Angiotensin-[1–7] attenuates kidney injury in experimental Alport syndrome

Angiotensin-[1–7] (Ang-[1–7]) antagonize the actions of the renin-angiotensin-system via the Mas receptor and thereby exert renoprotective effects. Murine recombinant angiotensin-converting enzyme (ACE)2 was reported to show renoprotective effects in an experimental Alport syndrome model; however, the protective effect of direct administration of Ang-[1–7] is unknown. Here, we used Col4a3−/− mice as a model of Alport syndrome, which were treated with saline or Ang- [1–7]; saline-treated wild-type mice were used as a control group. The mice were continuously infused with saline or Ang-[1–7] (25 μg/kg/h) using osmotic mini-pumps. Col4a3−/− mice showed increased α-smooth muscle actin (SMA), collagen, and fibronectin expression levels, which were attenuated by Ang-[1–7] treatment. Moreover, Ang-[1–7] alleviated activation of transforming growth factor-β/Smad signaling, and attenuated the protein expression of ED-1 and heme oxygenase-1, indicating reduction of renal inflammation. Ang-[1–7] treatment further reduced the expression levels of inflammatory cytokines and adhesion molecules and attenuated apoptosis in human kidney cells. Finally, Ang-[1–7] downregulated TNF-α converting enzyme and upregulated ACE2 expression. Thus, treatment with Ang-[1–7] altered the ACE2-Ang-[1–7]-Mas receptor axis in the kidneys of Col4a3−/− mice to attenuate the nephropathy progression of Alport syndrome.

We observed decreased expression of TACE and increased expression of ACE2 in the Col4a3 −/− mice kidney, and an improvement by the administration of Ang- [1][2][3][4][5][6][7]. TACE activity is known to induce cleavage and loss of tissue ACE2, thereby further exacerbating Ang II harmful effects. Such ACE2 regulation by TACE is caused by enzymatic cleavage, not by the regulation of gene transcription 16 . Therefore, uncoupling between tissue protein level and mRNA occurs. Previously, protein expression and activity of ACE2 were reduced in the 7-week-old Col4a3 −/− mice kidney, but mRNA level was not changed 13 . Unfortunately, our study did not check that mRNA levels of ACE2 did not change after Ang- [1][2][3][4][5][6][7] administration. Therefore, in our study, the decrease of ACE2 expression and recovery by Ang- [1][2][3][4][5][6][7] administration in the Col4a3 −/− mice kidney may be caused by TACE enzymatic cleavage, but also by various cytokines activated by TACE in the inflammation state.
In conclusion, Ang- [1][2][3][4][5][6][7] presents anti-fibrotic, anti-inflammatory and anti-apoptotic action via recovery of altered RAS in experimental AS mice. Thus, Ang- [1][2][3][4][5][6][7] shows good potential as a treatment for inhibiting the progression of CKD. Experimental animals and protocols. Wild-type (WT) and Col4a3 −/− mice on a congenic 129X1/SvJ background were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and only male mice were used in this study. The mice were housed at the animal care facility at the Chonnam national university medical school, and fed mice standard diet with ad libitum access to water. We only used male mice in this study. Tail tip genotyping was performed to verify the genotype for Col4a3 −/− mice by using the following primers: common, 5′-CCA GGC TTA AAG GGA AAT CC-3′; WT reverse, 5′-TGC TCT CTC AAA TGC ACC AG-3′; and mutant reverse, 5′-GCT ATC AGG ACA TAG CGT TGG-3′.
Urinary NGAL and microalbuminuria measurement. Urine of mice was collected by maintaining mice in individual metabolic cages for the last 3 days of the experiment. Mice were maintained in individual metabolic cages for the last 3 days of the experiment to allow urine collection. Urine samples were centrifuged at 8000 g (2020) 10:4225 | https://doi.org/10.1038/s41598-020-61250-5 www.nature.com/scientificreports www.nature.com/scientificreports/ for 5 minutes immediately after collection. Urinary level of NGAL were measured with a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA), according to manufacturer's instruction. Urinary microalbumin was measured using the turbidimetric immunoassay method (Olympus AU 5431, Toshiba TBA-200FR autoanalyzer, Tokyo, Japan), while urinary creatinine was measured using the Jaffe method. Urinary albumin excretion was estimated as the albumin-to-creatinine ratio in milligrams of albumin per gram of creatinine.
Semiquantitative immunoblotting. Western blot analysis was performed as previously described 4,34 .
Kidney tissues were homogenized in ice-cold isolation solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM ethylenediamine tetraacetic acid (EDTA), 8.5 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride (pH 7.2). The homogenates were centrifuged at 4000 × g for 15 minutes at 4 °C to remove whole cells, nuclei, and mitochondria. The total protein concentration was measured by bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, USA). All samples were adjusted to reach the same final protein concentrations. They were then dissolved at 65 °C for 15 minutes in SDS-containing sample buffer and stored at −20 °C. To confirm equal loading of proteins, an initial gel was stained with Coomassie blue. SDS-PAGE was performed on 9 or 12% polyacrylamide gels. The proteins were electrophoretically transferred onto nitrocellulose membranes (Hybond ECL RPN3032D; Amersham Pharmacia Biotech; Little Chalfont, UK) using Bio-Rad Mini Protean II apparatus (Bio-Rad; Hercules, CA, USA). The blots were blocked with 5% milk in PBS-T (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, and 0.1% Tween-20 at pH 7.5) for 1 hour; incubated overnight at 4 °C with primary antibodies; and incubated with secondary anti-rabbit, anti-mouse, or anti-goat horseradish peroxidase-conjugated antibodies thereafter. The immunoblots were then visualized using an enhanced chemiluminescence system. Protein levels were quantified using densitometry. The relative intensities of immunoblot signals were measured by densitometry using Scion image for windows software (Scion Corporation, 2000-2001. version Alpha 4.0.3.2. MD, USA) and were expressed as fold changes relative to control. Primary and secondary antibodies used in immunoblottings are listed in Table S1.

Real-time polymerase chain reaction (Real-Time PCR).
Polymerase chain reaction analysis was performed as previously described 35 . Renal cortex was homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and then dissolved in distilled water. The RNA concentration was determined by the absorbance read at 260 nm (Ultraspec 2000; Pharmacia Biotech, Cambridge, UK). The mRNA expression of inflammatory cytokines and adhesion molecules was determined by real-time PCR. cDNA was made by reverse transcribing 5 μg of total RNA using oligo (dT) priming and superscript reverse transcriptase II (Invitrogen, Carlsbad, CA, USA). cDNA was quantified using Smart Cycler II System (Cepheid, Sunnyvale, CA, USA) and SYBR Green was used for detection. Each PCR reaction was done in 10 pM forward primer, 10 pM reverse primer, 2X SYBR Green Premix Ex Taq (TAKARA BIO INC, Seta 3-4-1, Japan), 0.5 μl cDNA and H2O to bring the final volume to 20 μl. Relative levels of mRNA were determined by real-time PCR, using a Rotor-GeneTM 3000 Detector System (Corbette research, Mortlake, NSW, Australia). Sequences of primers are listed in Table S2.
The PCR was performed according to the following steps: (1) 95 °C for 5 minutes; (2) 95 °C for 20 seconds; (3) 58 to 60 °C for 20 seconds (optimized for each primer pair); (4) 72 °C for 30 seconds to detect SYBR Green. Steps 2-4 were repeated for additional 40 cycles, while at the end of the last cycle temperature was increased from 60 to 95 °C to produce a melt curve. Data from the reaction were collected and analyzed with the Corbett Research Software. The comparative critical threshold values from quadruplicate measurements were used to calculate the gene expression, with normalization to GAPDH as an internal control. Melting curve analysis was performed to enhance specificity of the amplification reaction.
Histology. Preparation and staining of the kidney tissue proceeded as previously described 35 . Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3 μm-thick sections. Hematoxylin and eosin (H&E) staining was performed to assess the histological morphology. The kidney tissue section slides were incubated in Gill's hematoxylin for 5 min, washed with tap water, incubated in 95% ethanol, and stained with eosin and phloxine for 1 min. Subsequently, the sections were dehydrated in ethanol and xylene, and were mounted with Canada balsam. For Masson's trichrome staining, after deparaffinization with xylene, the sections were treated with Bouin's solution at 56 °C for 30 min and were washed under running tap water until the sections were clear. The sections were subsequently stained with Weigert's hematoxylin, followed by staining with Biebrich Scarlet/Acid Fuchsin solution for 10 min and washing with distilled water. The sections were incubated with phosphotungstic acid/phosphomolybdic acid solution for 10 min and were treated with Aniline Blue solution for 15 min. They were subsequently incubated with acetic acid for 1 min and were dehydrated with ethanol and xylene. Collagen depositions, nuclei, and muscle fibers were stained blue, black, and red, respectively. Primary and secondary antibodies used in immunohistochemistry are listed in Table S3. Apoptosis of tubular epithelial cells was detected with TUNEL staining with ApopTag Plus Peroxidase In Situ Apoptosis Kit (Sigma-Aldrich), according to the manufacturer's instruction.
Cell culture and reagents. In vitro studies were conducted as previously described 35 . In short, human renal proximal tubular epithelial cells (HK-2 cells, American Type Culture Collection, Manassas, VA, USA) were used for in vitro study. HK-2 cells were cultured and passaged every 3~4 days in 100-mm dishes containing combined Dulbecco's modified Eagle's (DMEM) and Hams F-12 medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). HK-2 cells were then incubated in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C for 24 h, and sub-cultured until 70-80% confluence. HK-2 Cells were plated onto 60-mm dishes in a medium containing 10% FBS and incubated for 24 hours. The cells were then incubated in DMEM-F12 medium with serum free FBS