The Biological Roles of Urea: A Review of Preclinical Studies

Biosynthesis of Urea (Urea Cycle)

The urea cycle is a series of reactions that result in the production of urea from ammonia. It was discovered in 1932 by Hans Krebs and Henseleit. Protein catabolism produces ammonia, which is highly toxic to the body organs. Most organisms eliminate ammonia directly to the external environment. However, in some animals including humans, ammonia is converted to urea before its clearance.[37] The production of urea is controlled by a series of enzymes within the cytosol and the mitochondria of cells.[3839] In the mitochondria, ammonia combines with CO₂ using energy from adenosine triphosphate (ATP) to form carbamoyl phosphate. This combines with ornithine to form citrulline. Citrulline diffuses out of the mitochondria into the cytosol, where it combines with aspartate to form arginine succinate, which breaks down to form arginine and fumarate. Arginine receives water to form ornithine and urea. Ornithine re-enters the mitochondria to initiate another cycle, while urea is excreted in urine.

Uremia

This occurs when there is a high level of urea in the blood. The causes of elevated urea in the blood could be classified into three different categories, which include prerenal, renal, and postrenal causes. In prerenal causes, an increased production of urea in the liver through high-protein diets can lead to uremia. More so, a decreased blood pressure, shock, and dehydration could also reduce the clearance of urea, thereby increasing its blood concentration.[40] The renal causes may be due to a decrease in kidney functions, and the factors may include acute kidney disease and CKD, tubular necrosis, and many other related kidney diseases.[41] The post-renal causes occur due to the obstruction in the urinary outflow of urea. This could be due to enlarged prostate tumor of the bladder or severe infection of the urinary tract.[42]

Assessment of Urea

Urea is the primary metabolite of dietary protein and turnover of tissues. To determine its blood concentration, the whole urea is assayed and in some cases, only the nitrogen components of urea are measured. The normal range of urea nitrogen is between 5 and 20 mg/dL. This range varies due to several factors which include protein content of the diet, protein catabolism, water content of the body, liver urea biosynthesis, and urea excretion in the kidney.[43] Several methods exist for analyzing urea. The recent techniques are automated, and they produce reproducible and reliable results. Urea nitrogen can be measured using a diacetyl or Fearon reaction, which produces a yellow chromogen product when added to urea.[44] The quantification occurs through photometry procedure. Urea concentration can also be assayed using the enzymatic procedure which involves the breakdown of urea to ammonia and carbonic acid by the enzyme urease.[23] The absorbance is then measured at a specific wavelength. Blood urea nitrogen (BUN) test measures the amount of nitrogen in the blood that is derived from the waste product urea; however, the assay could be performed using serum and not whole blood.[45]

Literature Search

A PubMed search was carried out for articles published before 2019 using the phrase, “urea application in experimental animals.” A total number of 1142 articles were screened using the title and abstract. Thirteen articles met the inclusion criteria, while 1129 articles were excluded [Figure 1]. The search was mainly for research articles, and no clinical trials, case reports, and case series were reviewed. The information recorded from these publications included the test substance, amount injected, animal model, number of animals, duration of treatments, and effects. The research publications were categorized based on the following:

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Figure 1

A systematic guide for the selection of articles

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1. The toxicity of urea in experimental animals

2. The therapeutic effects of urea in experimental animals.

Urea Toxicity in CKD

Urea concentration increases during CKD.[464748] Many studies administered urea in nephrectomized or CKD animals to further increase its concentration in order to study its effects.[46471849505152] Table 1 gives a list of seven articles which were reviewed, and the following observations were made. Different nitrogenous compounds (arginine, aspartic acid, and urea) were administered to experimental rats of different sample sizes. The results showed retention of nitrogen compounds in the blood, and this may likely imply a buildup of toxic products that should be excreted in urine.[46] However, using animals of the same sample size would have guaranteed more valid and reliable results. We observed that urea induces the production of intracellular reactive oxygen species (ROS) in cells such as renal tubular cells, vascular endothelial cells, adipocytes, vascular smooth muscle cells, and beta cells in CKD mice.[4748] Most of the parameters were investigated using modern experimental techniques, which ensures validity of the studies. These ROS are highly reactive radicals that can damage cellular components. Increased ROS production in excess of antioxidants leads to oxidative stress, which has been reported in the etiology and pathogenesis of many diseases like diabetes, cancer, Alzheimer's disease, and many more.[535455] Many of the effects reported in Table 1 are due to increased ROS production, which occurs in animals with chronic diseases. The high concentration of urea in the plasma (uremia), which occurs in CKD, could result in a defect in carbohydrate, protein, and lipid metabolism.[56] ROS production has been shown to increase modification of insulin-signaling molecules and reduce insulin-stimulated insulin receptor substrate (IRS) and Akt phosphorylation and glucose transport in uremic animals, which therefore creates a state of insulin resistance.[4857] Uremia has also been reported to cause carbamylation of intracellular proteins which are responsible for the altered mitochondria ROS production. Trecherel et al.[58] studied in vitro expression of proapoptotic proteins (Bcl-2–associated death [BAD] promoter) in smooth muscle cells exposed to urea, and it was confirmed that urea has the potential to induce apoptosis and cell death in smooth muscle cells and plays a role in atherogenesis and progression of atherosclerosis, thus promoting cardiovascular disease. We observed that a high urea concentration administered to nephrectomized dogs was lethal and sufficient to cause death of animals. The symptoms exhibited by the animals included gastrointestinal disturbances like vomiting, diarrhea, nausea, weakness, and others.[49] There was also electrolyte imbalance, which has been reported to play a critical role in the pathogenesis of many diseases.[49] The hypernatremia, hyperkalemia, and hyperchloremia observed may be due to the inability of the kidney to excrete these ions. Although these ions are necessary for nerve and muscle functions, their excess concentration in the blood could be deleterious. In addition, the excess bicarbonate ions caused by urea could result in metabolic acidosis, which is characterized by increase in blood pH. In one of the studies conducted using CKD zebrafish, a decrease in the volume of the olfactory epithelium was shown to occur in uremic fish [Table 1], even though the mechanisms of this have not been ascertained.[50] The decrease may be due to olfactory cell degeneration or apoptosis induced by high urea level. The olfactory epithelium is the sensory system of smell, and therefore, any disruption to the olfactory cells may interfere with the ability to detect airborne substances in the environment.

Patients with CKD suffer from depression due to the accumulation of urea. This observation was further confirmed in mice and could be as a result of inhibition of the mTORC1-S6k signaling pathway and loss of synapses in the medial prefrontal cortex of the brain.[51] Furthermore, other studies on urea showed that it is capable of causing disruption of the intestinal barrier and proposed that the mechanism of this effect involves diffusion of urea into the gut and its conversion by microbial urease to ammonia.[5359] The impaired integrity of the intestinal epithelia potentially supports leakage of bacterial intestinal toxins into the body fluid and systemic inflammation. The deposition of extracellular cell matrix (ECM) by increased ROS production and protein kinase C activation has widely been reported in CKD.[60] The ECM causes endothelial dysfunction and disturbances in the ultrafiltration processes of the renal tubular cells, as reported in the wild-type CKD mice exposed to urea.[52] This causes an increase in blood urea with increase in signs of toxicity. This elevated urea level may be because of carbamylation reaction, a posttranscriptional modification where urea derived from its catabolism (isocyanic acid) tampers with the function and structural integrity of proteins.

Therapeutic Actions of Urea in Experimental Animals

According to a study conducted by Sweeny et al.[61], urea was shown to improve animal feed supplementation. It was reported to increase nutritional value and aids the digestion of low-energy crop stubbles. Emmanuel et al.[62] also studied the effects of different levels of urea supplementation on nutrient intake and growth performance in growing camels fed roughage-based complete pellet diets and urea was confirmed to influence digestibility, feed intake, growth rate, feed efficiency, and economics.[636465] Urea has been confirmed to have an effect on plasma sodium lower than 135 mEq/L (hyponatremia), according to a clinical research trial performed by Rondon-Berrios et al.[66] Patients who had syndrome of inappropriate ADH secretion (SIADH) as the only cause of hyponatremia were treated with oral urea as the sole drug therapy for hyponatremia[67] The mechanism of effectiveness of urea on hyponatremia caused by SIADH is based on the pharmacokinetics of urea; it is a partially effective due to its permeability into the muscle tissues, connecting tubule, cortical collecting duct, and medullary collecting duct. Urea has been shown to create a positive sodium balance, which contributes to improvement of plasma sodium.[68] This study was further supported by Van Reeth and Decaux[69] who reported that urea administration ameliorates hyponatremia and decreases brain damage in rats. Thiourea, a regulator of leptin hormone secretion from adipocytes, plays an important role in the progression of obesity and hepatic steatosis. In a study carried out by Liang et al.,[70] administration of thiourea (a similar compound to urea) in animals was observed to modulate triglyceride metabolism and decrease body weights, total cholesterol, high- and low-density lipoproteins.[70] Even though urea and its derivatives have been reported to have many therapeutic effects, their antitumor effects have also been widely reported [Table 2]. In a study carried out by Vedarethinam et al.,[71] it was reported that 1,3-bis-((3-hydroxynaphthalen-2-yl) phenylmethyl) urea (1,3-BPMU) has cytotoxic and growth inhibitory effects on hepatocellular carcinoma cells (HEP-G2).[71] They also stated that a flow cytometry analysis shows that 1,3-BPMU causes early and late apoptosis. This compound is a derivative of urea, and the effects are mediated by urease. Another urea derivative, URD12, has been confirmed to exhibit cytotoxic activity against the K562 human leukemia and KB human mouth epidermal carcinoma cell lines. Wang et al.[72] further studied URD12 in In vivo and in vitro assays using BGC-823 human gastric carcinoma, SMMC-7721 human hepatoma, and HepG2 human hepatocellular carcinoma cell lines [Figure 2]. URD12 was confirmed to inhibit the growth of tested tumor cell lines with no effects on the weight, spleen, and thymus. Several other studies have also reported that urea has therapeutic applications. Evidence suggest that topical urea may impact epidermal permeability and barrier function. Rawlings et al.[73] Susanne et al.[73] confirmed this myth by conducting a placebo-controlled, double-blinded study in healthy human volunteers, after which urea significantly improved cutaneous barrier function as there was a significant decrease in transepidermal water loss. Dermal therapists have shown considerable interest in urea due to its properties. Urea has been confirmed to have a great effect on xerosis.[74] It has a good hydrating effect by improving the water-holding capacity of the stratum corneum and the skin barrier function and regulates skin surface pH. With appropriate urea concentration, reduction of stratum corneum integrity together with decreased protease activity was not observed.[75] According to a comparative study conducted by Kiane de Kleijne et al., Caduff et al.,[76] urea showed great environmental benefits. Urea production from basic oxygen furnace gas (BOFG) avoids greenhouse gas emissions, and net emission savings could be achieved especially when combined with excess carbon-dioxide transport and storage.

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Figure 2

Summary of findings on the biological roles of urea

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