Intrarenal renin angiotensin system revisited: role of megalin-dependent endocytosis along the proximal nephron

Abstract

The existence of a local renin angiotensin system (RAS) of the kidney has been established. Angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), angiotensin receptors, and high concentrations of luminal angiotensin II have been found in the proximal tubule. Although functional data have documented the relevance of a local RAS, the dualism between biosynthesis and endocytotic uptake of its components and their cellular processing has been incompletely understood. To resolve this, we have selectively analyzed their distribution, endocytosis, transcytosis, and biosynthesis in the proximal tubule. The presence of immunoreactive AGT, restricted to the early proximal tubule, was due to its retrieval from the ultrafiltrate and storage in endosomal and lysosomal compartments. Cellular uptake was demonstrated by autoradiography of radiolabeled AGT and depended on intact endocytosis. AGT was identified as a ligand of the multiple ligand-binding repeats of megalin. AGT biosynthesis was restricted to the proximal straight tubule, revealing substantial AGT mRNA expression. Transgenic AGT overexpression under the control of an endogenous promoter was also restricted to the late proximal tubule. Proximal handling of renin largely followed the patterns of AGT, whereas its local biosynthesis was not significant. Transcytotic transport of AGT in a proximal cell line revealed a 5% recovery rate after 1 h. ACE was expressed along late proximal brush-border membrane, whereas ACE2 was present along the entire segment. Surface expression of ACE and ACE2 differed as a function of endocytosis. Our data on the localization and cellular processing of RAS components provide new aspects of the functional concept of a "self-contained" renal RAS.

FIGURE 1.

Renal AGT mRNA and AGT protein expression in normal SD and AGT transgenic rats. A, AGT transcript in PST (S2 and S3 segments) of control SD rat kidney by ³⁵S-labeled radioactive in situ hybridization; S1 segments and glomeruli (G) show background signal. B and C, double staining for AGT transcript (nonradioactive in situ hybridization; B) and AGT protein immunoreactivity (IR; fluorescence immunohistochemistry; C) in AGT-transgenic rat showing mutually exclusive distribution of signals. Dashed lines mark the border between medullary rays (signal in B) and cortical labyrinth (signal in C). D, HRP bright field staining of AGT in control SD rat kidney; AGT is localized in the subapical compartments of PCT as exemplified in its initial portion at the urinary pole of a glomerulus. E, ¹²⁵I-labeled AGT is detected by autoradiography in initial PCT; signal is indicated by arrowheads. Signal distribution was similar in D and E. Scale bar in A, D, and E, 20 μm; scale bar in B and C, 100 μm.

FIGURE 2.

Quantitative RT-PCR analysis of AGT mRNA expression from isolated single-nephron preparations. The percentages of AGT mRNA relative to the respective GAPDH mRNA abundances are shown for proximal S1, S1/S2, and S3 segments. *, p < 0.001.

FIGURE 3.

Renal AGT protein expression of megalin conditional knock-out mice. A, double immunofluorescence staining of megalin (red) and AGT (green) in Cre(+) kidney revealing mosaic distribution of megalin signal in a single PCT profile. Megalin-positive cells show subapical AGT distribution below the BBM, whereas megalin-deficient cells are devoid of AGT staining. Western blot for AGT from BBM fractions (B), plasma (C), and urine (D) of Cre(−) and Cre(+) mice. Note that in Cre(+), AGT is reduced in BBM (B), whereas urinary AGT excretion is strongly increased (D). Arrows mark the respective, specific bands for AGT. The values are the means ± S.E. (n = 5). *, p < 0.05; bar, 10 μm.

FIGURE 4.

Megalin binds AGT. A, silver staining and Western blot of megalin pulldown assay using GST, GST-RAP, and GST-AGT, showing that megalin binds to RAP and AGT. B, pulldown assay using Myc-tagged LBR 1–4 and the Myc-tagged C terminus (C-term) of megalin. Protein fragments of Myc-tagged LBR and C terminus (left, B). As opposed to the C terminus, all four LBR bind to GST-RAP (middle, B) and GST-AGT (right, B).

FIGURE 5.

Intracellular localization of AGT in the proximal tubule. A–D, double immunofluorescence staining of AGT (green) with anti-clathrin antibody staining clathrin-coated pits or vesicles (red, A), anti-EEA1 antibody staining early endosomes (red, B), anti-Rab11 antibody staining recycling endosomes (red, C), and anti-LAMP-2 antibody staining lysosomes (red, D). In the merge pictures, sites of double staining are indicated by arrowheads. The dotted lines mark the position of the apical plasma membrane beneath the brush-border membrane and the basal lamina, respectively, as indicated. Bar, 5 μm.

FIGURE 6.

Proximal tubular processing of AGT and Ang II. Opossum kidney cells cultured in dual chamber devices were incubated apically with 10 μg of AGT or 10 μg of Ang II for 1 h. Samples from the lower chambers were analyzed. A, in comparison with control, the amount of transcytosed AGT is significantly reduced upon RAP or colchicine administration (Western blot evaluation). B, transcytosed Ang II is significantly lower upon RAP, valsartan, or colchicine administration (competitive ELISA). The values are the means ± S.E. (n = 6). *, p < 0.05.

FIGURE 7.

Proximal tubular renin expression in megalin conditional knock-out mice. A, double immunofluorescence staining of AGT (green) and renin (red) in Cre(−) kidney shows consistent colocalization of both proteins in intracellular vesicles (arrowheads). B, double staining of megalin (red) and renin (green) in Cre(+) kidney shows that megalin-deficient cells are devoid of renin. C–E, renin abundances from BBM fractions (C), plasma (D), and urine (E) in Cre(−) versus Cre(+) (Western blots). Arrows point to renin (lower, 43-kDa band); the upper band corresponds to the 53–55-kDa prorenin range. Only the urinary renin levels differ significantly. The values are the means ± S.E. (n = 5). *, p < 0.05; bar, 10 μm.

FIGURE 8.

ACE and ACE2 expression in megalin conditional knock-out mice. A, double immunofluorescence staining of ACE in Cre(−) kidney. The lower dotted lines indicate the border between cortical labyrinth and medullary rays/outer stripe; signal is high in the latter. B, in PST of Cre(+) kidney, megalin-deficient cells exhibit reduced ACE signal in BBM compared with megalin-expressing cells. C, BBM fractions show an overall mild reduction of ACE in Cre(+) compared with Cre(−) by Western blot. D, double immunofluorescence staining of ACE2 in Cre(−) kidney. The lower dotted lines indicate transition from outer stripe to inner stripe of outer medulla. E, in Cre(+) kidney, megalin-deficient cells exhibit increased ACE2 signal in BBM compared with megalin-positive cells. F, BBM fractions show higher ACE2 abundance in Cre(+) compared with Cre(−) by Western blot. The upper dotted lines mark the renal capsule. The values are the means ± S.E. (n = 5). *, p < 0.05. Scale bar in A and D, 20 μm; scale bar in B and E, 10 μm.