATPase (B, E, H, and K) are shown in C (LETO rats), F (OLETF rats), I (OLETF + olmesartan), and L (OLETF + HRH). The calculated AGT protein expression in S3 is shown in M. Each value was expressed as a fold-increase against the average of protein level of LETO rats, which was taken as 1. * P < 0.05 vs. LETO + vehicle. Magnification, x100 (scale bar, 200 μm). High magnification images of C, F, I, and L are also attached. AGT: angiotensinogen; LETO: Long-Evans Tokushima Otsuka; OLETF: Otsuka Long-Evans Tokushima Fatty; HRH: hydralazine, reserpine, and hydrochlorothiazide; ecto-ATPase: ecto-adenosinetriphosphatase.

Figure 8

AGT mRNA localization in the kidney cortex proximal tubule segment (S)s of patients with diabetes. Little AGT mRNA was detected in S1 in control subjects (A) and in patients with diabetes (B). AGT mRNA in S2 did not differ significantly (E) between control subjects (C) and patients with type 2 diabetes (D). Data are presented as a fold-increase compared with the levels of control subjects. Green; AGT mRNA, Red; segment-specific marker. Dotted lines indicate the edge of glomeruli. Magnification, x200 (scale bar, 100 μm). AGT: angiotensinogen; DN: diabetic nephropathy; G: glomeruli.

Figure 9

AGT protein localization in the kidney cortex proximal tubule segment (S)s of patients with diabetes. AGT protein expression in S1 was significantly greater (C) in patients with type 2 diabetes (B) compared with control subjects (A). AGT protein expression in S2 did not differ significantly (F) between and control subjects (D) and patients with type 2 diabetes (E). Data are presented as a fold-increase compared with the levels of control subjects. * P < 0.05 vs. control subjects. Green; AGT protein, Red; segment-specific marker. Magnification, x200 (scale bar, 100 μm). AGT: angiotensinogen; DN: diabetic nephropathy.