Hypertension and diabetes are the leading causes of chronic kidney disease (CKD) (Hamrahian and Falkner, 2017). The pathogenesis of CKD in hypertension is multifactorial, involving genetic alterations, oxidative stress, endothelial dysfunction, and renin-angiotensin-aldosterone system (RAAS) disturbances (Mennuni et al., 2014). These factors promote inflammation, fibrosis, and both a reduced nephron count and a lower glomerular filtration rate (GFR) (Schlondorff, 2008). CKD is associated with a worsening cardiovascular prognosis and constitutes a serious health and social condition. Thus, there is a continuous search for new ways of protection in experimental models of hypertension.

Cardiovascular homeostasis is tightly bound to proper protein turnover, which is controlled by the ubiquitin-proteasome system (UPS). Proteasomes are multisubunit protease complexes that degrade damaged proteins in all parts of the cells (Pagan et al., 2013). The proteasomes specific to the heart involve a number of proteins with various biological impacts, such as atrogin (known as muscle atrophy F-box) participating in myocardial remodeling (Li et al., 2004); murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 participating on myocardial hypertrophy modulation (Chatterjee et al., 2011); the calcineurin-nuclear factor of the activated T cells (NFAT) pathway that mediates cardiac remodeling (Tang et al., 2010); sarcomere-associated protein MuRF1 associated with heart failure (Willis et al., 2009); or Nedd4 containing E3 ligase controlling the biological impact of multifunctional vascular endothelial growth factor (VEGF) (Murdaca et al., 2004). In the kidney, Nedd4L/Nedd4-2 seems to participate in distal nephron salt sensitivity (Ishigami et al., 2020), and ubiquitin-conjugating enzyme E2 contributes to HUWE1-mediated degradation of tubulointerstitial fibrosis (Wang et al., 2022). Pharmacological interference with proteasomes is emerging as a potential approach to influence various pathological processes.

Lactacystin is a proteasome inhibitor that blocks the hydrolysis and degradation of intracellular proteins by the UPS (Craiu et al., 1997). Chronic lactacystin administration induces a mild but significant rise in systolic blood pressure (BP) along with fibrotic remodeling of the left ventricle (LV) in Wistar rats (Simko et al., 2017), and hypertrophy of the aorta in L-NAME (L-NG-nitro arginine methyl ester)-induced hypertension (Vrankova et al., 2010). Several hypothetical mechanisms for hemodynamic alterations and heart and vessel damage by lactacystin have been proposed, such as enhanced oxidative stress (Vrankova et al., 2010; Huseby et al., 2016; Parajuli, 2019), decreased NO bioavailability (Simko et al., 2017; Sharma et al., 2020), sympathetic nervous system activation (Congo Carbajosa et al., 2015) and modification of various cytosolic, nuclear, and myofibrillar protein turnovers (Mearini et al., 2008). However, data on lactacystin’s renal effects are not available.

The aim of this study was to test whether lactacystin induces structural and fibrotic rebuilding of the kidneys. Furthermore, we sought to determine whether melatonin prevents these potential alterations. Melatonin (N-acetyl-5-methoxytryptamine), the main product of the pineal gland, not only regulates biological circadian rhythms (Reiter et al., 2020), but exerts various pleiotropic protective effects on the heart, vasculature (Reiter et al., 2010; Simko et al., 2019, 2020; Domínguez-Rodríguez et al., 2021; Tobeiha et al., 2022) and kidneys (Russcher et al., 2012; Hrenák et al., 2013; Hrenak et al., 2015; Shi et al., 2019). The effects of melatonin were compared with captopril, a classical angiotensin-converting enzyme (ACE) inhibitor, which exerts a well-established antiremodeling action in several organs and various pathologies (Pfeffer et al., 1992; Pechánová et al., 1997; Simko et al., 2010, 2014; Repová-Bednárová et al., 2013) including lactacystin-induced LV remodeling (Simko et al., 2017).

Six weeks of lactacystin administration raised the average SBP. In kidneys, lactacystin reduced glomerular density, increased the glomerular tuft area, and enhanced the hydroxyproline concentration. Lactacystin likewise elevated the intraglomerular proportion and the sum of Col I and Col III, the tubulointerstitial proportion of Col I and the sum of Col I and Col III without an effect on vascular/perivascular collagen. Six weeks of captopril treatment reduced SBP, while melatonin had no effect. Both melatonin and captopril raised glomerular density, reduced the glomerular tuft area, and decreased hydroxyproline concentration in the kidneys. Both drugs reduced the proportion and sum of intraglomerular and tubulointerstitial Col I and Col III.

Since the UPS controls the protein turnover of both regulatory and structural proteins, it becomes an attractive target for pharmacological interventions. Lactacystin represents the classical, first discovered proteasome inhibitor, reported in 1991 (Ōmura and Crump, 2019). Among other effects, lactacystin is considered to be an inhibitor of nuclear factor kappa B (NF-κB) transcription factor. Since NF-κB is assumed to be a checkpoint for hypertrophic growth mediated by humoral factors, inhibitors of NF-κB, such as lactacystin, are considered a possible way of protection. The net effect of lactacystin is challenging to estimate for two reasons: first, NF-κB is not only an essential factor for proliferation (Bellas et al., 1995); it may also interfere with nitric oxide synthesis, having the opposite effect (Vrankova et al., 2010; Simko et al., 2017). Moreover, lactacystin not only specifically blocks the degradation of the NF-κB inhibitor IκBα but can also modify other proteins involved in signaling processes (Vrankova et al., 2010).

In line with this, surprisingly, lactacystin did not reduce but increased blood pressure and fibrotic rebuilding in the left ventricle (Simko et al., 2017). Thus, lactacystin administration-induced BP elevation was recently characterized as a novel model of experimental hypertension (Vrankova et al., 2010; Simko et al., 2017; Sharma et al., 2020). Proteasome inhibition may cause hypertension either because of an increased endogenous protein inhibitor of neuronal nitric oxide synthase, leading to decreased NO bioavailability in the paraventricular nucleus (Sharma et al., 2020), or from tyrosine hydroxylase upregulation and activation in the hypothalamus and brainstem (Congo Carbajosa et al., 2015), both resulting in increased sympathetic outflow. Bearing in mind the remodeling of the heart (Simko et al., 2017) and aorta (Vrankova et al., 2010) in this model, it seems to be of importance to disclose whether lactacystin could act in a similar pro-proliferative way in hypertensive kidneys.

In this experiment, chronic lactacystin treatment was associated with the loss of glomeruli, indicated by decreased glomerular density and simultaneous glomerular hypertrophy, reflected in increased glomerular tuft area. Some authors consider the reduction of nephron density to be a risk factor for hypertension and CKD progression (Kanzaki et al., 2020). Furthermore, lactacystin treatment was associated with enhanced hydroxyproline concentration and site-specific fibrotic rebuilding of renal tissue. Published data suggest that chronic hypertension leads to the accumulation and dysregulation of extracellular matrix in the kidneys, resulting in renal fibrosis (Schlondorff, 2008). The fibrotic changes in the kidneys are common 1) in the glomerulus—glomerulosclerosis; 2) in the tubulointerstitium—interstitial fibrosis; and 3) in the vessels—arteriosclerosis and perivascular fibrosis (Bülow and Boor, 2019). The most abundant collagens expressed in the kidneys are types I and III. Col I forms long, thick, and stiff fibrils, decreasing tissue compliance (Sopakayang et al., 2012). In the early stages of renal fibrosis, Col I deposits in the glomerulus, tubulointerstitial space, and arterial wall (Alexakis et al., 2006). Col III is mainly found in softer tissues and is more distensible than Col I (Silver et al., 2001). In renal fibrosis, the expression of Col III increases in both interstitium and glomeruli (Alexakis et al., 2006). In the present study, an excessive deposition of Col I and Col III was observed in the glomerulus and tubulointerstitial space in lactacystin-treated hypertensive rats.

ACE inhibitor captopril reduces fibrosis associated with target organ damage. Indeed, captopril decreased the concentration of soluble collagen in the LV of rats with combined continuous light and L-NAME-induced hypertension (Simko et al., 2010), attenuated LV collagen deposition in SHR (Zhao et al., 2015), and reduced LV fibrosis in mice with transverse aortic constriction (Zhang et al., 2019) and Sprague-Dawley rats with L-NAME-induced hypertension (Sonoda et al., 2017). Similarly, in kidneys captopril reduced interstitial renal fibrosis in neonatal dogs with partial urethral obstruction (PUO) (Shirazi et al., 2007), ameliorated fibrosis in rats with PUO (Shirazi et al., 2014), and downregulated interstitial fibrosis in rats with unilateral ureteral obstruction (Hosseinian et al., 2019). In line with these data, our results show reduced intraglomerular, tubulointerstitial, and perivascular collagen accumulation and renal hydroxyproline concentration after 6 weeks of captopril administration in lactacystin-treated rats. In this experiment, captopril reduced the proportion and sum of intraglomerular and tubulointerstitial Col I and Col III, lowered the sum of vascular/perivascular Col I and Col III and increased the vascular/perivascular ratio of Col I to Col III, while reducing the kidney hydroxyproline concentration.

Melatonin exerts a vast number of pleiotropic protective effects on various tissues, including fibrosis amelioration. The antifibrotic effects of melatonin have been observed in the LV of lactacystin-treated rats (Simko et al., 2017), of SHRs (Simko et al., 2009), and in the LV and aorta of 24-h continuous light-exposed rats (Repová-Bednárová et al., 2013; Simko et al., 2014). In the kidneys, melatonin reduced renal fibrosis in mice with unilateral ureteral obstruction (Li et al., 2020), adenine-induced CKD mice (Yoon et al., 2020), diabetic mice (Li et al., 2019; Fan et al., 2020), prenatally diclofenac sodium injected rats (Khoshvakhti et al., 2015), and in human renal proximal tubule epithelial cells on a high glucose diet (Han et al., 2020). Accordingly, in this study, 6 weeks of melatonin treatment prevented intraglomerular and tubulointerstitial fibrosis development, alongside reduced renal hydroxyproline concentration in rats administered lactacystin. Although melatonin failed to reduce SBP in this experiment, its antifibrotic effects were likely a result of melatonin’s direct, local actions, such as obvious antioxidant and radical scavenging actions (Simko et al., 2009; Reiter et al., 2018), modulation of the sympathetic and renin-angiotensin system (Simko and Paulis, 2013), and increasing NO bioavailability and antiproliferative action (Paulis and Simko, 2007; Simko and Paulis, 2007, 2013; Simko and Pechanova, 2009; Domínguez-Rodríguez et al., 2021).

The lactacystin administration induced fibrotic rebuilding of the kidneys. Both captopril and melatonin reduced the proportion and sum of intraglomerular and tubulointerstitial Col I and Col III and hydroxyproline concentration in the kidneys. Although captopril more prominently reduced Col I and Col III accumulation in the vascular/perivascular area compared to melatonin, the Col I/Col III ratio rose. Considering the stiffness of Col I, the findings suggest that captopril might cause renal capillaries to become more rigid, altering renal perfusion. On the contrary, melatonin did not significantly change the renal vascular/perivascular fibrosis while maintaining a normal Col I/Col III ratio. Since it concurrently reduced glomerular fibrosis, melatonin may play a role in maintaining adequate glomerular perfusion and filtration. Thus, melatonin may be comparable or superior to ACE inhibition because of its distinct effect on renal collagen composition, potentially contributing to improved renal perfusion and filtration rate.

In a model of lactacystin-induced hypertension and organ damage, both melatonin and captopril raised glomerular density, reduced the glomerular tuft area, and lowered the kidney hydroxyproline concentration. Both drugs reduced the proportion and sum of intraglomerular and tubulointerstitial Col I and Col III. As melatonin failed to reduce SBP, its antifibrotic effects were supposedly delivered by melatonin’s direct, local pleiotropic effects.

Considering the fibrotic remodeling of the left ventricle observed in previous works and the site-specific fibrotic rebuilding of the kidneys observed in this study, it seems reasonable to suggest that chronic treatment with lactacystin may be a perspective model of cardiorenal damage. Furthermore, we showed that melatonin, similar to captopril, is a promising means of protection against hypertensive kidney damage.

It would be of interest to correlate the renal histopathologic findings with immunohistochemical analysis of growth factors, angiogenic factors, and markers of collagen turnover in the kidneys. Indeed, according to the literature, there is a correlation between the histopathological evidence of renal fibrosis and profibrotic renal tissue markers, such as transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), and vascular endothelial growth factor A (VEGF-A) (Lopes et al., 2019). Moreover, the inhibition of matrix metalloproteinase (MMP)-9 (Wang et al., 2019) and decreased expression of MMP-1 (Nazneen et al., 2002) resulted in decreased histological evidence of renal fibrosis. However, these analyses were beyond the scope and possibilities of the present histopathological study.