The clinical efficacy of cGMP-specific sildenafil on mitochondrial biogenesis induction and renal damage in cats with acute on chronic kidney disease

Abstract

Background

Mitochondrial biogenesis (MB) induction has recently emerged as potential therapeutic approaches in kidney pathology and the mitochondria-targeted therapies should be investigated to improve treatment of animals with kidney diseases. This study aimed to investigate the effects of MB induction with sildenafil citrate on the cGMP/NO pathway, glomerular filtration, and reduction of kidney damage and fibrosis (TGF-β/SMAD pathway) in cats with acute on chronic kidney disease (ACKD). Thirty-three cats were divided into the non-azotemic (healthy) group (n:8) and the ACKD group (n:25), comprising different breeds, sexes, and ages. Sildenafil citrate was administered to the non-azotemic and ACKD groups (2.5 mg/kg, PO, q12 hours) for 30 days. Serum and urine NO, MDA, NGAL, KIM-1, TGF-β1, IL-18, FGF 23, PGC-1α and cGMP concentrations were measured.

Results

Serum cGMP concentrations increased (P < 0.05) in the non-azotemic group during the 2^nd (median 475.99 pmol/mL) and 3^rd (median 405.01 pmol/mL) weeks of the study, whereas serum cGMP concentrations decreased in the ACKD group during the 4^th(median 188.52 pmol/mL) week compared to the non-azotemic group (P < 0.05). No difference was observed in serum biomarker concentrations except NO, which increased in the 4^th week (P < 0.05). The urinary concentrations of NO, MDA, PGC-1α, TGF-β1, NGAL, KIM-1, IL-18, and FGF 23 in the ACKD group were found to be higher compared to those in the non-azotemic group from the 1^st to the 4^th week (P  < 0.05). In the ACKD group, the urine PGC-1α concentration in the 2^nd (median 6.10 ng/mL) week was lower compared to that in the 0 and 1^st (median 7.65 and 7.21 ng/mL, respectively) week, and the NO concentration in the 3^rd (median 28.94 µmol/mL) week was lower than that in the 0^th (median 37.43 µmol/mL) week (P < 0.05).

Conclusions

While sildenafil citrate has been determined to induce a low level of MB and to have a beneficial effect on glomerular filtration, it is observed to be ineffective in mitigating renal damage and fibrosis via the TGF-β/SMAD pathway in cats with ACKD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-024-04345-9.

Background

Permanent mitochondrial dysfunction leads to early renal fibrosis, molecular markers of fibrosis such as transforming growth factor beta 1 (TGF-β1) and alpha smooth muscle actin (α-SMA) increase, and urine osmolality decreases in the experimental acute kidney injury (AKI) model [1]. It is highlighted that mitochondrial dysfunction mediates AKI, and restoring mitochondrial function after kidney injury may significantly aid in the recovery from renal injury. Mitochondrial function restoration is called mitochondrial biogenesis (MB) and MB induction has a protective effect against damage in AKI and chronic kidney disease (CKD) cases [2–4]. AKI is defined by sudden damage to the renal parenchyma and may be accompanied by an abrupt decrease in glomerular filtration rate (GFR). CKD is defined by derangements in kidney structure or function lasting a long period of time. AKI and CKD are closely related, as AKI contributes significantly to the progression of CKD, while CKD increases the risk of AKI [5]. Animals with stable CKD may experience an acute decline in renal function, termed acute on CKD (ACKD) [6]. The pathogenesis, clinical signs, and laboratory findings of ACKD are similar to those of AKI, which often makes differentiation difficult. Although ACKD is believed to be common in cats, its etiology, progression, and short- and long-term prognosis remain largely uncharacterized, in contrast to the well-defined profiles of feline CKD and AKI. This similarity and the presence of azotemia can make it difficult to differentiate ACKD from AKI in some cases. However, weight loss is more indicative of CKD and, when present, may serve as a useful marker in distinguishing ACKD from AKI [7].

In veterinary clinical practice, traditional parameters such as serum urea and creatinine, used to evaluate kidney function, can be affected by various factors and do not reflect real-time changes in glomerular filtration rate (GFR) or indicate the presence of AKI [8]. The use of more sensitive serum and urinary biomarkers (e.g. symmetric dimethylated arginine, SDMA; neutrophil gelatinase-associated lipocalin, NGAL; kidney injury molecule-1, KIM-1; interleukin-18, IL-18; serum fibroblast growth factor 23, FGF 23) has been recommended for accurate and early diagnosis treatment follow-up and prognosis evaluation of kidney damage/diseases, recently [9–12]. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) is the main regulator of MB in high energy-dependent organs [13] and PGC-1α expression is found in kidney proximal tubule cells, and MB is also essential for tubular function [2, 4, 14]. Also, it was demonstrated that cGMP is important for the regulation of MB in renal tubules, that sildenafil citrate increases the mass of functional mitochondria in the renal cortex by increasing ATP levels in the tissue and stimulates MB in renal tubule cells in vivo and in vitro [4].

Sildenafil citrate (cGMP-specific Phosphodiesterase 5 (PDE-5) inhibitor) induces MB in primary cultures of renal proximal tubule cells, also in folic acid-induced AKI, specific PDE-5 inhibitors increase cyclic guanosine monophosphate (cGMP) and are an inducer of MB both in vivo and in vitro. This potential effectiveness of PDE-5 inhibitors and utilization is recommended in AKI and diseases characterized by mitochondrial dysfunction and MB suppression [4]. Mitochondrial dysfunction has recently been linked to the pathophysiology of various kidney diseases. Alterations in mitochondrial biogenesis result in apoptosis, mitophagy, and defects in energy metabolism. Removal of damaged mitochondria is essential to prevent renal damage caused by oxidative stress. Mitophagy, the selective autophagy of mitochondria, facilitates the recycling of damaged mitochondrial components and plays a critical role in the regulation of oxidative stress. Thus, the relationship between mitochondrial dysfunction and renal fibrogenesis arises from the inability of mitochondria to meet the high metabolic demands of the kidney, resulting in increased production of reactive oxygen species and subsequent oxidative stress [5, 10, 13]. These are key pathophysiological mechanisms underlying the role of mitochondrial dysfunction in kidney diseases [5]. Therefore, mitochondrial dysfunction is significant in the pathogenesis of various kidney diseases, and mitochondria potentially serve as therapeutic targets, necessitating further investigation [15]. This study aimed to investigate the effects of MB induction with sildenafil citrate on the cGMP/NO pathway and glomerular filtration, as well as to assess the efficacy of sildenafil citrate in reducing or preventing kidney damage and fibrosis through the TGF-β/SMAD pathway in cats with ACKD.

Methods

This study was approved by Selçuk University Faculty of Veterinary Medicine Local Ethics Committee (14/04/2020). Also, informed consent was obtained from all of the owners.

Animals

A total of 33 cats of various breeds, sexes, and ages were included in the study. These cats were admitted to Selcuk University Faculty of Veterinary Medicine and İstanbul University - Cerrahpasa Faculty of Veterinary Medicine animal hospital.

Cats with ACKD

Twenty-five owned cats of different breeds, ages, and sexes diagnosed with ACKD and exhibiting clinical signs such as lethargy, vomiting, anorexia, oliguria, and the presence of weight loss. ACKD cats were selected based on a continuous decline in kidney function, characterized by the presence of proteinuria and hyperphosphatemia, alongside an acute insult marked by sudden renal parenchymal damage and reduced renal function. International Renal Interest Society (IRIS) criteria were employed to assess and monitor the extent of renal function reduction. Accordingly to IRIS criteria cats with SDMA concentration > 18 µg/dL and serum creatinine concentration > 1.6 mg/dL, UPC > 0.4 and SpG < 1.035 were included in the ACKD group. Additionally, ultrasonographic evaluation revealed differences in renal size, heightened cortical echogenicity, slight perirenal fluid accumulation, medullary rim sign, and pyelectasia. Furthermore, measurements of serum SDMA and creatinine concentrations, urine specific gravity (SpG), and urine protein-to-creatinine ratio (UPC) were conducted [11, 16–18]. Thus, cats with CKD exhibiting clinical and ultrasonographic findings consistent with ACKD due to an acute insult, and whose renal impairment was evaluated using IRIS criteria, were classified as azotemic [5, 7, 10, 19].

Cats were excluded from the study if they exhibited conditions such as increased urine specific gravity (SpG), elevated serum total protein levels, and elevated packed cell volume/hematocrit levels indicating pre-renal azotemia due to factors like fluid loss, bleeding, hypovolemia, or dehydration unrelated to kidney disease. Also, cats with diseases other than or concurrent with renal disease causing renal azotemia, as well as cats with SpG > 1.035 and hyperkalemia indicating post-renal azotemia due to lower urinary tract disease or symptoms of urinary tract obstruction, were excluded. Moreover, cats with uncontrolled hypertension and requiring urgent hypertension intervention were also not included in the study. As a result, although inflammatory, ischemic, pyelonephritis, or other causes are listed in the etiology of ACKD, 25 cats were included in the ACKD group of the present study without etiological classification, based on the aforementioned IRIS criteria, laboratory results, and ultrasonographic findings [6, 7, 20].

Healthy or non-azotemic cats

Eight healthy cats of different breeds, ages, sexes, and owned by volunteer hospital staffs were included in the study. The cats had no history of disease and no abnormal laboratory or ultrasound findings. According to the IRIS criteria, cats with SDMA concentration < 18 µg/dL and serum creatinine concentration < 1.6 mg/dL, UPC < 0.4, and SpG 1.035–1.060 were considered non-azotemic.

Ultrasonographic examinations

B-mode ultrasonographic examination (Esaote MyLab X7, Italy) of the urinary system in all the cats was performed in the supine position, after clipping the hair of the abdominal area and applying acoustic ultrasound gel, using a 2.5–7.5 MHz microconvex probe. Ultrasonographic examination included the evaluation of the internal structure of the kidneys, parenchymal echogenicity changes, the presence of mass, fluid, cysts and mineralization, morphology of the ureters, urinary bladder and urethra, left/right kidney dimensions and corticomedullary differentiation [21, 22].

Laboratory examinations

Venous blood (using tubes with and without anticoagulant) and urine samples were obtained with an appropriate method causing minimal patient stress from all the cats before treatment (0) and during the treatment period, weekly, for 30 days. To obtain serum samples for serum biochemistry profiling and biomarker analysis, blood samples collected in tubes without anticoagulant were allowed to clot at room temperature for 15 min and then centrifuged at 2000 × g for 10 min. Serum biochemistry profiling was performed within 45 min after sampling. Serum biochemistry profiling included blood-urea nitrogen (BUN), creatinine (Cr), phosphorus (P), SDMA concentrations measurements using an autoanalyzer (IDEXX Catalyst ONE). The remaining portions were transferred to eppendorf tubes and stored at -20 °C until the day of biomarker analyses (approx. 6 months). Urine samples were collected aseptically from all the cats by ultrasound-guided cystocentesis (3 mL). Urinalysis included urine dipstick (SpG, pH, leukocyte, erythrocyte, protein, glucose, nitrite, urobilinogen, bilirubin) analysis, urine total protein and creatinine concentration measurements (uTP; pyrogallol red-molybdate method, uCr; jaffe method) (within 10 min after collection using URIT-31 autoanalyzer) [23, 24]. In addition to urine dipstick analysis, SpG and protein levels were evaluated using a refractometer. Furthermore, the presence of leukocytes, casts, and erythrocytes was investigated through microscopic examination of the urine sediment (after centrifugation at 1500 g for 10 min), using a light microscope (Olympus, Japan) at 40x magnification. The remaining portions were transferred to eppendorf tubes and stored at -20 °C until the day of biomarker analyses (approx. 6 months).

Serum and urine biomarker measurements

Serum and urine Nitric oxide (NO), Malondialdehyde (MDA), NGAL, KIM-1, TGF-β1, IL-18, FGF 23 and PGC-1α concentrations were measured using feline-specific ELISA kits (Bioassay Technology Laboratory, Shangai, China), and cGMP concentrations were measured using non-specific universal ELISA kits (Bioassay Technology Laboratory, Shangai, China) according to the manufacturer’s instructions. Measurement of adsorbents was carried out on a microplate reader device (ELx800 Absorbance Microplate Reader, United States). The reported intra-assay and inter-assay coefficients of variation for NO, MDA, NGAL, KIM-1, TGF-β1, IL-18, FGF 23 and PGC-1α concentrations were < 8% and < 10%, respectively. For cGMP it were < 10% and < 12%. Minimum detectable concentrations were 0.96 umol/mL for NO, 0.29 nmol/mL for MDA, 0.24 ng/mL for NGAL, 0.043 ng/mL for KIM-1, 3.13 ng/L for TGF-β1, 12.39 ng/L for IL-18, 0.08 ng/mL for FGF 23, 0.21 ng/mL for PGC-1α and 4.26 pmol/mL for cGMP.

Treatment protocol

Within the scope of the treatment protocols of the present study, no medications such as angiotensin converting enzyme inhibitors, aldosterone antagonists, calcium channel blockers, or beta blockers were used for systemic hypertension, which is one of the important complications in cats with CKD, due to the effects of sildenafil on blood pressure. Sildenafil citrate was administered to the non-azotemic and ACKD groups at a dose of 2.5 mg/kg (orally, twice a day with 12 h intervals) for 30 days [25]. Alongside sildenafil citrate administration, renal supportive treatment was provided to the ACKD group. This treatment included resolving dehydration, correcting metabolic acidosis and electrolyte disorders (through isotonic/polyionic fluids and lactated Ringer’s solution administration), and addressing complications such as those related to anemia, metabolic bone disease, and uremia [26, 27]. Sildenafil citrate treatment started at the same time as the standard of care treatment. To avoid affecting biomarker concentrations, all cats were fed the same renal diet (Hill’s^® k/d) for nutritional standardization throughout the study, and dietary regulations (adjusting sodium, phosphorus, and protein intake) were recommended at the end of the study [26].

Statistical analyses

Statistical analyzes were performed using SPSS 25.0 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). The distribution of the data was checked with the Kolmogorov-Smirnov test. The data determined to be nonparametrically distributed were presented as median (min-max). Mann Whitney U test was utilized to evaluate the differences between the groups in the same sample weeks and the differences were indicated with an asterisk (*). Friedman test was utilized to evaluate the differences between time periods within the group. Wilcoxon test was performed for parameters with the test result P < 0.05. Receiver operator characteristic (ROC) analysis, with the area under the curve (AUC), was also performed in order to evaluate the clinical efficacies of serum and urine biomarker concentrations in monitoring the response to sildenafil citrate administration. The optimal cut-off points were those with the least number of misclassifications, and for these the sensitivity and specificity were determined. It was accepted that AUC of 0.5 suggests no discrimination (i.e., the ability to diagnose patients with and without the disease or condition based on the test), 0.6–0.8 was considered acceptable, 0.8–0.9 excellent and > 0.9 outstanding [28]. The statistical significance level for all the data was accepted as P < 0.05.

Results

Compared to the non-azotemic group, the ACKD group exhibited higher concentrations of BUN, Cr, and SDMA, as well as higher urine SpG and UPC ratio between weeks 1–4 (P < 0.05). Within the ACKD group, decreases (P < 0.05) were observed in serum P and UPC levels during sildenafil citrate administration, along with a decrease in serum BUN concentration during the 1^st and 3^rd weeks of the study. No difference was observed in serum P concentrations between ACKD and non-azotemic groups (P > 0.05). A decrease was observed in the serum P concentrations of the ACKD group from the 1^st week of the study (P < 0.05). The results for all IRIS criteria are presented in Supplementary File 1.

An statistically significant increase in serum median cGMP concentrations in the non-azotemic group was determined (P < 0.05) in the 2^nd and 3^rd weeks of the study, and a decrease in serum median cGMP concentration in the ACKD group was determined in the 4^thweek of the study compared to the non-azotemic group (P < 0.05). The serum median NO concentration in the ACKD group was statistically significant higher than that in the non-azotemic group during the 1^st week of treatment (P < 0.05). Additionally, a difference (P < 0.05) in serum NO concentration was observed in the ACKD group during the 4^th week. No differences were observed between groups in terms of serum median PGC-1α, MDA, NGAL, KIM-1, TGF-β1, IL-18 and FGF 23 concentrations. Comparison data of the serum biomarker concentration measurements were presented in Table 1.

Table 1.

Comparison data of serum biomarker concentrations

Groups	ACKD Group (n:25) (median (min-max)					Non-azotemic Group (n:8) (median (min-max)
Weeks	0	1	2	3	4	0	1	2	3	4
sNO (µmol/mL)	17.35 (12.19–42.07)b	18.67 (13.64–46.38)*b	17.95 (6.38–60.66)b	20.68 (11.73–53.01)b	18.61 (8.91–45.66)a	16.48 (14.07–20.08)	14.32 (11.87–17.01)	15.14 (7.64–23.13)	17.38 (15.65–23.60)	15.83 (13.07–19.46)
sMDA (µmol/mL)	1.92 (1.19–3.06)	2.00 (1.23–3.38)	2.15 (0.19–2.94)	2.07 (1.50–2.93)	1.98 (0.24–3.03)	2.04 (1.56–2.35)	1.76 (0.22–2.94)	2.00 (0.26–2.58)	1.74 (0.16–2.49)	2.05 (1.12–2.38)
sNGAL (ng/mL)	9.38 (6.37–12.83)	9.11 (2.15–18.47)	9.32 (4.72–19.83)	10.08 (4.25–15.45)	8.99 (4.36–13.92)	10.00 (7.61–11.43)	8.55 (4.70-11.95)	11.49 (7.69–13.95)	11.47 (6.43–16.08)	10.07 (9.14–10.44)
sKIM-1 (ng/mL)	4.24 (2.70–8.28)	3.81 (1.91–6.26)	3.81 (2.29–5.41)	3.75 (2.90–5.45)	3.81 (2.57–5.08)	3.77 (0.26–4.79)	3.81 (2.61–6.30)	4.11 (3.25–5.27)	4.38 (3.19–4.84)	3.38 (2.58–3.46)
sTGF-β1 (ng/L)	72.37 (46.75–99.70)	75.58 (9.08-123.97)	73.14 (28.43-114.57)	72.83 (24.99-104.72)	71.91 (24.31-450.99)	69.09 (30.15–90.73)	75.26 (62.37–93.10)	72.61 (56.08–86.03)	76.47 (60.69–98.49)	55.19 (50.95–74.44)
sIL-18 (ng/L)	240.84 (155.88-476.13)	238.85 (87.63-464.45)	252.55 (159.59-672.67)	269.43 (165.01-438.57)	262.62 (120.49-380.84)	260.50 (126.23-290.75)	222.54 (186.44–330.90)	241.51 (178.25-349.68)	303.51 (187.47-385.63)	262.16 (226.93-304.05)
sFGF 23 (ng/mL)	1.46 (1.10–2.06)	1.40 (0.42–2.09)*	1.36 (0.81–1.97)	1.43 (0.16–2.62)	1.56 (0.34–9.34)	1.37 (1.03–1.61)	1.70 (1.16–2.21)	1.45 (1.29–2.24)	1.71 (1.32-2.00)	1.26 (0.67–1.44)
sPGC-1α (ng/mL)	3.59 (1.78–5.38)	3.20 (0.81–5.52)	3.41 (0.55-6.00)	3.47 (2.02–5.09)	3.57 (0.34–13.48)	3.50 (1.84–4.70)	3.53 (1.94–5.80)	3.69 (2.43–4.85)	3.87 (1.26–7.02)	4.18 (3.90–5.38)
scGMP (pmol/mL)	232.46 (75.22–897.00)	298.23 (71.96–910.60)	370.23 (77.37-1606.69)	254.30 (74.96-1197.81)	188.52 (78.14-1412.64)*	221.68 (111.74-760.91)a	472.09 (86.21-1261.92)ab	475.99 (78.50-1473.67)b	405.01 (79.46-1671.93)b	869.25 (740.94-1101.18)ab

sNO (Serum nitric oxide), sMDA (Serum malondialdehyde), sNGAL (Serum neutrophil gelatinase-associated lipocalin), sKIM-1 (Serum kidney ınjury molecule-1), sTGF-β1 (Serum transforming growth factor beta-1), sIL-18 (Serum interleukin-18), sFGF 23 (Serum fibroblast growth factor 23), sPGC-1α (Serum peroxisome proliferator-activated receptor gamma coactivator 1-alpha), scGMP (Serum cyclic guanosine monophosphate). Differences between groups for the same sample weeks were indicated with *. Differences between weeks within the group were shown with a, b, c

Urinary median concentrations of NO, MDA, PGC-1α, TGF-β1, NGAL, KIM-1, IL-18 and FGF 23 were statistically significantly higher in the ACKD group compared to the non-azotemic group during weeks 1–4 (P < 0.05). In the ACKD group, the urine median PGC-1α concentration in the 2^nd week was statistically significantly lower compared to that in the 0-1^st week, and the NO median concentration in the 3^rd week was lower than that in the 0^th week (P < 0.05). Comparison data of the urine biomarker concentration measurements were presented in Table 2.

Table 2.

Comparison data of urinary biomarker concentrations

Groups	ACKD Group (n:25) (median (min-max)					Non-azotemic Group (n:8) (median (min-max)
Weeks	0	1	2	3	4	0	1	2	3	4
uNO (µmol/mL)	37.43 (16.35–49.87)*a	35.28 (26.11–60.54)*ab	29.47 (17.16–52.65)*ab	28.94 (9.36–60.10)*b	35.38 (9.79–58.17)*ab	8.94 (2.00-16.85)	11.33 (9.49–17.84)	12.37 (6.60-16.89)	10.65 (6.93–16.53)	14.28 (7.94–26.63)
uMDA (µmol/mL)	4.85 (2.64–9.85)*	4.89 (3.14–8.55)*	4.68 (2.02–7.05)*	4.65 (1.05–7.40)*	4.94 (2.83–7.15)*	1.81 (1.23–2.46)	2.20 (1.30–2.87)	1.81 (1.25–2.90)	1.84 (1.08–2.69)	2.43 (1.54–3.12)
uNGAL (ng/mL)	24.66 (15.29–43.74)*	24.72 (19.02–42.17)*	21.72 (8.04–37.93)*	21.20 (4.67–33.20)*	26.93 (16.82–32.60)*	7.25 (5.53–9.76)	9.92 (5.74–17.53)	8.60 (4.13–42.02)	7.22 (3.42–33.81)	16.02 (10.64–28.24)
uKIM-1 (ng/mL)	7.52 (4.19–15.06)*	7.67 (5.28–14.15)*	7.20 (3.98–11.50)*	6.56 (1.81–12.48)*	7.53 (2.67–10.46)*	2.96 (1.63–4.26)	3.08 (1.83–106.40)	3.01 (2.01–5.41)	2.69 (1.32–4.42)	3.68 (2.21–6.19)
uTGF-β1 (ng/L)	141.57 (98.75-206.98)*	149.72 (117.90-207.34)*	128.67 (80.10-175.06)*	132.78 (52.25-201.55)*	155.55 (105.33-202.09)*	50.27 (35.84–62.53)	61.14 (43.42–77.04)	65.76 (45.03–83.21)	51.81 (41.54–75.37)	50.77 (33.08-107.37)
uIL-18 (ng/L)	421.90 (240.22-603.28)*	428.75 (267.63-582.95)*	424.14 (209.67-619.63)*	388.20 (127.85-562.66)*	464.74 (262.54-631.34)*	154.08 (99.15-174.05)	168.29 (97.89-217.33)	157.11 (119.93-238.27)	138.66 (108.97-225.95)	168.22 (144.99-317.97)
uFGF 23 (ng/mL)	3.11 (1.50–4.39)*	3.07 (1.79–5.71)*	2.55 (1.23–4.25)*	2.77 (0.85–5.28)*	3.03 (1.00-4.19)*	0.80 (0.58–1.09)	0.97 (0.78–2.04)	0.90 (0.63–1.77)	0.79 (0.49–1.46)	1.17 (0.91–1.94)
uPGC-1α (ng/mL)	7.65 (3.54–10.18)*b	7.21 (5.03–16.57)*b	6.10 (2.17–9.91)*a	6.26 (1.26–12.24)*ab	6.79 (1.44–10.79)*ab	2.27 (1.27–3.31)	2.80 (2.40–5.18)	2.46 (1.22–4.51)	1.76 (1.10–3.93)	3.71 (1.88–5.56)
ucGMP (pmol/mL)	196.69 (26.25-653.11)	158.43 (23.45-689.34)	191.59 (28.96-1018.81)	115.77 (23.33-892.75)	117.24 (23.10-440.17)	100.16 (38.28-159.99)	233.05 (26.48-342.81)	239.07 (85.33-538.62)	155.18 (26.06-521.43)	70.93 (32.48-429.35)

uNO (Urinary nitric oxide), uMDA (Urinary malondialdehyde), uNGAL (Urinary neutrophil gelatinase-associated lipocalin), uKIM-1 (Urinary kidney ınjury molecule-1), uTGF-β1 (Urinary transforming growth factor beta-1), uIL-18 (Urinary interleukin-18), uFGF 23 (Urinary fibroblast growth factor 23), uPGC-1α (Urinary peroxisome proliferator-activated receptor gamma coactivator 1-alpha), ucGMP (Urinary cyclic guanosine monophosphate). Differences between groups for the same sample weeks were indicated with *. Differences between weeks within the group were shown with a, b, c

ROC analysis of serum biomarkers to monitor the recovery rate between diseased and healthy cats following sildenafil citrate administration, based on MB induction, revealed that the NO concentration had acceptable clinical performance (AUC = 0.691) at a cut-off value of 16.23. It was observed that other serum biomarkers had low clinical performance in monitoring the response to sildenafil citrate administration (AUC < 0.6). ROC analysis results of serum biomarkers are given in Table 3, ROC curves are given in Fig. 1.

Table 3.

ROC analysis results of serum biomarkers

Parameter	AUC	Std. Error	P value	Asymp. %95 CI		Cut-off	Sensitivity	Specificity
				Lower bound	Upper bound
sNO (µmol/mL)	0.691	0.055	0.002	0.583	0.799	16.23	71.2	54.5
sMDA (µmol/mL)	0.566	0.063	0.296	0.443	0.689	1.89	61	45.5
sNGAL (ng/mL)	0.407	0.063	0.141	0.283	0.531	9.63	42.4	36.4
sKIM-1 (ng/mL)	0.513	0.063	0.842	0.388	0.637	3.49	71.2	36.4
sTGF-β1 (ng/L)	0.507	0.061	0.909	0.388	0.627	60.82	71.2	30.3
sIL-18 (ng/L)	0.448	0.061	0.413	0.330	0.567	266.66	39	62.7
sFGF 23 (ng/mL)	0.442	0.062	0.360	0.322	0.563	1.36	64.4	36.4
sPGC-1α (ng/mL)	0.436	0.063	0.313	0.313	0.559	3.56	47.5	42.4
scGMP (pmol/mL)	0.386	0.064	0.071	0.260	0.512	150.52	67.8	30.3

AUC (Area under curve), Std. Error (Standard error), CI (Confidence interval), sNO (Serum nitric oxide), sMDA (Serum malondialdehyde), sNGAL (Serum neutrophil gelatinase-associated lipocalin), sKIM-1 (Serum kidney ınjury molecule-1), sTGF-β1 (Serum transforming growth factor beta-1), sIL-18 (Serum interleukin-18), sFGF 23 (Serum fibroblast growth factor 23), sPGC-1α (Serum peroxisome proliferator-activated receptor gamma coactivator 1-alpha), scGMP (Serum cyclic guanosine monophosphate)

Fig. 1.

ROC curves of serum biomarkers. sNO (Serum nitric oxide), sMDA (Serum malondialdehyde), sNGAL (Serum neutrophil gelatinase-associated lipocalin), sKIM-1 (Serum kidney ınjury molecule-1), sTGF-β1 (Serum transforming growth factor beta-1), sIL-18 (Serum interleukin-18), sFGF 23 (Serum fibroblast growth factor 23), sPGC-1α (Serum peroxisome proliferator-activated receptor gamma coactivator 1-alpha), scGMP (Serum cyclic guanosine monophosphate)

ROC analysis of urine biomarkers to monitor the recovery rate between diseased and healthy cats following sildenafil citrate administration, based on MB induction, revealed that all biomarkers except cGMP exhibited outstanding clinical performance (AUC > 0.9). ROC analysis results of urine biomarkers are given in Table 4, and ROC curves are given in Fig. 2.

Table 4.

ROC analysis results of urine biomarkers

Parameter	AUC	Std. Error	P value	Asymp. %95 CI		Cut-off	Sensitivity	Specificity
				Lower bound	Upper bound
uNO (µmol/mL)	0.992	0.006	0.000	0.980	1.000	16.17	100	86.1
uMDA (µmol/mL)	0.983	0.010	0.000	0.963	1.000	1.99	100	55.6
uNGAL (ng/mL)	0.911	0.040	0.000	0.833	0.990	13.60	96.9	77.8
uKIM-1 (ng/mL)	0.953	0.029	0.000	0.897	1.000	3.97	100	77.8
u TGF-β1 (ng/L)	0.998	0.002	0.000	0.993	1.000	78.99	100	99.4
uIL-18 (ng/L)	0.994	0.005	0.000	0.983	1.000	208.14	100	88.9
uFGF 23 (ng/mL)	0.987	0.007	0.000	0.973	1.000	1.22	100	75
u PGC-1α (ng/mL)	0.966	0.016	0.000	0.935	0.996	4.09	90.6	88.9
ucGMP (pmol/mL)	0.485	0.061	0.807	0.366	0.604	163.66	51.6	58.3

AUC (Area under curve), Std. Error (Standard error), CI (Confidence interval), uNO (Urinary nitric oxide), uMDA (Urinary malondialdehyde), uNGAL (Urinary neutrophil gelatinase-associated lipocalin), uKIM-1 (Urinary kidney ınjury molecule-1), uTGF-β1 (Urinary transforming growth factor beta-1), uIL-18 (Urinary interleukin-18), uFGF 23 (Urinary fibroblast growth factor 23), uPGC-1α (Urinary peroxisome proliferator-activated receptor gamma coactivator 1-alpha), ucGMP (Urinary cyclic guanosine monophosphate)

Fig. 2.

ROC curves of urinary biomarkers. uNO (Urinary nitric oxide), uMDA (Urinary malondialdehyde), uNGAL (Urinary neutrophil gelatinase-associated lipocalin), uKIM-1 (Urinary kidney ınjury molecule-1), uTGF-β1 (Urinary transforming growth factor beta-1), uIL-18 (Urinary interleukin-18), uFGF 23 (Urinary fibroblast growth factor 23), uPGC-1α (Urinary peroxisome proliferator-activated receptor gamma coactivator 1-alpha), ucGMP (Urinary cyclic guanosine monophosphate)

Discussion

In the present study, the effects of sildenafil citrate on the cGMP/NO and TGF-β/SMAD pathways, as well as its clinical effectiveness on glomerular filtration, kidney damage, and fibrosis, were investigated comparatively. Thus, the clinical efficacy of sildenafil citrate in inducing MB and improving glomerular filtration, as well as reducing and/or preventing kidney damage and fibrosis, was evaluated using serum SDMA and creatinine concentrations, urine SpG, and UPC ratio in cats with ACKD, defined based on IRIS criteria, laboratory findings, and ultrasonographic evaluations. The results of the present study demonstrated that sildenafil citrate induces MB in non-azotemic cats and positively affects glomerular filtration in cats with ACKD during the 30-day treatment protocol. Although mitochondria-focused therapeutic alternatives have only been investigated for a short period, multiple ongoing promising clinical trials are examining their effects on short- and long-term outcomes. In the present study, it was determined that sildenafil citrate is not effective in reducing or preventing kidney damage and fibrosis in cats with ACKD via the TGF-β/SMAD pathway during a 30-day follow-up.

Sildenafil citrate is a PDE-5 inhibitor. It causes relaxation of vascular smooth muscles by increasing cGMP and inhibits PDE-5, which catalyzes the breakdown of cGMP [29]. It induces the increase of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) proteins through NO production. NO increases cGMP formation by activation of guanylate cyclase, stimulates cGMP protein kinase G, opens mitochondrial ATP-sensitive potassium (KATP) channels, reduces intracellular calcium ion levels, and causes dilatation of arteries. Additionally, sildenafil citrate inhibits apoptosis via NO-induced Bcl-2 upregulation [30, 31]. Experimental studies have reported that sildenafil contributes to the improvement of AKI cases by activating the MB pathway and reducing renal KIM-1 expression [4]. Similarly to sildenafil, tadalafil has renal protective effects as well [32, 33]. It provides a decrease in urinary NGAL and KIM-1 excretions, can be used in the prophylactic treatment of ischemic AKI [33], and has antioxidant, anti-inflammatory [34] and anti-apoptotic [35] properties through the cGMP/NO process [36]. In the present study, sildenafil citrate, which was used to develop cellular protective treatments to reduce and/or stop the progression to renal failure and fibrosis on the basis of the pathogenesis of AKI and CKD development, was observed to be well tolerated in cats, and no hematological or biochemical side effects were observed as reported in previous studies [34–40]. It was determined that as a result of 30-day use of sildenafil citrate, the statistically significant increased serum cGMP concentrations in the non-azotemic group in the 2^nd and 3^rd weeks of the study (P < 0.05) indicates that MB stimulation was achieved [4, 29–31]. However, serum cGMP concentrations were lower in the ACKD group compared to the non-azotemic group and decreased at 4^thweek of the study (P < 0.05), indicate that MB stimulation was not achieved in the ACKD group. In the cats with ACKD, urinary NO and MDA concentrations as well as increased urinary expressions of selected renal biomarkers (NGAL, KIM-1, IL-18, FGF 23) (P < 0.05) indicate loss of renal function, presence of oxidative stress and renal damage. We expected a reduction in urinary NGAL and KIM-1 excretion similar to that observed with tadalafil, another PDE-5 inhibitor [32, 33]. However, in the ACKD group, the lack of change in renal biomarker concentrations during 30 days of sildenafil citrate use did not support findings that sildenafil citrate reduces NGAL and KIM-1 expression and aids in the recovery of renal damage [4, 33–36].

PGC-1α is described as a master regulator of mitochondrial biogenesis, regulating the anti-oxidant defense [41] and playing an important role in maintaining kidney health [42]. Mitochondrial biogenesis via PGC-1α overexpression helped expedite recovery if applied post-injury [43]. Rasbach and Schnellmann [44] found that stimulating mitochondrial biogenesis by overexpressing PGC-1α prior to ischemic injury did not prevent injury or promote recovery, suggesting that the timing and amount should be fine-tuned. In the diabetic nephropathy as a CKD model was found to be TNF-α and IL-1β could strongly decrease the expression levels of PPARγ, and the expression of coactivators (including SRC-1, SRC-2, and PGC-1α). It was suggested that PPARγ and its coactivator PGC-1α actively participate in protecting against renal inflammation by regulating the NF-κB pathway, which highlights their potential as therapeutic targets for renal diseases [45]. In chronic kidney disease (CKD), TGF-β1 reduces PGC-1α expression via SMAD3, and PGC-1α has been suggested to play a direct role in the fibrotic response to repeated insults to the kidney. Researchers have suggested that renal fibrosis, which leads to CKD and ultimately end-stage renal disease, is largely dependent on the dysregulated expression of inflammatory and metabolic pathways [46]. This genetic relationship was further supported by a study that also found PGC-1α acts downstream of TGF-β1 [47]. In the present study, the increase in biomarker concentrations (NGAL and KIM-1) observed in the ACKD group between 1^st and 4^th weeks suggests that renal damage is ongoing, that increased PGC-1α expression via the TGF-β1/SMAD pathway cannot provide TGF-β1 downregulation, and that sildenafil citrate is ineffective in protecting against renal damage and fibrosis in cats with ACKD, and that it is likely to be dose- and time-related [44], ongoing inflammation, increased inflammatory mediators [45] and CKD stage.

These results demonstrate that sildenafil citrate induces MB induction in healthy cats and fails due to proximal tubular damage and fibrosis in cats with ACKD. Our study suggests that high urinary TGF-β1, PGC-1α, and serum FGF23 levels in cats with ACKD may be related to ongoing inflammation and increased inflammatory mediators [45]. Therefore, we consider that the increase in inflammatory mediators in cats with CKD in our study supports acute exacerbation and the diagnosis of ACKD.

It is emphasized that the effects of cGMP are terminated by PDE-5 inhibitors and that the effects of PDE-5 inhibitors significantly depend on the activity of the NO/GC/cGMP pathway [48]. It was reported that in the kidneys, NO has hemodynamic/non-hemodynamic effects that modulate tubular and glomerular function [49] and that NO modulates a wide range of cellular processes mediated by cGMP or cGMP-independent mechanisms. The majority of the biological effects of NO are regulated by activation of soluble guanylyl cyclase (sGC), which synthesizes cGMP, a second messenger for numerous intracellular signaling pathways [50, 51]. Therefore, it was reported that maintaining cGMP production is beneficial for preserving the function of the glomerular filtration barrier with sGC-targeted pharmacological interventions [52]. In the present study, based on the routine parameters used to evaluate glomerular filtration [11, 16, 17, 53], decreases in UPC, BUN and P concentrations observed throughout the study period (P < 0.05) as well as alterations in serum Cr and SDMA concentrations, indicate that sildenafil citrate positively affects glomerular filtration in cats with ACKD [52].

It was reported that in the AKI to CKD transition, the control of pro-chronic inflammation and fibrotic gene expressions occurs through the TGF-β/SMAD pathway, and with the activation of this pathway, reactive oxygen species stimulate tissue damage and inflammatory response [37, 39]. NGAL is an important biomarker that can be easily detected in blood and urine after AKI [9, 10, 54], and evaluation of urinary NGAL concentration in cats has been reported to be important for kidney disease [55, 56]. It was stated that KIM-1 is sensitive to ischemic damage of the proximal tubular segment and tissue KIM-1 level is a sensitive indicator of AKI in cats [57, 58]. The specificity and sensitivity of urine levels of IL-18, a pro-inflammatory cytokine, were found to be over 90% in AKI cases. IL-18 is specific for ischemic AKI and is not affected by nephrotoxins, chronic kidney disease, or urinary tract infections [9, 10]. It was stated that IL-8 and TGF-β1 concentrations are higher in cats with CKD compared to healthy ones, and a positive correlation was determined between serum creatinine and TGF-β1 concentrations [59]. Also, elevated TGF-β1 concentrations were associated with cardiac, hepatic, pulmonary and renal fibrosis [60–62]. FGF 23 was demonstrated as a biomarker of damage and prognosis and a potential treatment target in AKI syndrome [12, 63, 64]. FGF 23 emerges as a promising biomarker for early detection of chronic renal failure progression in cats, potentially serving as a marker or mediator linked to azotemia, hyperphosphatemia, and renal secondary hyperparathyroidism in felines experiencing chronic renal failure [65–69]. Compared to the non-azotemic cats, in the cats with ACKD in the present study, it was interpreted that increased urinary NO and MDA expressions were associated with the development of oxidative stress [37–39, 70], increased urinary NGAL, KIM-1 and IL-18 expressions were associated with renal damage [4, 9, 10, 57–59, 71, 72], increased urinary PGC-1α, TGF-β1 and FGF 23 expressions were associated with decreased MB activity, PGC-1α downregulation, fibrosis development during CKD progression [38, 65, 68, 69, 73]. As FGF 23 promotes phosphaturia, its levels increase progressively beginning in early CKD, presumably as a physiological adaptation to maintain normal serum phosphate levels or phosphorus balance. In the present study, the rise in serum FGF 23 levels over the 4-week period may also be associated with the body’s regulation response to phosphorus balance [65].

Urine is a promising biological fluid due to its noninvasive sampling method and broad diagnostic biomarker framework [74], and urinary biomarker analyzes are seen as an interesting potential source with their cheap cost and ability to use large amounts in a short time [75]. It was highlighted that urine biomarker analysis may be utilized as tissue markers in the characterization of renal toxicity and the relationships between histological lesions and markers following the administration of nephrotoxic substances [71]. Especially in cancer and kidney diseases, the abundant presence of specific biomarkers in urine was shown as another advantage of urine utilization [76]. In a previous study, it was speculated that urinary markers might be more sensitive for true histological damage, whereas serum levels of markers might be more sensitive for changes in clearance. Also, in line with this assumption, it was highlighted that use of serum/plasma samples might reflect overall disease severity and multiorgan failure than kidney damage only [77, 78]. Comparative ROC analysis in the present study (Tables 3 and 4) shows that the use of urine samples provides more useful clinical information than the serum samples in the evaluation of MB induction, renal function, damage and fibrosis in the cats with ACKD. Although it is difficult to obtain urine samples for biomarker evaluation in oliguric/anuric patients, the low sensitivity and specificity of serum biomarker samples in the evaluation of MB induction, renal function, damage and fibrosis should be taken into consideration [79].

Limitations of this study include the fact that MB induction was investigated only through the cGMP/NO pathway, and changes in glomerular filtration, kidney damage, and fibrosis were assessed solely through the TGF-β/SMAD pathways. In addition, sildenafil citrate was not used in different dosage regimens in different groups, and the effect of long-term use and follow-up was not conducted. The lack of hematological parameter evaluations could be counted as a limitation. Additionally, histopathological evaluation was not performed, and the etiology of ACKD cases was not investigated. Furthermore, although the inclusion criteria for the study were based on IRIS and provide in-depth information on the subject, the diagnostic and prognostic performances of the aforementioned parameters need to be investigated in a heterogeneous cat population.

Conclusion

As a result, it was determined that sildenafil citrate caused a low level of MB induction and positively affected glomerular filtration but was not effective in reducing and/or preventing renal damage and fibrosis in cats with ACKD, as evaluated via the cGMP/NO and TGF-β/SMAD pathways. Also, it was determined that in the cats with ACKD, increased urinary NO and MDA expressions are due to oxidative stress, increased urinary NGAL, KIM-1 and IL-18 expressions are due to renal damage, increased urinary PGC-1α, TGF-β1 and FGF 23 expressions can provide specific information in the evaluation of MB activity, fibrosis development and CKD progression. Although the present results are promising, it was concluded that further research is needed for the clinical and prognostic uses of aforementioned parameters and pharmacokinetic-based studies for the sildenafil citrate dosing regimen in feline patients.

Electronic supplementary material

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Abbreviations

MB

Mitochondrial biogenesis

ACKD

Acute on chronic kidney disease

AKI

Acute kidney injury

CKD

Chronic kidney disease

GFR

Glomerular filtration rate

SpG

Urine specific gravity

IRIS

International Renal Interest Society

UPC

Urine protein/creatinine ratio

BUN

Blood-urea nitrogen

CR

Creatinine

P

Phosphorus

SDMA

Symetric dimethyl arginin

sNO

Serum nitric oxide

sMDA

Serum malondialdehyde

sNGAL

Serum neutrophil gelatinase-associated lipocalin

sKIM-1

Serum kidney ınjury molecule-1

sTGF-β1

Serum transforming growth factor beta-1

sIL-18

Serum interleukin-18

sFGF 23

Serum fibroblast growth factor 23

sPGC-1α

Serum peroxisome proliferator-activated receptor gamma coactivator 1-alpha

scGMP

Serum cyclic guanosine monophosphate

uNO

Urinary nitric oxide

uMDA

Urinary malondialdehyde

uNGAL

Urinary neutrophil gelatinase-associated lipocalin

uKIM-1

Urinary kidney ınjury molecule-1

uTGF-β1

Urinary transforming growth factor beta-1

uIL-18

Urinary interleukin-18

uFGF 23

Urinary fibroblast growth factor 23

uPGC-1α

Urinary peroxisome proliferator-activated receptor gamma coactivator 1-alpha

ucGMP

Urinary cyclic guanosine monophosphate

Author contributions

M.M., M.E.O., B.D. design of the study, M.M., M.E.O., B.D., M.I, A.N. writing of the manuscript, M.C.K., B.B., M.K.D., S.I., S.S.I, D.Z.T. contributed to the acquisition and analyses.

Funding

This study was supported by The Scientific and Technological Research Council of Türkiye (TUBITAK, 120O988) and The Scientific Research Projects Coordinatorship of Selcuk University (SUBAP, BAP, 22401056).

Data availability

All data generated or analyzed during the current study are included in this article. The data supporting the findings of this study are available from the corresponding author, MM, upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by Selçuk University Faculty of Veterinary Medicine Local Ethics Committee (14/04/2020). Also, informed consent was obtained from all of the owners.

Consent for publication

All authors: We approve the article and give permission for submission and publication.

Competing interests

The authors declare no competing interests.