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. 2021 Aug;473(8):1229-1246.
doi: 10.1007/s00424-021-02598-z. Epub 2021 Jul 6.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

Marta Figueiredo  1 , Arezoo Daryadel  1 , Gabin Sihn  2 , Dominik N Müller  2   3   4 , Elena Popova  2 , Anthony Rouselle  2 , Genevieve Nguyen  5 , Michael Bader  6   7   8   9 , Carsten A Wagner  10
Affiliations

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

Marta Figueiredo et al. Pflugers Arch. 2021 Aug.

Abstract

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

Keywords: Endocytosis; H+-ATPase; Low molecular weight proteins; Lysosome; Proximal tubule.

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Figures

Fig. 1
Fig. 1
Generation of the Atp6ap2 shRNA rat model and efficiency of Atp6ap2/(P)RR) knock-down. A Structure of the transgene construct, pTet-sh, made of two expression cassettes. A first one carries a tetracycline operator (tetO) sequence and expresses an shRNA against Atp6ap2 under the control of the human H1 promoter. A second cassette consists of a tetracycline repressor (tetR) cDNA followed by a polyadenylation site (pA), and is driven by the CAGGS promoter. Primers TetOfw and CAGGSrv (arrows) were used for genotyping of rats. B Genotyping by PCR performed on newborn rat tails with a 195-bp PCR product characteristic of the transgenic animals (Sh). C Comparative expression of tetracycline repressor (TetR) protein between transgenic (Sh) and control (Wt) rats, as studied in various tissues by western blot. GAPDH was used as loading control. D RT-qPCR analysis of total mRNA of kidneys from rat Wt and Sh demonstrates successful knock-down of (P)RR/Atp6ap2 mRNA (n = 6/genotype). E Immunohistochemistry for the H+ATPase a4 subunit (green), ATP6ap2 (red), and DAPI (blue) in kidney sections from Wt and Sh rats. Yellow overlay is seen in the Wt sections due to colocalization of a4 and ATP6ap2 while in sections from Sh rats only green staining for a4 is visible. Scale bar size: 50 µm. F Renal morphology of Sh and control animals. Semi-thin Sect. (200 nm thick) were stained with Toluidine Blue, scale bar size 100 µm
Fig. 2
Fig. 2
Preserved fluid-phase endocytosis but impaired receptor-mediated endocytosis in (P)RR/ATP6ap2 deficient rats. A Albuminuria in Sh rats detected by Coomassie blue staining of SDS-Page gels loaded with urine samples normalized to creatinine (7 mg/mL) BSA (7 mg/mL) was loaded as positive control. Bar graph summarizing data (n = 4 in each animal group). Student’s t-test *p < 0.05. B Elevated vitamin D binding protein (VDBP) in urine of Sh rats detected by immunoblotting of urine samples normalized to creatinine (7 mg/mL). Bar graph summarizing data from n = 4/genotype. C Immunohistochemistry for dextran-FITC (10 kDa, green), DAPI (blue), and actin/phalloidin (red) in kidney slices from Wt and shRNA rat 10 min after injection (scale bar size 50 µm). D Quantification of dextran-FITC in the cytoplasmic fraction of the control and Sh rats kidney homogenate. FITC-dextran was normalized to total protein content. E Immunohistochemistry for human transferrin (red), megalin (green), and DAPI (blue) in Wt and Sh rats kidney 10 min and 40 min after injection showed strong residual staining of transferrin after 40 min in Sh rat kidneys (see insert) (scale bar size 50 µm). F Western blotting for human transferrin in Wt and Sh rat urine 10 min after injection. Urine samples were normalized to creatinine (7 mg/mL). Bar graph summarizing data from n = 4/genotype. Statistical analyses were performed using Student’s t-test **p < 0.01
Fig. 3
Fig. 3
Altered expression of proteins involved in receptor-mediated endocytosis. A Brush border membrane preparations of control and Sh rat kidneys were used for blotting for H+ATPase a4 (ATP6V0a4), B2 (ATP6V1B2), and A (ATP6V1A) subunits as well as for NaPiIIa, NHE3, megalin, and cubilin. B Densitometries were adjusted to β-actin (loading control). Statistical analysis using Student’s t-test (n = 6/genotype or n = 4/genotype for NaPiIIa). *p < 0.05 ***p ≤ 0.001
Fig. 4
Fig. 4
Generation of kidney-epithelial cell-specific ATP6ap2 ablation in mouse. A RT-qPCR analysis of total mRNA of kidneys, lungs, and hearts from Wt and Flox/Pax8 + mice treated with 1 mg/mL doxycycline (low dose). Atp6ap2 mRNA abundance was normalized to HPRT. Statistical analysis was performed using Student’s t-test (Wt/Pax8 + : n = 4 and Flox/Pax8 + : n = 5) ***p < 0.001. B Western blotting for ATP6ap2 in total membrane preparations from the kidney of Wt and Flox/Pax8 + mice and summary of data as bar graph. Statistical analysis was performed using Student’s t-test (Wt/Pax8 + : n = 4 and Flox/Pax8 + : n = 5) *p < 0.05. C Immunohistochemistry for ATP6ap2 (red), AQP2 (green), and DAPI (blue) in kidney sections from Wt/Pax8 and Flox/Pax8 + mice with proximal tubules (upper panels) and medullary collecting ducts (lower panels). Scale bar size 100 µm
Fig. 5
Fig. 5
Albuminuria and low molecular weight proteinuria in the absence of the ATP6ap2. Urine samples were normalized to creatinine. Bovine serum albumin (BSA, 7 mg/mL) was loaded as positive control. A Albumin was detected by Coomassie blue staining whereas B vitamin D binding protein (VDBP) or C (Pro)cathepsin B was revealed by immunoblotting. Data were summarized as bar graphs (n = 4/genotype. Student’s t-test *p < 0.05, ***p < 0.001
Fig. 6
Fig. 6
Delayed receptor-mediated endocytosis in ATP6ap2 deleted mice. WT/Pax8 + and Flox/Pax8 + mice were coinjected with dextran-FITC (10 kDa) and human recombinant transferrin. Kidneys were collected 10 and 40 min after injection. A Immunohistochemistry for dextran-FITC,10 kDa (green), DAPI (blue), and actin/phalloidin (red) in kidney slices from Wt and Flox/Pax8 + mice 10 and 40 min after injection. B Immunohistochemistry for human transferrin (red), DAPI (blue), and megalin (green) in kidneys from Wt and Flox/Pax8 + mice 10 and 40 min after injection. C Immunohistochemistry for Lamp-1 (green) and human transferrin (red) 40 min after injection. Scale bar size 50 µm for all pictures
Fig. 7
Fig. 7
Deletion of the ATP6ap2 alters expression of major proteins in the endocytic pathway. A Brush border membrane preparations from kidneys of Wt/Pax8 + and Flox/Pax8 + mice were blotted for the H+ATPase a4 (ATP6V0a4), a2 (ATP6V0a2), EII (ATP6V1EII), B2 (ATP6V1B2), and A (ATP6V1A) subunits as well as for NHE3, megalin, and cubilin. B Densitometries were normalized to β-actin (loading control). Student’s t-test (n = 4 per group), **p ≤ 0.01, ***p ≤ 0.001
Fig. 8
Fig. 8
Accumulation of autophagosomes in ATP6ap2 deficient mice. A, B Western blotting for total and phosphorylated mammalian target of rapamycin (mTOR) and the LC3B subunit of the autophagosome and densitometry summarizing results. Student’s t-test (n = 4 per group), **p ≤ 0.01. C Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (LC3-B) (red) and DAPI (blue) in kidneys from Wt and Flox/Pax8 + mice insert shows higher magnification. Scale size bar 100 µm
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