Introduction
Urolithiasis, commonly referred to as kidney or renal stone disease, is characterized by the accumulation of crystals within the renal tract. In recent times, it has emerged as the third most widespread issue affecting the urinary system, exerting a significant impact on public health over the past two decades [
1]. While kidney stones are often perceived as non-life-threatening, they can have lethal consequences. These stones progressively damage the kidneys and can ultimately lead to chronic kidney disease [
2]. Furthermore, the financial load associated with stone disease is considerable, with the expenses related to stone treatment and management rivaling those of combined treatments for prostate and bladder cancers [
3].
Urinary stone disease has an ancient history in humans, with documented cases dating back to the Egyptian era. The historical record of urolithiasis is extensive, with evidence found in Egyptian mummies and references in ancient texts such as the Asutu in Mesopotamia, the Sushruta Samhita, and the Aphorisms of Hippocrates [
4]. In contemporary times, urolithiasis remains a formidable challenge within the human population, with prevalence rates varying across different regions. Notably, it affects about 1–19% of individuals in Asia, 7–13% in North America, 5–10% in Europe, 4% in South America, and a striking 20–25% in Middle Eastern countries [
5].
The epidemiology of urolithiasis is influenced by various factors, including geography, climate, ethnicity, diet, genetics, and demographic characteristics such as age, sex, and race [
6]. The increased prevalence of kidney stones in developed countries is associated with elevated consumption of salt and protein, as well as the prevalence of metabolic syndrome. In contrast, in developing countries, it can be linked to malnutrition and inadequate water intake [
5]. Therefore, there is a pressing need for novel medical prophylaxis strategies to address stone formation in the face of these diverse contributing factors.
To date, urolithiasis remains a challenging condition to treat effectively, owing to its intricate pathophysiology and multifaceted etiology. Across different ethnic groups worldwide, the historical use of various medicinal plants has garnered attention for their potential in managing urolithiasis. Many of these botanical resources have demonstrated promising effects. Significantly, medicinal plants often present an economically viable and regarded as a safe alternative for intervention. Recent investigations have assessed the antiurolithiatic efficacy of numerous plant species, as documented in studies [
7‐
9].
C. proximus, a member of the Poaceae family, is a traditional medicinal plant in Egypt known as “Halfa Bar.” It has gained recognition within Egyptian traditional medicine for its dual functionality as both a diuretic and a renal antispasmodic [
10]. Particularly in the southern regions of Egypt, this plant plays a significant role in traditional medicine, where it is utilized as a diuretic to enhance urine flow and as a remedy to aid in the expulsion of small urinary tract stones [
11].
C. proximus serves as a valuable source of bioactive metabolites, encompassing terpenoids, and phenols. These compounds have found applications in pharmaceutical drug production and the cosmetics industry, as reported by Malin et al. in 2018 [
12]. Moreover, recent studies conducted by Yagi et al. in 2020 [
13] and Moglad et al. in 2020 [
14] have shed light on its efficacy in combating human breast cancer and human colon adenocarcinoma cell lines, revealing promising anticancer properties in addition to its established antimicrobial activity.
P. crispum, known as Parsley, belongs to the Apiaceae family and is commonly referred to as “Baqdunis.” It boasts a rich history of medicinal utilization across European, Mediterranean, and Asian regions [
15]. One of its distinctive features is the presence of small, dark seeds that contain a notably higher concentration of volatile oil when compared to its stems or leaves [
16]. Parsley has garnered global recognition owing to its remarkable medicinal properties, primarily attributed to its potent antioxidant activity. This antioxidative potential can be ascribed to the presence of bioactive compounds like tocopherol, flavonoids, and carotenoids [
17]. The therapeutic potential of parsley and its derivatives extends to a range of kidney-related ailments, making it a valuable candidate for complementary or alternative treatments [
18]. Research has revealed its anti-inflammatory properties, edema mitigation capabilities, anti-hypertensive effects, anti-diabetic properties, and antimicrobial activity. Additionally, parsley enhances antioxidant defenses, modulates enzyme activities, elevates glutathione levels in the kidney, and contributes to the restoration of kidney tissue following nephrotoxicity [
19].
GA is a viscous exudate obtained from the umbrella-shaped branches of
Acacia seyal and
Acacia senegal. This substance is extracted either by making an incision on the branches to allow the exudate to flow or by harvesting naturally occurring exudate, which subsequently hardens upon exposure to air. GA is predominantly sourced from regions such as Sudan, Chad, and Nigeria [
20]. Historically, GA has been employed to address a range of medical conditions, including chronic renal disease and stomach pain. Recent investigations have illuminated various pharmacological and medical attributes associated with GA. These encompass anti-inflammatory properties and its capacity to safeguard nephrons [
21]. Notably, GA stands out for its exceptional antioxidant activity. Moreover, research indicates that high doses of GA can enhance renal function, modulate specific serum minerals, bolster antioxidant enzyme activity, and prevent renal damage in rat models afflicted with diabetic kidney failure, all while maintaining safety [
22].
Therefore, the quest for antilithiatic drugs derived from natural sources, known for their efficacy and minimal side effects, has gained substantial momentum. Plant-based formulations, in particular, are regarded as a promising avenue due to their perceived safety and affordability. In this context, the primary objective of this study is to examine the protective effects of an ethanolic extracts obtained from C. Proximus and P. crispum seeds, along with GA, and its nanogel emulsion, against urolithiasis induced in rats by EG and AC.
The evaluation of this study encompasses several facets, including an assessment of the mitigating effects on the antioxidant machinery within renal tissue, specifically catalase (CAT), reduced glutathione (GSH), and Malondialdehyde (MDA) levels. Additionally, the study examines serum toxicity markers, encompassing nitrogenous waste products such as blood urea nitrogen (BUN), uric acid, urea, and creatinine. Mineral levels, including calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), and potassium (K), are also scrutinized. Furthermore, urinary markers, including urine pH, urine volume, creatinine clearance, uric acid, Ca, Mg, P, Na, K, and oxalate, are part of the assessment. The methodology extends to microscopic examination of urine, histopathological examination of kidney tissue, and immunohistochemistry examination. These comprehensive evaluations aim to provide a comprehensive understanding of the protective effects of C. proximus and P. crispum seeds ethanolic extracts/GA emulsion and its nanogel emulsion form, against EG and AC-induced urolithiasis in rat models.
Discussion
Due to contemporary lifestyles, urolithiasis remains a pervasive global medical challenge, and its prevalence is steadily escalating, evidently manifesting a pronounced tendency towards high recurrence rates. However, the utilization of medicinal plants holds profound significance on a global scale, serving not only as standalone remedies but also as complementary adjuncts to conventional medicinal approaches [
44,
45]. The diverse components of medicinal plants, including seeds, leaves, flowers, fruits, stems, and roots, have consistently emerged as reservoirs of bioactive compounds [
46,
47].
The ingestion of ethylene glycol by rats has long been a pivotal experimental model for investigating nephrolithiasis. However, the deposition of kidney crystals following ethylene glycol administration can be inconsistent. To consistently induce high rates of kidney crystal deposition, researchers have employed the combination of ammonium chloride with ethylene glycol. When male SD rats were subjected to treatment with 0.75% EG and 1% AC, nearly all exhibited the deposition of calcium oxalate crystals in the kidneys [
48]. Ethylene glycol undergoes oxidation to oxalic acid via non-specific dehydrogenase activity, leading to hyperoxaluria, a crucial factor in urolithiasis. Furthermore, EG undergoes metabolic processes leading to the formation of calcium oxalate monohydrate, resulting in renal mitochondrial toxicity closely resembling that observed in clinical calcium oxalate renal calculi [
49]. In this study, male rats were specifically chosen to ensure the efficient formation of calcium oxalate stones. It has been documented that the male sex hormone testosterone has been observed to promote the formation of calcium oxalate crystals in adult rats treated with EG [
50]. Furthermore, Research indicates that in the EG-induced animal model, the duration of stone deposition in female rats is comparatively shorter than in male rats [
51]. Therefore, the present study predominantly employs male rats.
In this study, urolithiatic rats demonstrated a significant reduction in food intake, feed efficiency ratio, and body weight gain compared to the normal control group, indicating preliminary signs of renal toxicity, consistent with previous research findings [
52]. However, administration of both the emulsion and nanogel emulsion significantly alleviated these detrimental reductions. This preventive effect on declining body weight can be attributed to their capability to counteract the toxic effects caused by ethylene glycol and ammonium chloride. EG’s final metabolite, oxalic acid, predominantly impact the kidneys, leading to acute poisoning symptoms [
53]. The induction of renal stones in urolithiatic animals resulted in decreased food consumption, contributing to the decline in their body weight. Additionally, it is noteworthy that the urine of urolithiatic rats exhibited a significant presence of oxalate crystals, a phenomenon observed in various previous studies [
54,
55].
Furthermore, the urolithiatic control group exhibited significant changes, marked by a notable decrease in urine volume and pH, coupled with an increase in the count of urinary calcium oxalate crystals. Additionally, this group demonstrated a substantial elevation in the urinary excretion of calcium, phosphate, and oxalate ions, along with a notable reduction in excreted magnesium levels compared to the normal control group. Following ethylene glycol administration, evident induction of hyperoxaluria and nephrotoxicity were observed. This was substantiated by histopathological examinations, which revealed oxalate crystal deposition within renal tubule lumens, accompanied by inflammatory responses and necrotic changes. This evidence was further supported by a significant elevation in immunohistochemical levels of inflammatory mediators such as TNF-α, as well as indicators of cytotoxicity and cell death induction, notably cleaved caspase-3. These findings are consistent with previous studies reporting tubular hypertrophy, tubulointerstitial damage, and extensive calcium oxalate crystal deposition in kidney tubules across various urolithiatic rat models [
52].
The primary mechanism behind EG-induced stone formation is attributed to hyperoxaluria, leading to increased renal excretion and retention of oxalate [
56]. This disrupts the equilibrium between lithogenesis promoters such as phosphate, oxalate, calcium, uric acid, and inhibitors like magnesium [
57]. The heightened urinary phosphate excretion, in combination with oxalate stress, fosters an environment conducive to stone formation by primarily generating calcium phosphate crystals, thus promoting calcium oxalate deposition [
58]. Improperly acidic or alkaline urine directly affects the solubility of various metabolites and salts in the body. Alkaline urine tends to reduce the solubility of calcium phosphate products, whereas an acidic pH in urine encourages the formation of stones containing calcium oxalate, uric acid, and cystine. Research has emphasized the pivotal role of urinary pH in kidney stone formation, suggesting that maintaining a urine pH around 6 on the pH scale significantly decreases the risk of stone development. Investigations on the risk of calcium oxalate crystallization across varying urine pH levels, involving individuals with and without a history of recurrent calcium oxalate stones, have shown that the highest risk of crystallization occurs within the pH range of 4.5 to 5.5 [
57]. Consequently, the observed elevation in oxalate, calcium, and uric acid levels in this study triggered crystallization and subsequent precipitation of calcium oxalate within nephrons, causing damage to renal epithelial cells. These findings collectively highlight the intricate interplay of factors contributing to urolithiasis pathogenesis.
The administration of either the emulsion or nanogel emulsion had noticeable effects, including increased urine volume and pH, a reduction in urinary calcium oxalate crystals, decreased excretion of calcium, oxalate, and phosphate, and enhanced elimination of urinary magnesium. These effects might arise from the dual functionality of
C. proximus, known for its diuretic and renal antispasmodic properties [
10]. Moreover, the potent antioxidative activity found in parsley seeds, attributed to bioactive compounds like flavonoids and carotenoids [
17], potentially enhances antioxidant defenses, raises kidney glutathione levels, and aids in kidney tissue restoration following nephrotoxicity [
19]. Additionally, the anti-inflammatory properties of GA and its ability to protect nephrons might also contribute to these observed effects [
21].
Regarding the clinical aspect, our urolithiatic animal models displayed notably increased serum levels of creatinine, BUN, urea, and uric acid compared to the normal control group. These findings strongly suggest severe impairment of kidney function, primarily due to reduced clearance of waste products from the bloodstream to the urine. This is attributed to both diminished glomerular filtration rate and damage to renal tubular cells. The obstruction caused by stones within the urinary system significantly reduced the glomerular filtration rate [
54]. Concurrently, damage to proximal tubule cells hindered the efficient clearance of waste products, especially nitrogenous substances like blood creatinine, urea, and uric acid. Consequently, this led to an increased accumulation of these waste products within the bloodstream [
59,
60], intensifying renal damage and further compromising kidney function.
Elevated urinary uric acid levels were also notably observed in our urolithiatic animals, associated with an increased risk of stone formation due to its role as a crystallization promoter [
61]. This association is due to the ability of uric acid-binding proteins to interact with calcium oxalate, altering its crystallization kinetics, emphasizing its significant role in stone formation [
62]. However, it’s noteworthy that administration of either the emulsion or nanogel emulsion, along with EG and AC, significantly reduced serum concentrations of creatinine, urea, uric acid, and BUN compared to the urolithiatic rat group. These changes are attributed to an improvement in the clearance of blood creatinine, urea, and uric acid into the urine following intervention.
Our findings align with previous studies conducted by Al-Yousofy et al. (2017) [
63] and El-Nabtity et al. (2019) [
64]. These investigations focused on a rat model of stone formation induced by administering 0.75% ethylene glycol and 2% ammonium chloride in drinking water over 10 days. The experiments revealed that treatment with either ethanolic or aqueous extracts of
C. proximus or
P. crispum exhibited notable nephroprotective and antiurolithiatic effects. The treated groups showed significantly lower levels of creatinine, blood urea nitrogen, and calcium compared to the stone-induction group. These extracts demonstrated antiurolithiatic properties through various mechanisms: 1- Reduced Urinary Calcium Excretion: Decreasing calcium excretion in urine lowers the risk of calcium oxalate crystallization. 2- Increased Urinary pH: Elevating urinary pH levels creates an unsuitable environment for calcium oxalate crystallization. 3- Diuretic Effect: Inducing diuresis increased urine volume, reducing urine supersaturation with stone-forming compounds. 4- Nephroprotective Activity: Additionally, these extracts exhibited nephroprotective properties, shielding the kidneys from adverse effects associated with stone formation.
Moreover, Gumaih et al. (2017) [
65] evaluated the antiurolithiatic effect of parsley in rats with EG-induced urolithiasis, reported a significant reduction in serum urea, creatinine, uric acid, and electrolytes, along with a notable decrease in urinary calcium and proteins within the treated group. The authors attributed parsley’s nephroprotective effects to its antioxidant activity derived from its high content of flavonoids. These bioactive substances play a crucial role in preventing free radical damage induced by ethylene glycol. Furthermore, the therapeutic potential of GA in kidney diseases has been documented. Patients suffering from chronic kidney diseases who received GA demonstrated significant reductions in serum urea, creatinine, and levels compared to baseline and the control group [
66].
The exposure of renal epithelial cells to elevated oxalate levels and the presence of calcium oxalate crystals often result in excessive reactive oxygen species (ROS) generation, leading to cellular injury and inflammation [
67]. The administration of EG and AC heightened the levels of MDA, a marker of lipid peroxidation, while reducing reduced glutathione levels and CAT enzyme activity. However, the administration of either the emulsion or its nanoform showcased remarkable antioxidant activity, as evidenced by reduced MDA levels and an increase in antioxidant enzyme activities. These findings correspond with existing literature consistently reporting oxidative stress and lipid peroxidation in stone formation contexts [
68,
69]. In a study by Ali et al. (2020) [
66], GA exhibited a substantial impact on patients undergoing hemodialysis. Specifically, it significantly elevated total antioxidant capacity levels while notably reducing oxidative markers like MDA and C-reactive protein. These compelling findings robustly support the potent anti-inflammatory properties attributed to GA.
The GC-MS analysis of the ethanolic extracts of
P. crispum seeds combined with
C. proximus ethanolic extracts revealed a rich composition of phytochemicals and bioactive compounds. Major identified components included polyphenols, terpenoids, carotenes, porphyrin, milbemycin, fucoxanthin, and cyanocobalamin, recognized for their well-established antioxidant and anti-inflammatory properties. These ethanolic extracts exhibited a notable antiurolithiatic effect against calculi induced by EC and AC in this study. The observed efficacy in mitigating urolithiasis likely stems from the identified bioactive compounds within the extracts, highlighting their potential therapeutic relevance in addressing urolithiasis and promoting renal health. Previous research supports the inhibitory effects of terpenoids on the formation and size of calcium oxalate crystals [
70]. Additionally, porphyrin has shown anti-ROS enzyme-mimicking capabilities, alleviating ROS-induced cell apoptosis and improving renal function in acute kidney injury mice [
71]. Lutein, as reported by Hu et al. (2022) [
72], displayed antioxidant properties, associated with a lower mortality risk in the chronic kidney disease population due to high-level carotene dietary intake. Fucoxanthin, highlighted by Wang et al. (2020) [
73], emerged as a compound with significant antiurolithiatic properties, restoring antioxidant levels, regulating stone and renal markers, and preventing glomerular and tubular damage in lithiatic rats induced by Ethylene glycol. Milbemycins were found to be effective fungal growth inhibitors [
74]. Furthermore, vitamin B12 exhibited antioxidant properties, protecting cells and DNA from reactive oxygen species-induced damage, inversely correlating with the risk of kidney stone development [
75]. Additionally, Ke et al. (2023) [
76], emphasized the association between a higher Oxidative Balance Score, indicative of an antioxidant-skewed balance, and a reduced risk of kidney stones, particularly among specific population subgroups.
Our study investigated the anti-urolithic effects of
C. proximus and
P. crispum seed ethanolic extract/GA emulsion, and nanogel emulsion, against EG and AC-induced urolithiasis in rats. The findings revealed that treatment with either the emulsion or nanogel emulsion significantly prevented urolithiasis-related abnormalities, including decreased urinary excreted magnesium and non-enzymic antioxidant glutathione, as well as catalase activity. Additionally, both treatments reduced oxalate crystal numbers, excretion of urolithiasis promoters, renal function parameters, and lipid peroxidation, while improving histopathological changes. Notably, the nanogel emulsion exhibited superior effects compared to the emulsion, as evidenced by decreased renal crystal deposition score and reduced expression of TNF-α and cleaved caspase-3. This observation suggests that converting the treatment into a nano-sized form could potentially enhance its bioavailability, effectiveness, and therapeutic action. Previous literature has emphasized the excellent biocompatibility of lipid-based nanoparticles [
77]. The transformation of the emulsion into a nanogel emulsion improved the bioavailability of bioactive compounds, resulting in a more pronounced reduction in urolithiasis and nephrotoxicity.