All experiments involving animals were approved by the Ethics Review Committee for Animal Experimentation of the Hebei Medical University (Shijiazhuang, China (approval no. 20210909). Mice with global deletion of NPR1 were constructed by Beijing Biocytogen Co., Ltd. based on transcript-NM_008727.5. The structure of the Npr1 gene was showed in National Center for Biotechnology Information (NCBI) (ncbi.nlm.nih.gov/gene/?term=18160). The protocol used for the gene targeting experiments was as previously published (17). The study design is shown in Fig. 1A. In brief, single guide RNAs (sgRNAs) at the 5'- and 3'- target sites were designed by Wellcome Sanger Institute Genome Editing (2020 version, Wellcome Sanger Institute; Fig. 1B and C). Oligos were synthesized and ligated into the pCS-4G vector using the Gibson method (Fig. 1C) (18). The sequence of recombinant plasmid was verified by checking sequence after transforming the recombinant plasmid into Escherichia coli by CaCl₂ method (19). sgRNA 1 and 12 were selected to generate a ~4.0-kb chromosomal deletion (exon 1) at the extreme genome editing -WYS-006-A locus in the mouse genome according to CRISPR-associated protein 9 (Cas9)/sgRNA activity based on their activity and specificity (Fig. 1D). Cas9/sgRNA was microinjected into fertilized mouse ova to obtain male heterozygotes, which were mated with C57BL/6N female mice. The inactivated Npr1 gene was transferred to the first filial generation (F1).

Candidate sgRNA expression plasmid (CAS9P; Sigma-Aldrich) and the plasmid containing the CRISPR/Cas9 target sequence pUCA (Luc)-target reporter gene were transfected into fertilized C57BL/6N mouse ova. Because the Luc gene contained a termination codon and target sequence, the termination codon and target sequence are complementary repeat sequences of Luc at both ends. The termination codon leads to the early termination of Luc translation and expression of non-functional luciferase. The cleavage of the target site by nuclease triggers the DNA repair mechanism based on Single-Strand Annealing (SSA), and the complementary repeat sequence forms a complete luciferase coding sequence through recombination, thus expressing functional luciferase.

A total of 50 8-week-old male mice (including 17 C57BL/6N wild-type (WT) and 33 Npr1^+/- mice) with a mean weight of 20 g were housed in cages at 18-22˚C) and humidity (50-60%) and a 12/12-h light/dark cycle. They were provided with normal mouse diet ad libitum. GSK650394 (HY-15192, Sigma-Aldrich), a selective inhibitor of SGK1, was used to inhibit SGK1 activity. Dexamethasone sodium phosphate (cat. no. D424703, Aladdin) was used as a representative glucocorticoid to observe the effects on mouse heart and kidney function.

The mice were assigned to the following groups: i) WT (n=13); ii) Npr1^+/- (n=16); iii) WT + DEX (WT mice treated with 1 mg/kg dexamethasone intraperitoneal injection, n=4); iv) DEX + Npr1^+/- (Npr1^+/- mice treated with 1 mg/kg dexamethasone intraperitoneal injection, n=13) and v) Npr1^{+/- +} GSK650394 (Npr1^+/- mice treated with 10 mg/kg/day GSK650394 intraperitoneal injection once daily for 10 days, n=4). Mice were placed in metabolic cages for environmental acclimatization 3 days before the start of the experiments. Urine samples were collected for physiological analysis 12 h after the intraperitoneal injection of dexamethasone. Both dexamethasone and GSK650394 used physiological saline (0.9%) as the carrier and the WT and Npr1^+/- groups received physiological saline to reduce inter-group error. In the GSK650394-treated mice group, urine was collected within 12 h of the last injection of GSK650394. All urine was collected for evaluating excretion of urinary sodium, potassium, nitrogen and creatinine through automatic biochemical analyser (cat. no. AU5800, Beckman Coulter). Cardiac function was evaluated using echocardiography. After 12 h glucocorticoid treatment, the mice were sacrificed by cervical dislocation. The heart or kidney tissue was perfused and removed, then fixed overnight in 4% paraformaldehyde. Tissue was dehydrated in a 30% sucrose solution at 4˚C for overnight. Following this, the tissue was embedded in OCT and frozen in liquid nitrogen for later use. The fixed tissue was sectioned (5 µm thickness) at the maximum transverse diameter of the tissue in a -20˚C. Sections were taken for immunofluorescence (IF) and immunohistochemistry (IHC) staining. In addition, left ventricular heart and medullary kidney tissues were excised for western blotting.

The tail tip of each mouse was removed after anesthesia and placed the mouse tip in 0.5 ml centrifuge tube and adding 60 µl of digestive solution A [5 mM NaOH (cat. no. 1310-73-2; Millipore), 0.2 mM EDTA (cat. no. E8030, Solarbio)]. Next, 60 µl solution B (40 mM Tris HCl, pH 5.5, cat. no. T8230; Beijing Solarbio) was added in centrifuge tube and mixed by vortex shaking. The supernatant was used as PCR amplification template following centrifuge the tube at 13,400 x g, 4˚C for 2 min. EGE-WYS-006-A-WT-forward (F), EGE-WYS-006-A-mutant (Mut)-reverse (R) and EGE-WYS-006-A-WT-R sequences were mixed into the same tube for genotype identification. WT-F---Mut-R refers to the bands amplified by primers WT-F and Mut-R. WT-F---WT-R refer to bands amplified by primers WT-F and WT-R (Fig. S1). The following primer pairs were used for PCR: EGE-WYS-006-A-WT-F, 5'-CCCAAACTCCAGCGTTACCAGAAGC-3' and EGE-WYS-006-A-WT-R, 5'-CCTGAGATATTGAAAAGCTAGCCAC-3' and EGE-WYS-006-A-Mut-R, 5'-GACGAGGACCAAGACTGCAGGAAG-3'. The reaction system consisted of a total of 15 µl of mixed solution, including EGE-WYS-006-A-WT-F, EGE-WYS-006-A-WT-R and EGE-WYS-006-A-Mut-R 0.8 µl respectively, dd H₂O 12.1 µl, 5xGreen GoTaq buffer (M3005, Promega) 4 µl and amplification template 1 µl. The following conditions were used for PCR: Initial denaturation at 95 ˚C for 15 sec, annealing at 62 ˚C for 20 sec and extension at 72˚C for 30 sec for 30 cycles. Electrophoresis was performed using 1% agarose under conditions of 90 V for 30 min and visualized through gel imaging system (Image Quant LAS 4000, General Electric Company). For WT mice, there were theoretically two types of WT-F-Mut-R and WT-F-WT-R band; however, only WT-F-WT-R band was selected, with a size of 466 bp because the WT-F-Mut-R band exceeded the length of WT-F-WT-R and PCR products did not reach the expected length. For heterozygous mice, there were WT-F-WT-R and WT-F-Mut-R bands with sizes of 466 and 357 bp, respectively.

Western blotting analysis was conducted as previously described (20). Briefly, the tissue was harvested with a surgical blade and lysed with RIPA lysis buffer (cat. no. R0010; Beijing Solarbio) for 30 min at 4˚C. The samples were quantified by BCA) kit (cat. no. P0009; Beyotime Biotechnology) and heated in boiling water for 5 min and 30 ug total protein/lane was loaded and separated using SDS-PAGE on 10% gel and transferred onto PVDF membranes (cat. no. IEVH07850, Millipore). Membranes were blocked for 2 h with 5% milk at room temperature. Subsequently, membranes were incubated with the following primary antibodies: Anti-NPR1 (cat. no. ab14356; Abcam, 1:1,000); anti-SGK1 (cat. no. DF6188; Affinity Biosciences, Ltd.; 1:1000) and anti-β-actin (cat. no. 81115-1-RR, Proteintech, 1:10,000) overnight at 4˚C. Following primary incubation, membranes were incubated with Goat Anti-Rabbit IgG H&L (horseradish peroxidase) secondary antibody (cat. no. ab205718; Abcam, 1:1,000) for 1.5 h at room temperature. Bands were visualized using ultra-sensitive ECL chemiluminescence kit (P0018AS, Beyotime Institute of Biotechnology) by chemiluminescence imaging system (ChemiDoc XRS, Bio-Rad). Data were analyzed using Image J (version 1.8.0, National Institutes of Health).

The relative levels of the target proteins were visualized via IHC using primary antibodies against NPR1 (cat. no. ab14356; Abcam, 1:1000) and SGK1 (cat. no. ET1610-19; HUABIO, 1:1000) as previously described (21). The stained tissue was observed in four randomly selected fields of view using a light microscope (DM6B, Leica) to compare expression levels of these proteins. The magnification was x200.

IF analysis was performed as previously described (22). The heart tissues were frozen at -20˚C and cut into 5-µm-thick sections with freezing microtome to incubate with primary antibodies against NPR1 (cat. no. sc-137041, Santa Cruz Biotechnology, Inc.; 1:1000) and SGK1 (cat. no. ET1610-19; HUABIO, 1:1,000), and then reacted with secondary antibodies Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight™ 488 (cat. no. S6002, ReportBio,1:10000) and Mouse IgG (H&L) Dylight 549 conjugated (cat. no. S1411, ReportBio, 1:10,000). The stained tissue was observed in four randomly selected fields of view by fluorescence microscope (DM6 B, Leica) and compared expression levels of these proteins by Image J (version 1.8.0, National Institutes of Health). The magnification was x400 and x100.

Blood pressure was measured using the tail-cuff method by an individual blinded to the genotype (23). After 5 days of training, the blood pressure of each animal was measured as the mean of ≥4 sessions on each of 5 days.

Mice were anesthetized via inhalation of 1-4% isoflurane, the chest was depilated and echocardiograms were obtained using an echocardiograph (HP Sonos 2500 Ultrasound System; Hewlett-Packard, Inc.). A transducer (7.5 MHz) was utilized to obtain two-dimensional guided M-type trajectories of the left ventricular short axis cross-section at the papillary apex, as previously described (24).

All experiments were repeated at least four times. Data are presented as the mean ± standard error of the mean. The data were compared using unpaired t test or one-way ANOVA followed by Tukey's post hoc test. Statistical analysis was performed using SPSS 21.0 (IBM Corp). P<0.05 was considered to indicate a statistically significant difference.

The structure of the Npr1 gene was showed in National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/gene/?term=18160) and exon 1 (including the 5'-UTR) was deleted. The two sgRNAs were designed to be located at 2.6 kb upstream of ATG and downstream of exon 1, resulting in a 4.0-kb genome deletion, which was the goal of Npr1 knockout process (Fig. 1A). The mouse model was prepared using an EGE system based on CRISPR/Cas9. A total of eight sgRNA sequences were designed and synthesized into oligos that were ligated into the pCS-4G vector using the Gibson method (Fig. 1B and C). Two sgRNAs, EGE-WYS-006-A-sgRNA 1 and EGE-WYS-006-A-sgRNA 12, were screened using the CRISPR/Cas9 activity detection method (Fig. 1D).

In western blotting, lanes 1 and 2 had one band, representing the WT mice (n=2). Lanes 3, 4, 5, 6 and 7 had two bands (n=5), which were specific for the heterozygous mice. The two types of bands represented WT (n=4) and the heterozygous mice (n=7), respectively. There was no band in lane 8, which represented the negative control (Fig. 2A). The protein levels of NPR1 in the heart and renal tissue of Npr1^+/- mice decreased significantly compared with those in WT mice, as demonstrated through western blotting, IF and IHC assay (Fig. 2B-F). Compared with those in the WT group, the weight, heart rate and blood pressure of Npr1^+/- mice increased significantly, indicating that Npr1 heterozygosity caused changes in the physiological indices (Fig. 2G-I).

Cardiac function deteriorated in Npr1^+/- mice; left ventricular ejection fraction (EF%) and left ventricular shortening fraction (FS%) in Npr1^+/- mice decreased significantly, while left ventricular systolic and diastolic diameter increased in the Npr1^+/- mice compared with those in WT mice. Dexamethasone improved cardiac function and parameters in the Npr1^+/- mice (Fig. 3A-G). A characteristic increase in left ventricular diameter and volume was observed in the systolic and diastolic period after the Npr1 gene knockdown. However, these changes were suppressed by dexamethasone treatment (Fig. 3A-G). The heart size was also consistent with this trend (Fig. 3J). There was no significant difference between the stroke volume and cardiac output of Npr1^+/- and WT mice (Fig. 3H-I), suggesting that heart failure observed in Npr1^+/- mice was still at the compensatory stage.

The cumulative excretion of urinary sodium, potassium, nitrogen and creatinine in 12 h in Npr1^+/- mice decreased significantly compared with that in the WT mice. Dexamethasone abolished the effects of Npr1 knockdown on renal function (Fig. 4B, C, E and G).

The increase in serum creatinine and the decrease in creatinine clearance and urea nitrogen excretion in Npr1^+/- mice reflected impaired renal excretory function; dexamethasone reversed the impaired renal excretory function in these mice (Fig. 4D, F and G). Urine volume of the Npr1^+/- group did not change significantly; however, dexamethasone significantly increased urine volume compared with that in the WT group (Fig. 4A).

In the renal tissues of mice, upregulation of SGK1 expression was observed in the Npr1^+/- group compared with that in the WT group (Fig. 5A-C). However, dexamethasone treatment decreased SGK1 expression and increased NPR1 expression in Npr1^+/- mice (Fig. 5A-C).

To explore the association between the improvement of cardiorenal function caused by dexamethasone and downregulation of SGK1 expression, SGK1 inhibitor GSK650394 was used to inhibit expression of SGK1 and to observe whether the cardiorenal function of Npr1^+/- mice was improved. IHC analysis demonstrated that SGK1 inhibitor GSK650394 significantly inhibited expression of SGK1 (Fig. 6A). Compared with those in the Npr1^+/- mice, FS%, EF% and left ventricular diastolic diameter and volume in the Npr1^{+/- +} GSK650394 group decreased significantly, reflecting an improvement in cardiac function (Fig. 6B-F). Furthermore, urinary sodium and potassium excretion and creatinine clearance rate of the kidney increased in the Npr1^+/- + GSK650394 group, indicating renal function improvement (Fig. 6G-I).