In vivo and in vitro experimental models for urolithiasis pathophysiology research

Abstract

Objective

This review aims to investigate and establish potential in vitro and in vivo models for evaluating the anti-urolithiatic activity of therapeutic agents, exploring experimental approaches that can reliably simulate human stone formation and effectively assess the efficacy of anti-urolithiatic compounds.

Methods

Multiple in vitro and in vivo approaches were explored. In vitro methods included the​​ estimation of calcium oxalate by titrimetry, nucleation assays, aggregation assays, turbidimetric assays, and electron microscopy studies. Artificial stone models such as BegoStone and Ultracal 30 were fabricated to mimic the physicochemical characteristics of urinary calculi. In vivo models included ethylene glycol-induced, calcium oxalate/ammonium oxalate-induced, diet-induced, and infection-related models in rodents. Additionally, genetically modified animal models such as TRPV5 knockout, CLDN14 knockout, AGXT knockout, and URAT1 overexpression mice were discussed to study molecular pathways of urolithiasis. Parameters such as urinary oxalate, calcium levels, and histopathological evaluation of kidney tissues were used to validate stone formation and dissolution processes.

Results

In vitro models effectively demonstrate the processes of crystal nucleation, aggregation, and inhibition, allowing quantitative assessment of potential anti-urolithiatic activity. Electron microscopy provides detailed insights into crystal morphology and ultrastructural alterations. Artificial stones fabricated using BegoStone and Ultracal 30 closely replicate natural calculi hardness and composition, making them suitable for lithotripsy and dissolution studies. In vivo models successfully mimic human urolithiasis pathophysiology, particularly the ethylene glycol-induced rat model, which shows reproducible calcium oxalate crystal deposition in renal tissues. The application of genetic models highlights the role of specific transporters and enzymes in calcium and oxalate homeostasis.

Conclusion

A combination of in vitro and in vivo experimental models provides a comprehensive platform for evaluating the anti-urolithiatic potential of therapeutic agents. The integration of biochemical, morphological, and genetic analyses enhances the understanding of stone pathogenesis and development of novel anti-urolithiatic therapies.

1. Introduction

One of the most ancient, widespread, and often recurrent disorders, urolithiasis dates back to 4000 BC and was first discovered in Egyptian mummies. According to epidemiological research, urolithiasis is more frequent in males than in females, and it is more typically seen in both genders aged 20–40 years [1].

The disease known as urolithiasis is defined by the growth of stones in the urinary system, which includes the kidneys, ureters, bladder, or urethra. There are usually five different kinds of stones. The most common kind is calcium oxalate (CaOx), accounting for 80% of occurrences. Other common types are calcium phosphate (5%) and uric acid stones (4%) [2].

The oldest and most excruciating urologic condition, urolithiasis, affects 5–7 million people. The Greek terms “ouron” (urination) and “lithos” (stone) are the source of the phrase “urolithiasis” [3]. Fifty percent of the patients experience a recurrence within 5 years, and currently, no widely approved standard treatment can stop these recurrences [4].

Stone formation is a complex process which occurs due to the successive physicochemical events such as supersaturation, nucleation, growth, aggregation, and retention within the renal tubules [5].

Humans are not the only animals with urolithiasis. The stone disease also occurs in two- or four-legged animals (non-mammals include birds and turtles, and mammals include dogs and cats). Stone disease is a universal phenomenon throughout the animal kingdom; therefore, the use of animal models to study the disease may have the advantage of similar pathogeneses, and may also have benefits both for humans and animals. In this review, we examined the current types of animal models used for the stone disease, in order to share with the readers [6,7].

2. In vitro methods

2.1. Estimation of CaOx by titrimetry for urolithiasis [[8], [9], [10], [11]]

Titrimetric techniques can be used to estimate CaOx in the setting of urolithiasis or the development of urinary stones. Titration of calcium ions with ethylenediaminetetraacetic acid solution is a representative technique.

2.1.1. Materials and reagents

The materials and reagents are specified as follows: CaOx crystals (prepared using standard procedures), the test substance (evaluated for its urolithiatic activity), the titration solution (typically a standardized solution [e.g., KMnO₄] used for titration), the acid solution (usually H₂SO₄, which is used for the dissolution of CaOx crystals), and a suitable indicator (employed to signal the endpoint of the titration).

2.1.2. Procedure

2.1.2.1. Step 1: preparation of experimental kidney stones (CaOx stones) by homogeneous precipitation

Calcium chloride dihydrate (1470 mg) is dissolved in 100 mL of distilled water and sodium oxalate (1340 mg) is dissolved in 100 mL of 2 mol/L H₂SO₄. Both are mixed equally in a beaker to precipitate out CaOx with stirring. Equimolar solutions of calcium chloride dihydrate in distilled water and disodium hydrogen phosphate in 10 mL of 2 mol/L H₂SO₄ are allowed to react in sufficient distilled water in a beaker. The resulting precipitate is calcium phosphate. Both precipitates are washed by ammonia solution to remove traces of H₂SO₄. Precipitates are washed with distilled water and dried.

2.1.2.2. Step 2: preparation of egg-based semi-permeable membrane

The semi-permeable membrane of eggs is located between the albumin and yolk, and the calcified outer shell. A glass rod is used to puncture the apex of the eggs in order to extract the complete contents. After being properly cleaned with distilled water, empty eggs are left in a beaker containing 2 mg of hydrochloric acid for an entire night, resulting in total decalcification. After that, they are rinsed with distilled water, washed again with distilled water and then immersed in an ammonia solution for a while to neutralize any remaining acid traces, maintaining a pH of around 7.4 while being kept in a refrigerator.

2.1.2.3. Step 3: titrimetry-based CaOx estimation

The dissolution percentage of CaOx is evaluated by taking exactly 1 mg of CaOx and various concentrations of a sample, packing them together in a semipermeable egg membrane. This is allowed to be suspended in a conical flask containing 100 mL of 0.1 mol/L tris buffer. For the blank, only 1 mg of CaOx is used. For the standard, 1 mg of CaOx is used along with the standard drug, i.e., cystone. The conical flasks of all groups are kept in an incubator preheated to 37 °C for 2 h. The contents of the semipermeable membranes are removed​ from each group into separate test tubes, followed by the addition of 2 mL of 0.5 mol/L H₂SO₄ to each test tube. The mixtures are then titrated with 0.9494 mol/L KMnO₄ until a light pink color endpoint is obtained. The amount of remaining undissolved CaOx is subtracted from the total quantity used in the experiment at the beginning to know the total quantity of dissolved CaOx. Each milliliter of 0.9494 mol/L KMnO₄ is equivalent to 0.1898 mg of CaOx.

The choice of titration solution, acid solution, and indicator may vary based on the specific requirements of the experiment and the nature of the test substance.

This titrimetric method provides a quantitative assessment of the ability of a substance to influence the dissolution or inhibition of urinary crystals, offering valuable insights into its potential urolithiatic activity.

2.2. Nucleation assay for urolithiasis [[12], [13], [14], [15]]

A nucleation assay is used to assess the crystal formation in urine, which is a crucial early stage in the development of kidney stones. This assay measures the amount of solid crystals that precipitate out of the urine under conditions where dissolved chemicals, like calcium and oxalate, are present. The assay aids in understanding the mechanisms underlying stone formation and in the development of methods to avoid or minimize the risk of kidney stone formation by examining parameters that influence nucleation, such as supersaturation levels and the presence of inhibitors or promoters.

2.2.1. Chemicals

The chemicals used include the following: sodium chloride (NaCl), sodium citrate, buffer solutions (such as phosphate buffer), calcium chloride (CaCl₂), and magnesium salt (MgCl₂).

2.2.2. Procedure

The inhibitory activity of the samples on the nucleation of CaOx crystals is determined by a spectrophotometric assay. Crystallization is initiated by adding CaCl₂ (4 mmol/L) and sodium oxalate (50 mmol/L) solutions to the sample, both prepared in a buffer containing 0.05 mol/L Tris and 0.15 mol/L NaCl at pH 6.5 and 37 °C. The rate of nucleation is determined by comparing the induction time of crystals (time of appearance of crystals that reach a critical size and thus become optically detectable) in the presence of the sample to that of the control with no sample. The absorbance (optical density [OD]) is recorded at 620 nm, and the percentage inhibition is calculated as (1−OD_experimental/OD_control)×100.

The temperature, incubation time, and nucleating agent selection can all be changed based on the specific goals of the investigation. It may be necessary to introduce controls free of nucleating substances to observe the urine’s natural tendency to crystallize. For significant outcomes, the circumstances must be kept as close to physiological as feasible.

Designing and interpreting nucleation assays for urolithiasis always involve consulting with urology and crystallography experts, as well as pertinent scientific literature. In order to make significant conclusions about the factors influencing crystal formation in urine, this type of research requires careful consideration of the experimental circumstances and detailed analysis.

2.3. Estimation by the aggregation assay [14, [16], [17], [18]]

An important phase in the development of kidney stones is the propensity for crystal formation and growth, and the aggregation assay helps assess this process. The aggregation assay can offer insights into the mechanisms of stone formation and the efficacy of possible therapies or inhibitors aimed at preventing stone growth and recurrence by replicating circumstances in the urinary system.

2.3.1. Chemicals

The chemicals used include the following: CaCl₂, sodium oxalate (Na₂C₂O₄), phosphate buffer saline (PBS), MgCl₂, citrate, urine samples (human or synthetic), distilled water, potassium chloride (KCl), ammonium chloride (NH₄Cl), Tris buffer, hydrochloric acid (HCl), and sodium hydroxide (NaOH).

2.3.2. Procedure

The CaOx crystals are prepared by mixing both CaCl₂ and sodium oxalate solutions at 50 mmol/L. Both solutions are then equilibrated in a bath for 1 h at 60 °C. The solutions are then cooled to 37 °C and then are evaporated. The CaOx crystals are then dissolved in 0.05 mol/L Tris and 0.15 mol/L NaCl at pH 6.5 to a final concentration of 1 mg/mL. The absorbance at 620 nm is recorded. The rate of aggregation is estimated by comparing the slope of turbidity in the presence of the sample with that obtained in the control. The percentage inhibition is calculated as (1–S_i/S_c)×100, where S_i is the slope of the plot in the presence of inhibitor (sample) and S_c is the slope of the control plot (with no inhibitor).

2.4. Turbidimetric assay [[19], [20], [21], [22]]

A typical technique for determining the quantity of particles in a solution based on the degree of turbidity—the cloudiness or haziness of the solution—is the turbidimetric assay. Turbidimetric assay can be modified to assess the quantity or presence of crystals in urine in the setting of urolithiasis. An overview of the turbidimetric assay used to estimate urolithiasis is provided below.

2.4.1. Chemicals

The chemicals include the following: NaCl, buffer solutions (e.g., phosphate buffer), MgCl₂, and CaCl₂.

2.4.2. Procedure

For this, 1.0 mL of 0.025 mol/L CaCl₂ and 2 mL of Tris buffer (pH 7.4) are added to a test tube. Then 1.0 mL of 0.025 mol/L sodium oxalate is added. Formation of the turbidity occurs immediately after mixing of the above chemicals and then the measurement of turbidity formed (in terms of absorption at 620 nm in an ultraviolet-visible spectrophotometer) is started immediately up to a period of 10 min after the mixing. This control experiment can be done in replicates. Absorption is recorded and data obtained are used as the uncontrolled growth of the stone nucleus for the comparison of growth in the presence of the standard drugs and samples.

Depending on the particular objectives of a study, the temperature, duration of incubation, and other parameters can be changed.

Urine’s inherent turbidity can be taken into consideration by including controls devoid of crystals. There is a correlation between the degree of turbidity and either the severity of urolithiasis or the probability of stone formation. Guidance from urology and analytical chemistry specialists, along with consultation of relevant scientific literature, should be obtained when developing and interpreting turbidimetric assays for urolithiasis. In order to make significant conclusions about the factors influencing crystal formation in urine, this type of research requires careful consideration of the experimental circumstances and detailed analysis.

2.5. BegoStone powder and Ultracal 30 [[23], [24], [25], [26], [27]]

BegoStone powder (BEGO GmbH & Co. KG, Bremen, Germany) and Ultracal 30 (USG Corporation, Chicago, IL, USA), which are typically used to create dental stone models, can also be modified for use in experimental models of urolithiasis research to test therapies and simulate stone development.

2.5.1. Materials

The materials required include the following: BegoStone powder or Ultracal 30, filtered water, polytetrafluoroethylene molds, a pH meter (if necessary), urine samples (optional, used for simulating in vivo conditions), and inhibitors or promoters of crystallization (either one can be used).

2.5.2. Procedure

2.5.2.1. Preparation of stone models

Depending on the requirements of an experiment, equal parts of Ultracal 30 and BegoStone powder are weighed. The ingredients and distilled water are mixed in a mixing dish. The usual proportion is roughly 30 mL of water for every 100 g of powder; this ratio can be adjusted to get the right consistency. Using a spatula, the ingredients are thoroughly combined until the mixture is homogeneous. To create imitation stones, the mixture is poured into molds. Kidney stone size and shape can be replicated in these molds. The mixture should be given time to solidify and set, and the time required for this process could take many hours depending on the specific products used and the surrounding conditions. After setting, the stones should be removed from the molds. To improve simulation and strength, the stones are incubated at 37 °C for 24–48 h.

2.5.2.2. Experimental evaluation

For testing the effect of inhibitors or promoters, these are added to the urine or the fluid in which the stones are immersed. Common inhibitors include citrate and magnesium, while promoters may include additional calcium or oxalate. The stones should be periodically observed under a microscope to assess changes in crystal structure and aggregation; changes in stone weight, size, or surface characteristics should be measured to evaluate the impact of different conditions or treatments; a pH meter should be used to ensure that the fluid environment remains consistent with physiological conditions; and treated and untreated samples should be compared to evaluate the effectiveness of different interventions.

Adherence to safety guidelines and manufacturer recommendations is essential. Researchers should report the fabrication details, including powder-to-water ratios, to ensure the reproducibility and comparability of results across studies. Adjustments to the procedure may be necessary based on specific research goals or material variations. Comparisons should be made between treated and untreated samples to evaluate the effectiveness of different interventions.

3. In vivo methods

To investigate the pathophysiology of urolithiasis, test possible therapies, and comprehend the underlying mechanisms, in vivo animal models are essential. Research on urolithiasis has involved the use of a variety of animal species, including dogs, rats, mice, and rabbits. The following are a few popular in vivo techniques for researching urolithiasis in animals: chemical induction of stone formation by the ethylene glycol model—CaOx stones are formed when animals, usually rats or mice, are given ethylene glycol in their drinking water, and this popular model replicates some features of how stones are formed in humans; CaOx, CaCl₂, or ammonium oxalate model—urolithiasis in animals can be brought on by injecting drugs such as ammonium oxalate or a combination of CaCl₂ and oxalate; models induced by diet—high oxalate diets given to animals may exacerbate CaOx stone development, while excessive calcium intake in high calcium diets can promote stone formation when combined with other conditions; genetically modified models—the genetic basis of urolithiasis can be investigated using animals that have certain genetic alterations linked to pathways that generate stones such as transient receptor potential vanilloid 5 (TRPV5) knockout mice, CLDN14 knockout mice, AGXT knockout mice, and URAT1 overexpression mice; models of obstruction (ureteral ligation)—urinary stasis brought on by ureteric obstruction might result in kidney stones; models related to infections (infection-induced stone production)—in animals that are vulnerable, the introduction of bacteria or substances that cause urinary tract infections can encourage the production of stones; models of metabolic disorders—stones can occur on their own in animals with metabolic abnormalities such as hyperoxaluria or hypercalciuria.

3.1. Chemical induction of stone formation

3.1.1. Ethylene glycol model [[28], [29], [30], [31]]

By adding ethylene glycol to the drinking water of rats, the ethylene glycol model for urolithiasis is induced, which encourages the development of kidney stones and CaOx crystals. This extensively utilized model necessitates the cautious selection and housing of animals, along with consistent weight, water consumption, and health monitoring. At the conclusion of the study, a necropsy is conducted to examine the kidney and bladder tissues.

3.1.1.1. Mechanism of action

Alcohol dehydrogenase and aldehyde dehydrogenase in the liver quickly catalyze and convert ethylene glycol to glycolic acid; this is converted to glyoxylic acid, which is then further oxidized by glycolate oxidase and lactate dehydrogenase to oxalic acid or oxalate, hence resulting in hyperoxaluria. The primary risk factor for urolithiasis is hyperoxaluria.

3.1.1.2. Procedure

Healthy male Wistar rats weighing 120–200 g are used. They are divided into four groups, with six animals in each group. For a period of 28 days, Group I serves as the control. Groups II, III, and IV function as positive controls. Both the standard and test groups receive ethylene glycol (0.75% v/v, p.o.). Groups III and IV receive the test medicine (sample) and the standard medication cystone (750 mg/kg, p.o.). Urine and serum samples are collected from all animals on the 28th day, and all necessary tests and comparisons are made.

3.1.1.3. Advantages

Because the kidney is the primary target organ for ethylene glycol and the most sensitive organ, it is a commonly accepted model of urolithiasis for research. An organic solvent that is widely accessible is ethylene glycol. It deposits microcrystals and modifies oxalate metabolism, which is comparable in humans and rats.

3.1.1.4. Limitations

Oxalate-induced nephrotoxicity results in cellular damage, disrupting normal kidney function and potentially leading to further pathological complications.

3.1.2. CaOx, CaCl₂, or ammonium oxalate model [[32], [33], [34], [35], [36], [37]]

Both the CaOx and CaCl₂ model and the ammonium oxalate model are experimental methods used to induce urolithiasis in animal models, particularly rodents. These models involve the administration of substances that promote the formation of CaOx crystals and stones in the urinary tract, mimicking aspects of human kidney stone formation. Here is a summary of both models.

3.1.2.1. Mechanism of action

In these models, either CaOx or ammonium oxalate is administered to animals, leading to supersaturation of calcium and oxalate ions in the bloodstream. This results in the formation of CaOx crystals, which precipitate and aggregate within the renal tubules and urinary tract, ultimately leading to kidney stone formation. Over time, these crystals will grow larger, causing tubular obstruction and renal tissue damage.

3.1.2.2. Procedure

In order to have four groups, adult Wistar rats weighing between 120 g and 200 g each are randomly selected. As a preventive measure, Group I is given saline acting as the control, and Groups II, III, and IV serve as the positive control, standard (cystone 750 mg/kg, p.o.), and test groups, respectively. Groups II, III, and IV are injected with sodium oxalate 70 mg/kg, i.p. for 7 days. For the purpose of comparison, different biological samples are taken from every rat.

It is recommended that researchers tailor their protocols to the particular needs of their investigations, and that they adhere rigorously to ethical guidelines and laws pertaining to the use of animals in experiments. Research employing the ammonium oxalate model for urolithiasis necessitates ethical approval and consultation with veterinarians.

3.1.2.3. Advantages

It takes less time, is a reliable model, and deposits microcrystals through a driving force such as hyperoxaluria induced by ethylene glycol.

3.2. Models induced by diet [38,39]

High oxalate foods, known as oxalate compounds, have the ability to bond with calcium to form crystals, which may put vulnerable people at risk for kidney stones. The following foods have a lot of oxalates:

• •Deep, leafy greens: Swiss chard and spinach, beet greens, and collard greens;
• •Nuts and seeds: cashews, almonds, and peanuts;
• •Seeds: sesame and sunflower seeds;
• •Vegetables: beets, okra, sweet potatoes, leeks, and turnips;
• •Fruits: figs, grapes, kiwis, oranges, strawberries, blueberries, and raspberries;
• •Grains: bran flakes, wheat germ, quinoa, and buckwheat;
• •Additional foods: black and green tea, chocolate tea blends, goods made from soy (tofu and soy milk).

The modified lithogenic diet consists of 30% lactose-rich diet and 1% ethylene glycol. The 30% lactose-rich lab diet contains 3.68% sucrose, 30% lactose, 23.4% protein, 10% fat, 5.3% crude fibre, 6.9% ash minerals (calcium [0.95%], phosphorus [0.67%], and Mg [0.21%]), vitamin A 22 IU/g, vitamin D 4.5 IU/g, and vitamin E 49 IU/g.

3.2.1. Procedure

Healthy adult male Wistar rats of 150–200 g are divided into four groups. Group I serves as the control and is fed with regular lab diet. Groups II, III, and IV serve as the diseased control, standard, and test, respectively, and they are given the modified lithogenic diet for 28 days. Simultaneously, Groups III and IV are given a standard drug and test drug, respectively, from Day 1 to Day 28 as a preventive regimen. Various biological samples are collected at last for measurement and comparison.

3.2.2. Advantages

It is a non-nephrotoxic model and diet-induced urolithiasis is an effective model as it produces stable crystal deposition.

3.3. Genetically modified model

The genetic alterations, namely urate transporter 1 (URAT1) overexpression, alanine-glyoxylate aminotransferase (AGXT) knockout, CLDN14 knockout, and TRPV5 knockout, are linked to important proteins and pathways that are involved in renal physiology and kidney stone formation (urolithiasis). An outline of each modification’s significance in relation to urolithiasis is provided below.

The procedure for conducting is similar to the previous methods so we can see the significance of each type of model in urolithiasis.

3.3.1. TRPV5 knockout mice [40,41]

The kidneys express TRPV5, a calcium channel, especially in the distal convoluted tubules. It is essential for the reabsorption of calcium into the bloodstream from the urine. Mice lacking TRPV5 are expected to show different kidney functions when it comes to processing calcium, which could result in higher calcium levels in the urine. A frequent form of kidney stones called CaOx can occur when there is an elevated level of calcium in the urine.

3.3.2. CLDN14 knockout mice [40]

CLDN14, also known as claudin-14, is a tight junction protein that controls the paracellular movement of ions in the renal tubules, hence impacting the absorption of calcium. Reduced calcium reabsorption may result from enhanced paracellular calcium transport in CLDN14 knockout mice. Higher calcium concentrations in the urine as a result of this condition may increase the risk of CaOx stone development.

3.3.3. AGXT knockout mice [43,44]

The enzyme known as AGXT is involved in the metabolism of oxalate. It converts glyoxylate, an oxalate precursor, into less toxic substances. Mice lacking AGXT are expected to display compromised oxalate metabolism, resulting in elevated oxalate concentrations in their urine. The development of CaOx stones is linked to the elevated urine oxalate.

3.3.4. URAT1 overexpression mice [45,46]

URAT1 is a transporter that helps the kidneys reabsorb uric acid. Increased uric acid reabsorption due to overexpression of URAT1 may raise uric acid concentrations in the urine. Uric acid stones are known to occur when there is an increase in uric acid levels.

Researchers can better grasp the biochemical and genetic processes underlying kidney stone formation due to these genetic changes in mice. They aid in identifying potential therapeutic targets for urolithiasis treatment or prevention. Researching these models can shed light on the intricate interactions between multiple factors that affect renal physiology and kidney stone development.

3.4. Ureteral ligation method for urolithiasis in animal models [[47], [48], [49], [50]]

3.4.1. Mechanism of action

The obstruction leads to urine buildup in the kidney, resulting in hydronephrosis, which can impair kidney function and cause complications such as infection. Prompt treatments, such as pain management and stone removal, are crucial to relieve the obstruction and prevent kidney damage. Ureteral obstruction is a serious complication of urolithiasis that requires urgent medical attention to alleviate symptoms and prevent long-term complications.

3.4.2. Procedure

Rats are adapted for 1 week, given standard feeds (normal proteins, fats, and carbohydrates) and ad libitum drinking water. The rats are then divided into three groups randomly, and each group consists of 12 rats. The surgery uses a combination of ketamine (80 mg/kg body weight) and xylazine (10 mg/kg body weight) as the anesthetic agent. Group I is used as the control, treated with laparotomy and then closed back without organ manipulation. Group II undergoes laparotomy followed by ligation of the right ureter in the proximal part, 5 mm from the renal pelvis with surgical silk suture, and then the abdominal cavity is closed. Group III is treated with laparotomy, followed by ligation of the right ureter in the distal part, 5 mm from the urinary bladder with surgical silk suture, and then the abdominal cavity is closed. One week after surgery, three rats are taken from each group randomly, euthanized, and their kidneys are excised from the body. The right and left kidneys are then stored in tubes containing 10% formalin to make histopathological preparations. Similar treatment is carried out in the 2nd, 3rd, and 4th weeks postoperatively.

It is crucial to remember that ethical standards must be followed in all animal experiments including those involving surgery, and that researchers should put the welfare of the animals first. Any study involving animals must also abide by all applicable laws and obtain the necessary authorization.

3.4.3. Disadvantages

The main disadvantages include pain and discomfort, renal dysfunction, and complications such as pyelonephritis.

3.5. Models related to infections [[51], [52], [53]]

The mechanisms underlying the production of struvite stones (magnesium ammonium phosphate stones) are as follows:

3.5.1. Microbial involvement: bacterial infections, especially those caused by urease-producing bacteria (e.g., Proteus, Klebsiella, and Staphylococcus), are frequently linked to struvite stones.

Bacteria create urease that hydrolyzes urea, raising the pH of the urine and releasing carbonate and ammonium ions in the process. This produces an environment that is conducive to the development of struvite stones. This process is known as urease activity.

3.5.2. Stone formation and inflammation

The immune response initiates inflammatory pathways in response to an infection. Changes in the urine environment, such as variations in pH, calcium, and other ions, can be facilitated by chronic inflammation and lead to the production of stones.

Growth and nucleation of crystals can occur at nucleation sites, giving rise to a surface on which stones can form.

3.5.3. Urinary tract infections

The development of struvite stones may be linked to persistent or recurrent urinary tract infections. Preventing recurrence of stones requires effective treatment of the underlying infection.

3.5.4. Pyelonephritis and kidney abscesses

Stone formation may be facilitated by kidney infections, such as kidney abscesses or pyelonephritis. Stone growth may be accelerated by the development of biofilms and the presence of infected urine.

3.6. Models of metabolic disorders

Urolithiasis is a disease that causes solid crystals or stones to form in the urinary tract. Kidney stones can occur as a result of metabolic problems, and the primary cause of urolithiasis is the precipitation of materials such as calcium, oxalate, and phosphate in the urine. The following models represent metabolic diseases linked to urolithiasis.

3.6.1. Hypercalciuria [54,55]

The term “hypercalciuria” describes the high excretion of calcium in the urine. The connection to urolithiasis is that calcium phosphate or CaOx stones can develop as a result of elevated calcium levels in the urine.

3.6.2. Hyperoxaluria [56,57]

An elevated excretion of oxalate in the urine is known as hyperoxaluria. The connection to urolithiasis is that one of the most prevalent kinds of kidney stones is CaOx, which is created when high oxalate levels mix with calcium.

3.6.3. Cystinuria [58,59]

The overabundance of cystine excreted in the urine is a hallmark of a hereditary condition cystinuria. The connection to urolithiasis is that due to its weak solubility in urine, cystine stones may develop from a buildup of cystine.

3.6.4. Distal renal tubular acidosis (dRTA) [60,61]

The acid secretion of the distal tubules of the kidneys is compromised in dRTA. The connection to urolithiasis is that because of the alkaline pH of the urine due to dRTA, some forms of stones, such as calcium phosphate stones, are more likely to form.

3.6.5. Hypocitraturia [62,63]

The disorder known as hypocitraturia is characterized by a low level of citrate in the urine. The connection to urolithiasis is that citrate prevents crystals from forming. Low citrate concentrations can cause calcium salts to precipitate, which can aid in the development of stones.

Urolithiasis management and prevention depend heavily on an understanding of and attention to these metabolic abnormalities. In order to find underlying metabolic problems in patients with a history of kidney stones, metabolic tests may be performed. Medication, dietary adjustments, and lifestyle changes may also be used as therapeutic measures to lower the risk of kidney stone development. Seeking advice from a healthcare professional is crucial in order to develop a customized management plan and assessment.

4. Discussion

This review describes several approaches to estimate CaOx in the context of urolithiasis, including the use of certain stone powders such as BegoStone and Ultracal 30, titrimetric techniques, nucleation assays, and aggregation assays.

In the ethylene glycol model, rats are given ethylene glycol in their drinking water to induce urolithiasis, which results in the formation of CaOx crystals and kidney stones [28]. Changes in the amount of ethylene glycol and the length of exposure highlight how crucial it is to customize the study goals. CaCl₂ and ammonium oxalate models emphasize precision and consistency through the use of important components such as solution preparation and administration methods. Knockout and overexpression of URAT1, AGXT, TRPV5, and CLDN14 genes provide information about the proteins and processes involved in kidney stone formation [41,42]. Every genetic alteration has unique effects; for example, TRPV5 knockout may cause the development of CaOx stones [44]. By providing possible therapeutic targets, these genetic models contribute to the study of the genetic and metabolic processes causing urolithiasis [46]. By surgically ligating the ureter to cause kidney stones to form, the ureteral ligation approach necessitates anesthesia, precise placement, and postoperative care. Struvite stone production is facilitated by infections, especially those that involve urease-producing bacteria, as these can lead to microbial involvement, urease activity, and inflammatory reactions [51]. Some forms of kidney stones are linked to metabolic abnormalities such as hyperoxaluria, hypocitraturia, dRTA, cystinuria, and hyperuricemia. It is essential to comprehend these metabolic anomalies in order to manage urolithiasis [54].

The majority of these models have focused on producing hyperoxaluria. It should be noted that the majority of human kidney stone patients do not suffer from hyperoxaluria; therefore, this modelling has limited applicability. Modified gene expression studies have mainly been performed in other animal models besides the rat [27]. While there are resources available for rat transgenic studies (Rat Genome database, http://rgd.mcw.edu), comparatively there are considerably fewer for this application, which represents a limitation in the future use of this animal model [52].

Similarly, other hyperoxaluric rat models can be produced by inbreeding hyperoxaluric progeny with the administration of sodium oxalate or glycolic acid. In addition, a number of other models of urolithiasis have also been developed such as transgenic mice with selective knockout of osteopontin, Tamm-Horsfall protein, oxalate, Na⁺-phosphate, or cysteine [36]. Despite the genomic advantages of the rat model, its overall accuracy and consistency in relation to human kidney stone disease remain controversial among researchers. However, the hyperoxaluric rat model of urolithiasis represents a well-established, relatively economical model. Even though kidneys of the rats and humans have inherent differences, rat kidneys are smaller, weighing around 0.75–1.2 g, measuring 1.6 cm×1 cm×0.9 cm (unipapillary), while human kidneys weigh approximately 170 g, measuring 11 cm×6 cm×2 cm (multi-papillary); however, despite these gross differences, the cortex-medulla ratio (2:1) is similar in both species, and scientists have been utilizing these models of hyperoxaluric rats for decades [63].

In summary, the variety of models included in the text illustrates the intricacy of the study of urolithiasis. In order to enhance our knowledge of kidney stone formation and how to manage it, researchers must carefully select and modify models to meet​ study objectives ​and comply with​ ethical guidelines.

5. Conclusion

The sequence of events that leads to the formation of human urinary stone disease remains unclear. As shown here, multiple animal models have been used in an effort to better understand the pathophysiologic chain of events that ultimately leads to the formation of a urinary calculus. To date, the endpoints for each of these animal models have been the development of crystals seen either in urine or with histologic examination of the renal papilla. While crystals may be an early form of eventual aggregation and stone fragmentation, in clinical practice crystalluria does not necessarily equate to future stone formation. As such, future research is needed to determine whether the process of crystallization is independent of stone formation. The utility of animal models is required to understand the temporal development of urinary stone formation. Ultimately, it would be ideal if the urology and nephrology communities would agree on a few animal models, so that results would be complementary and comparable.

Author contributions

Study concept and design: Ranjith R. Kumar, Prakash.

Data acquisition: Manjunatha P. Mudugal, Suresh Janadri.

Data analysis: Uday R. Sharma, Archana Bandepalli Gangireddy, Surendra Vada.

Drafting of manuscript: Ranjith R. Kumar, Hari T. Babu.

Critical revision of the manuscript: Suresh Janadri.

Conflicts of interest

The authors declare no conflict of interest.