sGC stimulator (BAY 41‐8543) combined with PDE9 inhibitor (BAY 73‐6691) reduces renal fibrosis in 5/6 nephrectomized rats

Abstract

Renal fibrosis is closely related to the prognosis of chronic kidney disease (CKD). The increase in cGMP reduces renal fibrosis. Soluble guanylate cyclase (sGC) and phosphodiesterase (PDE) are key enzymes that maintain cGMP levels. BAY 41–8543 (1 mg/kg/day) and/or BAY 73–6691 (1 mg/kg/day) were used to treat 5/6 nephrectomized rats for 13 weeks. 5/6 Nephrectomy caused an increase in cystatin C, proteinuria and glomerulosclerosis and renal interstitial fibrosis. Neither sGC stimulation nor PDE9 inhibition alone improved kidney function and morphology, whereas BAY 41–8543 in combination with BAY 73–6691 attenuated renal interstitial fibrosis. This beneficial effect could not be explained by alterations in blood pressure and the renal immune system. BAY 41–8543 in combination with BAY 73–6691 had no effect on renal macrophage, CD4 + T‐cell and CD8 + T‐cell in the late‐stage of 5/6 nephrectomy. RNA sequencing revealed BAY 41–8543 in combination with BAY 73–6691 down‐regulated the expression of fibrosis‐related genes such as Collagen Type I Alpha 1, Collagen Type III Alpha 1 Chain and Collagen Type XIV Alpha 1 Chain. sGC stimulator combined with PDE9 inhibitor attenuated renal fibrosis in 5/6 nephrectomized rats by down‐regulating fibrosis‐related gene expression. This novel approach of using low‐dose combination therapies to minimize side effects while maintaining therapeutic efficacy offers a promising strategy for the treatment of CKD.

We investigated for the first time the potential renoprotective effects of a low‐dose non‐NO‐dependent sGC stimulator (BAY 41‐8543) in combination with a low‐dose PDE9 inhibitor (BAY 73‐6691) in a 5/6 nephrectomy model. This combination therapy significantly attenuates renal interstitial fibrosis by downregulating key fibrosis‐related genes (COL1A1, COL3A1, COL14A1). These findings highlight a new therapeutic strategy for CKD, emphasizing the importance of gene transcription changes as early indicators of therapeutic efficacy.

Plain English Summary.

This study explores a new treatment strategy for kidney disease, specifically chronic kidney disease (CKD). We tested a combination of two drugs that increase levels of a beneficial molecule called cGMP, known to reduce kidney fibrosis (scarring). Using a rat model of CKD, the combination treatment significantly decreased fibrosis and regulated fibrosis‐related genes without affecting blood pressure or immune cell levels in the kidneys. These findings suggest that combining these drugs at low doses may be an effective and safer option for reducing kidney fibrosis in CKD patients. Further studies are needed to confirm these results in human patients.

1. INTRODUCTION

Chronic kidney disease (CKD) is a chronic progressive disease with a high mortality rate. There are many causes for CKD, but almost all ultimately lead to glomerulosclerosis, tubular atrophy and interstitial fibrosis. The degree of renal interstitial fibrosis is closely related to the renal prognosis. ¹ Therefore, research aimed at treating or preventing renal interstitial fibrosis is of great significance for CKD patients.

Recent studies have demonstrated that the pathway [NO (nitric oxide) – sGC (soluble guanylate cyclase) – cGMP (3′,5′‐cyclic guanosine monophosphate) – PDE (phosphodiesterase) – GMP (guanosine monophosphate)] is strongly associated with renal fibrosis and prognosis. ² , ³ Renal injury causes impairment of NO formation and bioavailability and thus reduces cGMP production through the NO pathway. ⁴ cGMP is an important intracellular second messenger involved in many (patho‐)physiological processes in the kidney, including vasodilatation, antifibrosis, antiproliferation, anti‐inflammation and neurotransmission. ⁵ cGMP deficiency, on the other hand, accelerates renal deterioration. ⁶ sGC and PDE are key enzymes in the maintenance of cGMP levels and play an important role in controlling the strength and length of cGMP‐dependent cell signalling. sGC catalyses the conversion from GTP to cGMP, whereas cGMP‐specific PDE degrades cGMP to GMP. Therefore, enhancement of sGC and/or inhibition of PDE can ameliorate inadequate cGMP levels in pathological conditions, resulting in an improved renal outcome. In fact, many studies have demonstrated that sGC stimulators improve renal fibrosis and proteinuria in various renal diseases by increasing cGMP levels. ⁷ , ⁸ It has been suggested that an increase in renal PDE activity under pathological conditions accelerates cGMP degradation and thereby weakens the effect of sGC stimulators. ⁹ A combined application of sGC stimulators and PDE inhibitors could hypothetically produce significant effects at lower drug doses. At present, there are few studies on this drug combination. Both sGC stimulators and PDE inhibitors have been proposed to act in a dose‐dependent manner ¹⁰ , ¹¹ and the application of lower drug doses may mitigate side effects, especially for sGC stimulators. It is well known that sGC stimulators have a systemic vasodilatory effect and that this effect is dose‐dependent. ¹⁰ Furthermore, sGC stimulators have been shown to reduce blood pressure in normotensive rats. ¹² We, therefore, proposed that a low‐dose combination could optimize the therapeutic effect of sGC stimulators and PDE inhibitors. The phosphodiesterase 9 (PDE9) has the highest affinity of all PDE family members for specifically hydrolyzing cGMP and has been found to be expressed in the kidney. ⁹ To investigate this hypothesis, we combined the non‐NO‐dependent sGC stimulator BAY 41–8543 (1 mg/kg/d) with the potent and selective PDE9 inhibitor BAY 73–6691 (1 mg/kg/d) in a 5/6 nephrectomized rats and studied the reno‐ and cardio‐protective effects of this drug combination versus the use of a single drug.

2. MATERIALS AND METHODS

2.1. Experimental animals and protocol

The animal experiments were approved by the Animal Care and Use Committee of Jinan University, Guangzhou, China (IACUC‐20190830‐02). All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, as well as the National Research Council's Guide for the Care and Use of Laboratory Animals. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies. ¹³

Wistar rats (7 weeks; male) from Vital River Laboratory Animal Technology Co., Ltd (Beijing) were purchased. The rats were housed at room temperature (22–25°C), a humidity of 55 ± 5%, a 12‐hour light/dark cycle and supplied with standard rat chow and water. After 7 days of acclimatization, rats were randomly arranged into five groups: sham + placebo (10 animals); five‐sixths nephrectomy (5/6Nx) + placebo (15 animals); 5/6Nx + sGC stimulator (BAY 41–8543; 1 mg/kg/day, 15 animals); 5/6Nx + PDE9 inhibitor (BAY 73–6691; 1 mg/kg/day, 15 animals); 5/6Nx + 1 mg/kg/day sGC stimulator + 1 mg/kg/day PDE9 inhibitor (15 animals). To randomly assign groups, each rat was first given a unique identifier from 1 to 70. Then, 70 consecutive random numbers were selected from a random number table, with one number assigned to each rat. The rats were subsequently sorted in ascending order based on their assigned random numbers. Using this ordered list, the first 10 rats were assigned to the sham + placebo group, the following 15 to the 5/6Nx + placebo group and so on. To reduce surgical bias, the 5/6 nephrectomy procedure was consistently performed by the same experimenter. Additionally, outliers were identified and excluded using the ROUT method (Q = 1%) in GraphPad Prism 6 software. Both absolute and relative parameter values were included in the statistical analysis. During the course of the study, anaesthesia and gavage resulted in the death of some animals, so the actual number of animals in each group was as follows: Sham + PBO (n = 8), 5/6Nx + PBO (n = 15), 5/6Nx + BAY 41–8543 (n = 13), 5/6Nx + BAY 73–6691 (n = 14), 5/6Nx + BAY 41–8543 + BAY 73–6691 (n = 14). 5/6Nx was performed on rats under anaesthesia with 2,2,2‐tribromomethanol (500 mg/kg intraperitoneally) as follows: uni‐nephrectomy of the right kidney (Uni‐Nx) in week 1 followed by amputation of the poles of the left kidney in week 3. At the same time points, all other operations except nephrectomy were performed on rats in the Sham + PBO group. All rats received medication/placebo by gavage for 13 weeks from the time of surgery until 24 hours before euthanasia. The PDE9 inhibitor BAY 73–6691 ¹⁴ , ¹⁵ , ¹⁶ and the sGC stimulator BAY 41–8543 ¹⁰ , ¹⁷ , ¹⁸ were manufactured by Bayer, Pharmaceuticals AG (Wuppertal, Germany) and doses were selected based on previous studies. Both compounds were dissolved in 0.5% w/v hydroxypropyl methylcellulose aqueous solution and administered to rats by gavage. Rats in control groups (Sham + PBO and 5/6Nx + PBO) received 0.5% w/v hydroxypropyl methylcellulose aqueous solution. Urine and blood were collected before surgery and at the study endpoint. Urine was collected as follows: rats were individually placed in metabolic cages, where 24‐hour urine was collected and the volume was recorded. The urine was then centrifuged at 4°C for 10 minutes at 12000 rpm and the resulting supernatant was stored at −80°C for further analysis. Blood samples at baseline were collected from the retro‐orbital venous plexus under anaesthesia with isoflurane (3%) via chamber induction, whereas blood samples at the study endpoint were collected from the abdominal aorta under anaesthesia with 2,2,2‐tribromomethanol (500 mg/kg intraperitoneally). All blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C and the supernatant was stored at −80°C for further analysis.24 hours before euthanasia, we stopped pharmacological interventions and performed echocardiography (vevo 770TM‐230, VisualSonics, Canada). Blood pressure (BP) was measured before euthanasia. BP was measured using the tail‐cuff method (BP‐2000 Blood Pressure Analysis System, model BP‐2000‐RP‐4, Visitech Systems, U.S.A.). All rats were euthanized at week 18, after which blood and organ (kidney, heart) samples were collected. The kidneys were weighed and cut longitudinally into two halves: one half was fixed in 4% paraformaldehyde for histological analysis, and the other half was preserved with RNA Tissue Protection Reagent (No. 76106, Qiagen, Germany) at −80°C for later analysis. The hearts (ventricle and atria) were also weighed and divided into two parts: the apex of the heart was fixed in 4% paraformaldehyde for further histological analysis, and the rest was preserved with RNA Tissue Protection Reagent (No. 76106, Qiagen, Germany) at −80°C for later analysis.

2.2. Serum and urine analyses

Serum creatinine, urinary creatinine and serum troponin T concentrations were measured using an automated biochemical analyser (Siemens biochemical analyser and its Leadman reagent, Siemens, Germany). Urinary albumin (ab235642, Abcam, Cambridge, United Kingdom), serum Cystatin C (ab201281, Abcam, Cambridge, United Kingdom) and serum B‐type natriuretic peptide (BNP) (ab108816, Abcam, Cambridge, United Kingdom) levels were determined quantitatively using Enzyme‐Linked Immunosorbent Assay kit. Urinary albumin excretion rate (AER) and creatinine clearance rate (Ccr) were calculated as follows: AER (mg/24 h) = urinary albumin (mg/ml) * 24 h urine volume (ml); Ccr (ml/min) = [urinary creatinine (μmol/L) * urinary flow (ml/min)]/serum creatinine (μmol/L).

2.3. Echocardiography

One day before euthanasia, the rats were subjected to echocardiography by a diagnostic echocardiographer to collect data on the following parameters: interventricular septum in systole (IVSs), interventricular septum in diastole (IVSd), left ventricular end‐systolic dimension (LVESd), left ventricular end‐diastolic dimension (LVEDd), left ventricular posterior wall in systole (LVPWs) and left ventricular posterior wall in diastole (LVPWd). Further parameters were calculated using the following equations: Change in left ventricular posterior wall thickness (LVPW, %) = (LVPWs‐LVPWd)/LVPWd*100%; Change in interventricular septum thickness (IVS, %) = (IVSs‐IVSd)/IVSd*100%; Fractional shortening (FS, %) = (LVEDd‐LVESd)/LVEDd*100%; Stroke volume (SV, mm³) = 1.04*LVEDd^3–1.04*LVESd^3; Left ventricular ejection fraction (EF, %) = (1.04*LVEDd^3–1.04*LVESd^3)/(1.04*LVEDd^3)*100%.

2.4. Histology

After the hearts and kidneys were soaked in 4% paraformaldehyde for 48 hours, they were embedded in paraffin, then cut into 2 μm slices and stained with one of the following: Periodic Acid–Schiff staining (PAS), Sirius Red staining, Periodic Schiff‐Methenamine staining (PASM), Haematoxylin–Eosin staining (H&E) and Masson's Trichrome staining (Masson). All stained slices were examined under 400x magnification. The glomerulosclerosis index was assessed by two investigators using a subjective, semi‐quantitative scoring system (grades I‐IV) to evaluate the percentage of periodic acid Schiff‐positive areas within the glomerulus of PAS‐stained slices. On each PASM‐stained slice, 50 glomeruli were analysed using iViewer 6.3.6 (UNIC TECHNOLOGIES, INC., China) to assess the glomerular size. The percentage of cardiac/renal interstitial fibrosis was analysed using Sirius Red‐stained full‐scan images and using the ImageJ threshold method (National Institutes of Health, USA), as previously described. ¹⁹ Cardiomyocyte diameters were assessed by measuring 50 cardiomyocyte diameters on each H&E‐stained section and averaging them for further analysis. All analyses covered more than 80% of the area of each slice.

2.5. Immunofluorescence staining

We performed immunofluorescence analysis using the following specific antibodies: CD68 (primary antibody: GB113109, Servicebio, China, dilution 1:2000; secondary antibody: GB22303, Servicebio, China, dilution 1:500), CD8 (primary antibody: GB11068, Servicebio, China, dilution 1:500; secondary antibody: GB21303, Servicebio, China, dilution 1:300) and CD4 (primary antibody: GB11064–1, Servicebio, China, dilution 1:500; secondary antibody: GB21303, Servicebio, China, dilution 1:300). Frozen sections (4 μm) were placed in the primary antibody at 4°C overnight. Then, the tissues and cells were washed and incubated with a secondary antibody. After staining the nuclei with 4′,6‐Diamidino‐2‐phenylindole (DAPI), the tissues and cells were visualized. For each section, 12 randomized high‐power fields were examined using a fluorescence microscope at 200x magnification. Cytotoxic T cells (CD8 + cells/total cells) and helper T cells (CD4 + cells/total cells) were analysed using Aipathwell (Servicebio, China). The Relative fluorescence unit (RFU) of CD68 was assessed by ImageJ software (National Institutes of Health, USA).

2.6. RNA sequencing

Six kidney samples around the median serum creatinine concentration for each group were selected for RNA sequencing analysis using the HiSeq 4000 platform device (Illumina, Inc.; San Diego, California, USA). Differential expression was calculated applying the linear models approach incorporated in the limma software package using Tibco Spotfire version 6.5.2 (TIBCO Software, Palo Alto, USA). After adjusting for FDR (false discovery rate) with a threshold of p < 0.05 and log2 (Fold Change) > 1, fewer differentially expressed genes (DEGs) were identified. To more comprehensively assess transcriptome changes, we opted to use the more sensitive raw p‐value as the screening criterion. Therefore, genes with an absolute value of log2 (Fold Change) > 1 and p < 0.05 were considered as DEGs, using the 5/6Nx + PBO group as a control. Library preparation, sequencing and data analysis of messenger RNA (mRNA) were performed as previously described. ²⁰

2.7. Statistical analysis

Data are presented as means ± SEM. Statistical analysis was performed using GraphPad Prism 6 (GraphPad, La Jolla, California, USA). The identification and exclusion of outliers was performed using ROUT (Q = 1%) in the GraphPad Prism 6 software, which ultimately excluded only 4 outliers (2 from final serum troponin T; 1 from final serum BNP; 1 from Stroke volume). For comparisons between groups, normally distributed data were analysed using one‐way ANOVA with Dunnett's multiple comparisons test and non‐normally distributed data were analysed using Kruskal‐Wallis test with Dunn's multiple comparisons test. In all cases, differences are considered statistically significant when p < 0.05, with the significance level denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

3. RESULTS

3.1. 1 mg PDE9 inhibitor and/or 1 mg sGC stimulator did not improve renal function in 5/6 nephrectomized rats

Due to disease and operations resulting in the loss of some animals, the final number of animals in each group was as follows: sham + placebo (7 animals); 5/6Nx + placebo (9 animals); 5/6Nx + sGC stimulator (9 animals); 5/6Nx + PDE9 inhibitor (8 animals); 5/6Nx + sGC stimulator + PDE9 inhibitor (8 animals). After excluding deaths unrelated to the disease, the mortality rate for each group was: Sham + Placebo (12.50%), 5/6Nx + Placebo (40.00%), 5/6Nx + sGC stimulator (30.77%); 5/6Nx + PDE9 inhibitor (42.86%); 5/6Nx + sGC stimulator + PDE9 inhibitor (42.86%). Mortality analyses showed no significant differences between groups. The 5/6Nx rats exhibited a marked increase in serum cystatin C, serum creatinine and proteinuria, along with a significant reduction in creatinine clearance (Ccr) and body weight. Neither the 1 mg PDE9 inhibitor, the 1 mg sGC stimulator, nor their combination improved these impairments in renal function (Table 1, Figure 1). BP did not differ between groups (Table 1). The weights of the heart and kidneys of the 5/6 nephrectomized rats were not statistically different from those of the Sham + PBO group. In addition, serum troponin T and serum BNP, indicators of myocardial injury, were not elevated in 5/6Nx rats. Furthermore, echocardiographic data showed that 5/6 nephrectomy resulted in a slight elevation of FS, SV and EF, which 1 mg PDE9 inhibitor and/or 1 mg sGC stimulator treatment did not affect (Table S1).

TABLE 1.

Basic animal characteristics.

Parameters	Sham + PBO	5/6Nx + PBO	5/6Nx + 1 mg sGCs	5/6Nx + 1 mg PDE9i	5/6Nx + 1 mg sGCs + 1 mg PDE9i
Final data
Final Body Weight (g)	509.00 ± 18.09	464.11 ± 17.58	520.11 ± 19.05	477.38 ± 12.18	491.00 ± 21.80
Rel. Heart weight (10 −2 )	0.37 ± 0.01	0.39 ± 0.03	0.38 ± 0.02	0.44 ± 0.07	0.41 ± 0.03
Rel. Kidney weight (10 −2 )	0.41 ± 0.02	0.51 ± 0.06	0.58 ± 0.04	0.67 ± 0.07	0.52 ± 0.05
Final SBP (mmHg)	164.17 ± 7.06	163.00 ± 7.84	137.00 ± 18.59	203.00 ± 9.07	166.67 ± 24.10
Final DBP (mmHg)	86.67 ± 14.61	76.38 ± 13.89	46.00 ± 1.00	98.00 ± 4.58	46.67 ± 6.89
Final Serum creatinine (mg/dL)	0.27 ± 0.02 **	0.81 ± 0.10	0.79 ± 0.04	0.80 ± 0.09	0.88 ± 0.17
Final Serum Cystatin C (μg/L)	774.82 ± 79.02 ****	2519.30 ± 205.58	2112.37 ± 124.22	2349.03 ± 356.30	2439.01 ± 283.05
Final Ccr (ml/min)	2.82 ± 0.24 ****	1.14 ± 0.14	1.25 ± 0.09	1.10 ± 0.17	1.62 ± 0.40
Final AER (mg/24 h)	0.54 ± 0.21 *	66.40 ± 29.43	50.86 ± 8.96	79.23 ± 21.99	101.32 ± 27.61
Final Serum Troponin T (ng/L)	0.01 ± 0.003	0.04 ± 0.01	0.04 ± 0.01	0.07 ± 0.02	0.06 ± 0.02
Final Serum BNP (ng/mL)	0.20 ± 0.03	0.25 ± 0.05	0.29 ± 0.03	0.26 ± 0.04	0.38 ± 0.04
Change from baseline
Body Weight (g)	208.57 ± 20.75 *	149.11 ± 11.60	191.44 ± 16.66	119.13 ± 11.02	109.13 ± 17.29
SBP (mmHg)	26.50 ± 14.53	21.50 ± 6.92	9.25 ± 14.33	66.33 ± 2.19	33.00 ± 32.23
DBP (mmHg)	22.00 ± 19.86	−11.88 ± 9.27	−16.75 ± 21.76	40.00 ± 18.18	−35.67 ± 12.03
Serum creatinine (mg/dL)	0.06 ± 0.01 ***	0.61 ± 0.10	0.49 ± 0.04	0.58 ± 0.07	0.61 ± 0.15
Ccr (ml/min)	0.86 ± 0.28 **	−0.99 ± 0.27	−0.10 ± 0.22	−1.44 ± 0.61	0.13 ± 0.39

Note: Rel. heart weight (Relative heart weight) = heart weight / final body weight; Rel. kidney weight (Relative kidney weight) = kidney weight/final body weight; Ccr (Creatinine clearance rate) = [urinary creatinine (μmol/L) ^* urinary flow (ml/min)/serum creatinine (μmol/L); AER (Urinary albumin excretion rate) = Urinary albumin (mg/ml) ^* 24 h urine volume (ml); SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BNP: Serum B‐type natriuretic peptide; Sham: Sham operation; 5/6Nx: 5/6 nephrectomy model; PBO: Placebo; sGCs: soluble guanylate cyclase stimulator; PDE9i: Phosphodiesterase 9 inhibitor. Values are displayed as mean ± SEM.

^*p < 0.05.

^**p < 0.01.

^***p < 0.001.

^****p < 0.0001, significantly different from 5/6Nx + PBO.

FIGURE 1.

Effect on renal function compared to 5/6Nx + PBO group. (A) Final serum cystatin C (μg/L); (B) final Ccr (ml/min); (C) final AER (mg/24 h). AER: urinary albumin excretion rate; Ccr: creatinine clearance; PBO: placebo; sGCs: soluble guanylate cyclase stimulator; PDE9i: phosphodiesterase 9 inhibitor. Values are displayed as mean ± SEM. *p < 0.05; ****p < 0.0001, significantly different from 5/6Nx + PBO.

3.2. A combination of 1 mg PDE9 inhibitor and 1 mg sGC stimulator attenuated renal interstitial fibrosis in 5/6 nephrectomized rats

Histological analysis of the kidney showed that 5/6 nephrectomy severely damaged the renal morphology, causing renal interstitial fibrosis, glomerulosclerosis and increased glomerular size (Figure 2). Neither treatment with the PDE9 inhibitor nor sGC stimulator alone improved these indices, but the combination of the PDE9 inhibitor and sGC stimulator improved renal interstitial fibrosis (Figure 2B). Neither PDE9 inhibitor nor sGC stimulator alone or in combination had any effect on cardiomyocyte diameter or myocardial fibrosis (Figure S1).

FIGURE 2.

Effect on renal histology compared to the 5/6Nx + PBO group. (A) Representative micrographs of PASM and Masson staining of kidney sections from each group (X400, scale bar = 50 μm); label 1: glomerulus; label 2: renal tubules; arrow: renal interstitial fibrosis. (B) Renal interstitial fibrosis (%); (C) glomerulosclerosis score; (D) glomerular size (μm2). Sham: sham operation; 5/6Nx: 5/6 nephrectomy model; PASM: periodic Schiff‐Methenamine staining; PBO: placebo; sGCs: soluble guanylate cyclase stimulator; PDE9i: phosphodiesterase 9 inhibitor. Values are displayed as mean ± SEM. **p < 0.01; ****p < 0.0001, significantly different from 5/6Nx + PBO.

3.3. 1 mg of PDE9 inhibitor and/or 1 mg of sGC stimulator did not affect renal macrophage, CD4 + T cell and CD8 + T cell infiltration in 5/6 nephrectomized rats

No increase nor decrease in renal macrophage (Figure 3A and B), CD4 + T‐cell (Figure 3A and C) and CD8 + T‐cell (Figure 3A and D) infiltration after 5/6 nephrectomy was observed in this study. Neither PDE9 inhibitors nor sGC stimulators, alone or in combination, had any effect on these cells in the kidney. 5/6Nx did not affect cardiac CD4 + T cells (Figure S2A and S2C) but resulted in a decrease in cardiac macrophages (Figure S2A and S2B) and CD8 + T cells (Figure S2A and S2D). However, neither PDE9 inhibitors nor sGC stimulators, alone or in combination, influenced macrophages and CD8 + T cells in the heart.

FIGURE 3.

Effects on renal macrophage, helper T cells and cytotoxic T cells infiltration. (A) Representative micrographs of kidney sections from each treatment group (X200, scale bar = 100 μm); (B) macrophages (CD68+, RFU); (C) helper T cells (CD4 + cells/total cells, %); (D) cytotoxic T cells (CD8 + cells/total cells, %). Comparison with the 5/6Nx + PBO group; values are displayed as mean ± SEM; sham: sham operation; 5/6Nx: 5/6 nephrectomy model; PBO: placebo; sGCs: soluble guanylate cyclase stimulator; PDE9i: phosphodiesterase 9 inhibitor. RFU: relative fluorescence unit.

3.4. A combination of 1 mg PDE9 inhibitor and 1 mg sGC stimulator down‐regulated renal collagen‐related genes

Differentially expressed genes were screened for further analyses according to the guidelines described in “Methods” (absolute value of log2 [fold change] > 1 and p < 0.05). 5/6 nephrectomy up‐ or down‐regulated the expression of 507 genes relative to the normal group (Sham + PBO), whereas the combination group (1 mg PDE9 inhibitor and 1 mg sGC stimulator) up‐ or down‐regulated the expression of 299 genes relative to the 5/6Nx + PBO group (Figure 4). Forty‐four genes were regulated by both 5/6 nephrectomy and the drug combination, of which 23 genes were regulated by the combination (1 mg PDE9 inhibitor and 1 mg sGC stimulator) group alone, independently of the other treatment groups (Figure 4). Of these 23 genes, the combination group down‐regulated 7 collagen‐related genes, including COL1A1 (FC: −1.43, p = 0.007; Collagen Type I Alpha 1 Chain), COL3A1 (FC: −1.19, p = 0.007; Collagen Type III Alpha 1 Chain), COL14A1 (FC: −1.08, p = 0.009; Collagen Type XIV Alpha 1 Chain), LUM (FC: −1.51, p = 0.017; Lumican), ITGA11 (FC: −1.02, p = 0.008; Integrin Subunit Alpha 11), ADAMTS2 (FC: −1.14, p = 0.012; ADAM Metallopeptidase With Thrombospondin Type 1 Motif 2) and TGFB3 (FC: −1.13, p = 0.003; Transforming Growth Factor Beta 3).

FIGURE 4.

Differentially expressed genes in kidney tissue detected by RNA sequencing. (A) Venn diagram of the differentially expressed genes (p < 0.05 and an absolute value of log2[fold change] > 1) from RNA sequencing data. The numbers on the circles represent the quantity of differentially expressed genes. Independently of the 5/6Nx + 1 mg sGCs and 5/6Nx + 1 mg PDE9i groups, 23 differentially expressed genes co‐regulated in the 5/6Nx + 1 mg sGCs + 1 mg PDE9i group versus the 5/6Nx + PBO group were enumerated, including COL1A1 (FC: −1.43, p = 0.007), COL3A1 (FC: −1.19, p = 0.007), COL14A1 (FC: −1.08, p = 0.009), LUM (FC: −1.51, p = 0.017), ITGA11 (FC: −1.02, p = 0.008), ADAMTS2 (FC: −1.14, p = 0.012), TGFB3 (FC: −1.13, p = 0.003). (B, C, D, E) Volcano plots of RNA sequencing data displaying the pattern of gene expression values for every treatment group relative to the 5/6Nx + PBO group. The genes are coloured if they pass the thresholds for p < 0.05 and log2 (fold change) = ±1, orange if they are upregulated and blue if they are downregulated. COL1A1: collagen type I alpha 1 chain; COL3A1: collagen type III alpha 1 chain); COL14A1: collagen type XIV alpha 1 chain; LUM: Lumican; ITGA11: integrin subunit alpha 11; ADAMTS2: ADAM metallopeptidase with thrombospondin type 1 motif 2; TGFB3: transforming growth factor Beta 3. Sham: sham operation; 5/6Nx: 5/6 nephrectomy model; PBO: placebo; sGCs: soluble guanylate cyclase stimulator; PDE9i: phosphodiesterase 9 inhibitor.

4. DISCUSSION

5/6 nephrectomy severely impaired renal function and histology, including elevated serum cystatin C, proteinuria, glomerulosclerosis and interstitial fibrosis. Neither the low‐dose sGC stimulator nor the low‐dose PDE9 inhibitor treatment alone improved this renal injury functionally or histologically, whereas the combination of the low‐dose sGC stimulator and low‐dose PDE9 inhibitor attenuated renal interstitial fibrosis in 5/6 nephrectomized rats. The nephroprotective effect of a low‐dose sGC stimulator combined with a low‐dose PDE9 inhibitor was independent of BP and the infiltration of macrophages, CD4 + T cells and CD8 + T cells in the kidneys. Down‐regulation of fibrosis‐related gene expression (COL1A1, COL3A1, COL14A1, LUM, ITGA11, ADAMTS2, TGFB3) appears to be a key factor in the mechanisms of the combination of low‐dose sGC stimulator and PDE9 inhibitor in attenuating renal interstitial fibrosis.

Our results align with previous research highlighting the benefits of targeting the NO‐sGC‐cGMP pathway in renal diseases. ³ , ¹² , ¹⁸ Reviewing studies on the renoprotective effect of the sGC stimulator, most of them used a unilateral ureteral obstruction (UUO) model and rats were treated orally with BAY 41–8543 at a therapeutic dose of 10 mg/kg/d. In a rat model of UUO BAY 41–8543 (10 mg/kg/d, orally) reduced renal interstitial fibrosis and collagen IV mRNA expression. ¹⁸ , ²¹ , ²² In addition, BAY 41–8543 (4 mg/kg/d, intraperitoneal) attenuated renal fibrosis in a UUO mouse model. ²³ However, unlike these studies which often used higher doses of sGC stimulators/activators, our approach utilized lower doses, potentially reducing systemic side effects such as vasodilation and hypotension. ¹⁰ This low‐dose combination approach appears to be a novel and effective strategy, as evidenced by the significant reduction in renal fibrosis observed in our study. This supports the hypothesis that dual interventions are more effective in enhancing the effects of the NO‐sGC‐cGMP pathway. ²⁴ , ²⁵ A noteworthy phenomenon in our study was that renal function parameters such as serum creatinine, cystatin C levels and proteinuria were not immediately improved, but significant effects were observed at the level of gene transcription, especially in the expression of fibrosis‐related genes. RNA sequencing data revealed that the combination therapy downregulated several key fibrosis‐related genes, including COL1A1 (FC: −1.43, p = 0.007), COL3A1 (FC: −1.19, p = 0.007) and COL14A1 (FC: −1.08, p = 0.009). These genes are crucial components of the extracellular matrix, and their reduced expression likely contributes to the decreased fibrosis observed histologically. Activation of renal fibroblasts and increase in myofibroblasts promote extracellular matrix (ECM) formation and deposition. This ECM consists mainly of collagen (I, III, IV and V), fibronectin and laminin. ²⁶ Fibrosis can severely damage kidney morphology; therefore, the degree of interstitial fibrosis determines the prognosis of the patient's renal function. ²⁷ , ²⁸ Limiting the spread and duration of inflammation and consequent ECM deposition during disease progression protects as much residual renal tissue as possible. In addition to the downregulation of collagen genes, other significant changes included the reduced expression of LUM, ITGA11, ADAMTS2 and TGFB3. These genes are involved in the structural remodelling of the extracellular matrix and fibrotic processes. The broad‐spectrum downregulation of these genes suggests a comprehensive antifibrotic effect of the combination therapy at the molecular level, which might precede observable functional improvements.

In our present study, we administered drug therapy for 13 weeks immediately after 5/6 nephrectomy in rats. Pharmacological treatment was administered throughout the two main pathological stages of CKD, namely the early stage with an inflammatory response, and the late stage, in which fibrosis is predominant. The sGC stimulator and/or PDE9 inhibitor had no effect on the infiltration of predominantly inflammatory renal cells (macrophages, CD4 + T‐cells and CD8 + T‐cells), and the inflammatory cells of 5/6 nephrectomized rats did not differ from those of normal rats. Thirteen weeks after 5/6 nephrectomy, the inflammatory response is no longer the predominant pathology; therefore we did not observe the effect of the sGC stimulator and/or PDE9 inhibitor on inflammatory cells (macrophages, CD4 + T cells and CD8 + T cells) of renal tissue 13 weeks after 5/6 nephrectomy. This also fits with many of the current studies in this area. SGLT2 inhibitors have been shown to affect macrophage infiltration in CKD kidneys in several studies, but this conclusion is not confirmed in some studies beyond 8 weeks. ²⁹ , ³⁰ Renal interstitial fibrosis is usually associated with an inflammatory infiltrate consisting mainly of lymphocytes and macrophages, but the degree of inflammation does not fully correlate with fibrosis, especially in the later stages of CKD. ³¹

The clinical relevance of these findings is significant. CKD is a progressive disease with limited treatment options, primarily focused on managing symptoms rather than addressing the underlying fibrosis. The ability of the sGC stimulator and PDE9 inhibitor combination to attenuate fibrosis suggests a potential therapeutic strategy that could slow or even halt disease progression. Importantly, our findings indicate that this effect is achieved without significant changes in blood pressure or renal immune cell infiltration, highlighting the specificity and safety of the therapy. Given the delayed nature of the observed effects, it is crucial to recognize that gene transcription changes might serve as early indicators of therapeutic efficacy. This highlights the need for prolonged observation periods in clinical trials and the inclusion of molecular markers as endpoints to capture the early benefits of such therapies.

5. LIMITATIONS

Despite the promising results, our study has several limitations. The use of a single low‐dose combination limits the ability to fully explore the dose–response relationship and the potential for higher efficacy at different doses. Future studies should include a range of doses to determine the optimal therapeutic window. Additionally, while the 5/6 nephrectomy model is a well‐established model for studying CKD, it does not capture the full spectrum of CKD aetiologies seen in humans. Further research using other models of CKD and eventually clinical trials in human patients will be necessary to validate these findings and assess their applicability to human disease. Future research should focus on several key areas. Firstly, exploring different doses and combinations of sGC stimulators and PDE9 inhibitors could help optimize the therapeutic regimen. Secondly, studying the effects of this combination therapy in other CKD models, such as diabetic nephropathy or hypertensive nephropathy, would provide a broader understanding of its efficacy. Thirdly, investigating the long‐term safety and efficacy of this therapy in clinical trials will be crucial for translating these findings into clinical practice. Additionally, research into the specific molecular pathways affected by this combination therapy could yield further insights into its mechanisms of action and potential off‐target effects. Due to the limitations of the experimental conditions, the study did not validate the RNA sequencing results at the protein level, which would have been beneficial for further supporting the conclusions. Measuring blood and renal cGMP levels would aid in evaluating the effects of sGC stimulators and PDE9 inhibitors, thereby strengthening the conclusions.

6. CONCLUSIONS

In summary, we investigated for the first time the potential renoprotective effects of a low‐dose non‐NO‐dependent sGC stimulator (BAY 41‐8543) in combination with a low‐dose PDE9 inhibitor (BAY 73–6691) in a 5/6 nephrectomy model. This combination therapy significantly attenuates renal interstitial fibrosis by downregulating key fibrosis‐related genes (COL1A1, COL3A1, COL14A1, LUM, ITGA11, ADAMTS2, TGFB3). These findings highlight a new therapeutic strategy for CKD, emphasizing the importance of gene transcription changes as early indicators of therapeutic efficacy. The novel approach of using a low‐dose combination to mitigate side effects while achieving significant therapeutic effects represents a promising advancement in CKD treatment. Further research in preclinical and clinical settings is warranted to optimize this therapeutic strategy and explore its potential applications in other fibrotic diseases. By addressing the root cause of fibrosis, this approach has the potential to improve outcomes for CKD patients and offers a new direction for future research in renal and fibrotic diseases.

AUTHOR CONTRIBUTIONS

B.H. conceptualized and designed the study. X.C., Y.C., Z.Z. and H.W. performed the animal experiments. X.C., Y.C., D.D., M.MS.G. and T.K. performed the biological and histological analyses and statistical evaluation. X.C. analysed the final data set and wrote the initial manuscript. B.H. and B.K.K. supervised and with Y.L. and S.E. significantly amended the manuscript. All authors contributed to revising and editing the manuscript and approved the final version of the manuscript.

CONFLICT OF INTEREST STATEMENT

B.K.K. reports lecture fees and/or advisory board memberships and/or study participation from Astellas, Bayer, Boehringer Ingelheim, Chiesi, Riepharm, Pfizer, Sanofi, Servier and Vifor Pharma, all not related to the submitted work. D.D. and T.K. are research employees of Boehringer Ingelheim.

Supporting information

Table S1. Cardiac echocardiographic data.

Figure S1. Effects on cardiac histology compared to the 5/6Nx + PBO group. (A) Representative micrographs of PASM and Masson staining of heart sections from each group (X400, Scale bar = 50 μm); Blue arrow on PASM: Cardiomyocytes; Blue arrow on Masson: Myocardial fibrosis. (B) Cardiomyocyte diameter (μm); (C) Myocardial fibrosis (%). Sham: Sham operation; 5/6Nx: 5/6 nephrectomy model; PBO: Placebo; sGCs: Soluble guanylate cyclase stimulator; PDE9i: Phosphodiesterase 9 inhibitor. PASM: Periodic Schiff‐Methenamine staining. Values are displayed as mean ± SEM. ***p < 0.001, significantly different from the 5/6Nx + PBO group.

Figure S2. Effects on cardiac macrophage, helper T cell and cytotoxic T cell infiltration. (A) Representative micrographs of heart sections from each treatment group (X200, Scale bar = 100 μm); (B) Macrophages (CD68+, RFU); (C) Helper T cells (CD4 + cells/total cells, %); (D) Cytotoxic T cells (CD8 + cells/total cells, %). Values are displayed as mean ± SEM. **p < 0.01, significantly different from the 5/6Nx + PBO group. Sham: Sham operation; 5/6Nx: 5/6 nephrectomy model; PBO: Placebo; sGCs: Soluble guanylate cyclase stimulator; PDE9i: Phosphodiesterase 9 inhibitor. RFU: Relative fluorescence unit.

Chen X, Delić D, Liu Y, et al. sGC stimulator (BAY 41‐8543) combined with PDE9 inhibitor (BAY 73‐6691) reduces renal fibrosis in 5/6 nephrectomized rats. Basic Clin Pharmacol Toxicol. 2025;136(1):e14103. doi: 10.1111/bcpt.14103

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in figshare at https://doi.org/10.6084/m9.figshare.27178776.v1.