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Kidney stone a very common and frequently occurring disease, is also called as nephrolith. Kidney stones are formed when crystals aggregate and get deposited in the renal tissue and subsequently grow in size. When they get attached in the nephron, it is termed as nephrolithiasis. Process of kidney stone formation is called as urolithiasis. The production components of kidney stones, are calcium phosphate, uric acid, and oxalic acid etc.
The development of the stones is related to increased excretion of stone forming components or decreased urine volume. 80% of the kidney stones contain calcium oxalate (CaOx) as the major mineral mixed mostly with calcium phosphate (CaP) and sometime uric acid. Stone formation include crystal nucleation, growth, aggregation and their retention in the kidneys. These processes are inflect by a variety of urinary macromolecules which become integrated in the growing crystals and eventually turn into stones. Calcium phosphate is usually supersaturated in the renal tubule and CaP crystallites also exist, but not all CaP crystallites can form kidney stones, usually, the CaP crystallites are carried away by the liquid in the renal tubule, without the possibility of growing into stones. Only those left in the renal tubule can form kidney stones.
NEPHROLITHIASIS
The formation of crystal clumps in the urinary tract leads to the formation of kidney stones,in clinical term is also called as nephrolithiasis. Kidney stones sometimes show no symptoms or sometimes associated with one or several of the following like flank pain, gross blockage of one or both kidneys, and urinary infections. The stones forming components are calcium, uric acid, oxalate , phosphates, or cystine. Occasionally, calcium salts and uric acid are present in the same stone. Some very rare types of kidney stones include xanthine and triamterene stones.
2.1.1 General Pathophysiology
Kidney stones are formed when crystals grow into stones.Crystals form that is present in urine is supersaturated with salts (sodium urate, calcium oxalate, and cystine). Supersaturation occur when the amount of a compound in solution increases the solubility; at that point, there is a process that begins to remove this excess by crystal formation. This can be changed in following ways i.e. by changing the concentration of compound which is involved for crystallization or by changing the solubility of the compound.
2.2 RENAL FUNCTION
The physiological function of the kidneys is to defecate endogenous (e.g. creatinine, ureum and oxalate) as well as exogenous (like drugs) waste products and managing body homeostasis. Human kidneys are composed of 1-2 million nephrons, and these are the functional units. The nephron is a specialized structure which involved in both dilution and even in concentration of primary urine. Blood flow in the kidneys is 1.2-1.4 l per minute. Each day, 190 litres of fluid are filtered and concentrated to form urine. In this process, nutrients and salts are preserved and waste products are eliminated. Each nephron consists of different segments that perform exact functions. The blood enters the nephron in the glomerulus, where ultrafiltrate is formed. Ultrafiltrate has the same content as blood, but without macromolecules larger than 30 kilodalton. From nephron urine flows through the proximal tubules and Loop of Henle then to the distal tubules. In the proximal tubules glucose , water and sodium chloride are resorbed and return to the blood stream, with essential nutrients such as amino acids, bicarbonate, calcium, protein, potassium & phosphate. Inside the Loop of Henle, the concentrating process of urine proceeds and salt- and acid-base balance of blood is regulated in the distal tubule the. The ultimate urine is formed in the collecting ducts, which drain the urine into the calyces from where it enters the ureter
CRYSTAL FORMATION
Urinary supersaturation refers to a state in which stone salts are soluble at much higher concentrations in urine than in water. This phenomenon can be explained that urinary glycoproteins, glycosaminoglycans, magnesium, and citrate form complexes with these salts so that solution can be kept at much higher concentrations. Already at the starting of the 20th century, it was known that “crystalloids” are soluble in water, but at higher concentrations, they are soluble in urine as a result of “protective colloids,” such as mucins, albumin, nucleic acids & chondroitin sulfate [2]. It was demonstrated that urine is capable of inhibiting cartilage calcification in tibiae of ricketic rats [3]. The concept was born that stone formers may produce abnormal urinary crystals inhibitors [3]. Recently, it was proposed that proteins that are merged into the organic stone matrix may support their dissolution inside cells [4]. Despite these studies, we still do not know whether one of these substances is important in stone disease prevention or normal individuals whether excrete more or superior macromolecules [5]. As crystal formation in the kidney is common, for individuals crystals may be a problem who are predisposed to retain them in the kidney.
2.4 DIFFERENT TYPES OF CRYSTALS FORMING RENAL STONE
Kidney stones comprises of a variety of crystals such as calcium phosphate , calcium oxalate , uric acid etc. Many stones have a well – defined nucleus , with a granular and non – stratified appearance. Calcium phosphate crystals appear most frequently in both urine and stones i.e. 15-20% urinary stones.
2.4.1 Calcium Phospahte crystal
CaP crystals usually have a smooth surface, are hard and grow to a large size and can damage the kidney. When CaP becomes the main constituent i.e. more than 50% of stones, the stones are called CaP stones.
The pathogenesis of renal stone of calcium phosphate origin represents nidus formation and the subsequent development of the nidus into a stone. The nidus may form immediately by precipitation from supersaturated urine. A state of supersaturation of urine in terms of calcium and phosphate ions is essential for the development of a renal stone of calcium phosphate composition. A recent study has reported that the stone containing CaP occurrence is increasing over time [ 6, 7].
Idiopathic CaOx stone formers and CaP stone formers both are hypercalciuric, but CaP stone formers produce urine of higher pH, which favors CaP crystallization. Abnormal urine pH and calcium excretion rate are predominant findings in stone formers which play a important role in the pathogenesis of stone formation. Urine pH rises progressively with increasing CaP percentage in stones
Uric acid crystals mostly form in acidic urine, typically with a urine pH < 5.5. And these crystals can vary in both size and shape. Dietary factors are the major cause for formation of uric acid stones especially intake of high animal protein. Uric acid is a weak acid with pKa of 5.35 in urine. In acidic urine the undissociated form of uric acid predominates and is less soluble , leading to crystalluria and stone formation [13, 18].
Cystine crystals
Cystine crystals are monomorphic, colorless plates which look much similar to benzene rings. And are larger and recur more frequently more likely to cause chronic kidney disease.The formation of cystine stones is a consequence of increased levels of L-cystine in the urine because of flawed reabsorption of filtered cystine. This condition is result of an autosomal recessive disorder caused by mutations in one of the two genes, one on chromosome 2 and another one on chromosome 19, which code for components of the major proximal renal tubule cystine and dibasic amino acid transporter. This condition is exacerbated by the low solubility of cystine which favors formation of crystals that aggregate into stones. They are mostly found in acidic urine, typically with a urine pH < 6.0.
Calcium oxalate crystal
Calcium oxalate crystals are mostly found in acidic urine, occur as either bihydrated or monohydrated calcium oxalate. CaOx bihydrate crystals appear as colorless bipyramids of various sizes. Whereas calcium oxalate monohydrate crystals are colorless and are of several shapes, including ovoids, biconcave disks, rods and dumbbells. Approximately 80% of all stones contain calcium oxalate. CaOx stones can occur in both the bladder and kidney. They can be seen in individuals with high dietary oxalate intake, in patients with nephrolithiasis, and in patients with acute renal failure due to ethylene glycol ingestion.
Calcium carbonate crystals
Calcium crystals are of different size and often appear as large spheroids with radial fibers. They can also be seen as smaller crystals with round and sometimes ovoid shapes. They are colorless to yellow-brown and provide a brownish colour to the urine, when they occur in high concentration. They are usually large crystals and can be observed at low magnification (however, confirmation of crystal identity should always be observed under high magnification). These crystals are very common and seen in the urine of rabbits, horses, guinea pigs and goats.
Amorphous crystals
Amorphous crystals basically appear as aggregates of finely granular material but without any defining shape at the low microscopic level. They are comprised of urates, phosphates or xanthine. And are usually small crystals can be observed at high magnification (unless there are large amounts of them), i.e. they mimic bacteria.
Amorphous urates (Na, K, Mg, or Ca salts) are formed in acidic urine and have a yellow or yellow-brown colour with rhomboid shape. Xanthine crystals are usually present in the form of “amorphous” crystals. These crystals generally occur in Dalmations on allopurinol therapy for urate urolithiasis.. Small amorphous crystals sometimes can be confused with bacterial cocci, due to their shape, but can be easily distinguished by technique gram-staining. Degenerating crystals or cells can also resemble “amorphous” crystals.
Ammonium biurate crystals
Ammonium urate crystals appear as brown or yellow-brown spherical bodies with irregular protrusions. but in some urine samples, it was seen that they do not have irregular protrusions but have smooth borders and can resemble same as calcium carbonate. Crystals can be observed under low magnification, when seen in large number, however low numbers can seen at higher magnification only. Due to their potential pathologic importance, crystal identification should be verified by observation at high magnification. They are present in urine of any pH, & their formation is favored from neutral to alkaline urine. They are generally seen with amorphous urates. These crystals are more common in dogs and cats with congenital or acquired portal vascular anomalies, with or without concomitant ammonium urate uroliths.
Bilirubin crystals
Bilirubin crystals are formed from conjugated bilirubin (water soluble) and can as be seen as needle-like to granular crystals that are yellow in color. They most likely tend to precipitate onto other formed elements in the urine. Sometimes it was observed appearance of bilirubin crystals in cylindrical shape and is formed in association with droplets of fat, resulting in a flashlight appearance. They are usually small size crystals that are only observed at high magnification, if there are large aggregates of crystals it can be observed at low magnification. Bilirubin crystals are seen commonly in canine urine, especially those in highly concentrated models. They are less common in urine of other species. Bilirubin crystals (or a positive chemical reaction on the urine dipstick) in feline, bovine, equine, urine is an abnormal finding and the animal should be investigated for an underlying cholestatic process.
2.5 STONE FORMATION
Urinary supersaturations along with the formation of crystalline particles are responsible for the renal stone formation. Supersaturation is the major factor for crystallization in solutions like urine. The term supersaturation refers to a solution which contains more amount of the dissolved material that could be dissolved by the solvent under normal circumstances. The level of supersaturation of a salt is expressed as the ratio between the actual ion-activity product (AP salt) & the solubility product (SP salt). The ion-activity of a salt is calculated from the free ion concentrations and the activity coefficients equivalent to the charge of the ions in the salt. The conc. at which saturation is reached and crystallization starts and is called as thermodynamic solubility product (Ksp). Urine contains inhibitors of crystallization and can hold large concentration of solute above the Ksp, a metastable state. If the concentration of solute increases and reaches appoint where it cannot be further held in the solution, this concentration is termed as Kp, which is the point of formation of product in urine
2.5.1 Crystal Nucleation
Crystal nucleation is the formation of a solid crystal phase in a solution. It is one of the important step in the formation of kidney stones This process starts with the merger of stone salts in solution into baggy clusters that may increase in size when new components are added [24]. Nuclei form the first crystals but do not dissolve and show characteristic pattern (lattice structure). There are two types-
1. Homogenous nucleation – In this process, nuclei is formed in a pure solution.
2. Heterogenous nucleation – In this process, nuclei usually form on the existing surfaces.
In urine, heterogenous nucleation occur because stone diseases require adherence of crystal on the renal epithelial cell [25, 30].
In secondary nucleation, new crystals deposit on pre-existing crystal surfaces similar type & results in the mass production of crystals. Nucleation sites in urine can be epithelial cells, urinary casts, red blood cells, and other crystals. Crystal aggregation and attachment of crystals or aggregates to renal epithelial cells are very critical processes involved in stone formation.
Crystallisation represents the first phase of urinary stone formation. Stones formation is due to phase change in which dissolved salts condense into solids and this transformation is influenced by supersaturation [31]. If
• Supersaturation is less than 1, crystals of substance will dissolve but crystals can be formed and grow.
• Supersaturation is greater than 1 and urine supersaturation is greater than 1 is meta stable and excess dissolved substance will precipitate.
2.5.2 Crystal Growth
The driving force for crystallization is the potential energy of the atoms or molecules which gets reduced, when they form bonds with each other. The process starts with the nucleation stage. Some atoms or molecules in supersaturated urine starts forming clusters such that the bulk free energy of the cluster is less than that of the liquid. Crystal growth is determined by the physical properties of the material, the molecular shape and size of the molecule, pH , supersaturation levels, & defects that may form in the crystals structure.
When a crystal nucleus reaches a critical size and the relative supersaturation remains above 1, than by adding new crystal components to the nucleus, the overall free energy is decreased. This process is called crystal growth.
2.5.3 Crystal Aggregation
The process when crystals in solution joined together to form larger particles is called aggregation. The crystal aggregation is one of the most important process in stone formation. Aggregation of particles in a solution is determined by a sense of balance of forces, some with aggregating effects and some with disaggregating effects. A small interparticle distance increases the attractive force and promotes particle aggregation. Additionally, for stabilizing aggregate solid bridges formed by crystalline material connecting two particles. In different steps of stone formation, crystal aggregation is one of the most important step than nucleation and growth because aggregation occurs within seconds. It is a commonly belief that the process of calcium stone formation begins as the precipitation of CaP takes place inside the kidney [32, 33, 34].
In normal condition, repulsion occurs between the CaP crystals and tubular cells and it may result in elimination of small CaP crystal by dissolution or spontaneous passage in urine. Primary nucleation of CaOx crystal is induced by CaP. This event may cause formation of masses of crystals by growth and aggregation followed further by adherence of CaP crystals aggregates to the tubular surface. Newly formed crystals adhere to the tubular epithelial cell surface and the cellular responses that follow could results in crystal retention.
2.5.4 Crystal Retention
Kidney stone formation requires formation of crystals followed by their retention and accumulation in the kidney. It can be caused by the relationship of crystals with the epithelial cells lining the renal tubules. Crystal formation largely depends on the composition of the tubular fluid and it might also depend on the composition of the renal tubular epithelial cell surface. A non adherent surface of the urethra, distal tubules, bladder, collecting ducts, and the ureter may provide a natural defence mechanism against crystal retention, and may become defective when the anti-adherence properties are compromised [35,36].
RENAL CELL INJURY
In recent year injury of renal epithelial cells are induced by free radicals, which can promote the adhesion of urinary crystallites to cells, thus it become one of the research focuses [29, 30]. Uric acid, oxalic acid and phosphoric acid are the production component of kidney stones and all of these requires various oxidative process.
During kidney stone formation concentration of these components increases, along with enhanced oxidation, results in the production of many free radicals , like oxygen free radicals and hydroxy radicals (-OH).
Under normal condition, there are abundant antioxidants in renal tissue, like superoxide dismutase, catalase. Actually, these enzymes are effective free radical scavengers. But, under pathological condition ( after cells have been injured), the free radicals produced in-vivo have outstrip the scavenging capacity of the cells. Therefore, excessive free radicals injure renal cells, that result in the oxidation of unsaturated fatty acid in the cell membrane, and thus changes the, permeability, and fluidity of the cell membrane and leads to the dysfunction of the membrane, thus lead to the formation of stone.
At molecular level, there are series of changes of function and composition of the membrane when induced after the injury of renal epithelial cells caused by free radicals, such as the loss of membrane polarity, integrin translocation and phosphatidylserine valgus, resulting in the exposure of a large number of crystals to cells and crystal retention accordingly.
The cell debris falls off and enters the lumen after the renal epithelial cell is injured, including, disintegrated mitochondria, and some debris evacuate from the lysosomes phagocytosing cell debris to the extracellular space. The enormous amount of cell debris also provides a sympathetic condition for heterogeneous nucleation of crystals [31]. Calcium Phosphate (Cap) is the main component of kidney stones. Although Cap is usually supersaturated in the renal tubule and Cap crystallites also exist, not all the Cap crystallites can form kidney stones. Usually, the Cap crystallites are carried away by the liquid in renal tubule, without the possibility of growing into stones. And those left in the renal tubule can form kidney stones.
2.7 REACTIVE OXYGEN SPECIES (ROS)
ROS comprising free radicals, atoms or molecules with unpaired electrons, and their metabolites, are highly reactive and play a critical role as signaling molecules. But they can also produce chemical modifications of, and damage to proteins, lipids, carbohydrates and nucleotides [32, 33]. Major cellular ROS include superoxide anion (O2-•), nitric oxide radical (NO•), hydroxyl radical (OH•), and hydrogen peroxide (H2O2), which are generated by several pathways. O2-• anions are produced by NADPH oxidases, xanthine oxidase, lipooxigenase, cyclooxygenase, hemeoxygenase and as a byproduct of mitochondrial respiratory chain. Lipid radicals can also produce O2-•. NO• radicals are produced by the endothelial nitric oxide synthase (eNOS) mediated oxidation of L-arginine. In addition, eNOS can also produce O2-• rather than NO. The reaction between superoxide and nitric oxide can produce the highly reactive peroxynitrite ONOO-.
Cells are equipped with a number of scavenging systems to control ROS availability.These include superoxide dismutase (SOD) to eliminate O2-•, and glutathione (GSH) peroxidase (GPx) and catalase to detoxify H2O2. Superoxide has a short half-life and spontaneously converts to H2O2 which is long-lasting and far more reactive than superoxide ions. The reaction is noticeably enhanced by SOD. Moreover, in a more complex transition metal catalyzed reaction called metal catalyzed Haber-Weiss reaction, H2O2 yields an even more reactive hydroxyl radical, which is however, short lived and works at short range. Initially, superoxide anions donate single electrons to ferric ions resulting in molecular oxygen and ferrous ions. The Fenton Reaction between ferrous ions and H2O2, leads to the formation of OH•. H2O2 is subsequently metabolized to water via catalase or by glutathione peroxidase in the presence of reduced glutathione.
Under normal conditions the superoxide anions (O2-•), NO radicals (NO•) and their metabolites are generated by tightly controlled enzymes and serve as mediators in a variety of regulatory processes and signaling pathways including proliferation, activation or inactivation of regulatory biomolecules, and regulation of transcriptional activities. ROS regulate many calcium signals as well as such genes as c-fos, c-myc, and c-jun and transcription factor activation protein-1 (AP-1) and nuclear factor κB (NF-κB).
ROS and reactive nitrogen species (RNS) normally occur at steady state levels, generated when needed and then cleared by activities of various antioxidants and scavengers. But uncontrolled generation of the reactive oxygen or nitrogen species and/or a reduction in the endogenous antioxidant capacity creates oxidative stress (OS). Most cells respond to OS by boosting the levels of intracellular antioxidants such as glutathione. The oxidants can react with all the basic constituents of cells: lipids, carbohydrates, proteins and nucleic acids severely affecting their structure and function. Pathological changes may result from the damaging effects of ROS and from ROS-mediated changes in gene expression and signal transduction.
2.8 SOURCES OF ROS IN CAP NEPHROLITHIASIS
ROS are produced through the involvement of both mitochondria [34, 36-39] and NADPH oxidase [39-41]. NADPH oxidase is a major source of ROS in the kidneys [42, 43] , particularly in the presence of Angiotensin II [44]. NADPH oxidase consists of six subunits, the two transmembrane units, p22phox and gp91phox; and four cytosolic units, p47phox, p67phox, p40phox and the small GTPase rac1 or rac2 [45]. The two transmembrane units, gp91phox and p22phox and a flavin make cytochrome b558. The cytosolic units translocate to the membrane and assemble with the cytochrome to activate the enzyme.
ROS in response to oxalate and CaP crystals are in part produced with the involvement of NADPH oxidase through the activation of the rennin angiotensin system (RAS). Reduction of angiotensin production, by inhibiting the angiotensin converting enzyme as well as blocking the angiotensin receptor, increased renin expression, reduced osteopontin (OPN) expression, crystal deposition and ameliorated the associated inflammatory response [46-48]. NADPH oxidase inhibition by apocynin treatment reduced the production of ROS, urinary excretion of kidney injury molecule (KIM) and renal deposition of CaP crystals in hyperoxaluric rats [49]. Atrovastatin, which has been shown to reduce the expression of gp91phox and p22phox subunits of NADPH oxidase [50], also inhibited crystal deposition in rats with experimentally induced hyperoxaluria.
Mitochondria are generally the most common source of superoxide and H2O2 in most cells and tissues. Hyperoxaluria and CaP crystal deposition in rat kidneys causes mitochondrial damage. Treatment with taurine which has been shown to prevent oxidative injury of the mitochondria, reversed mitochondrial changes in the hyperoxaluric rat kidneys and decreased crystal deposition [51]. Selective probes, substrates and inhibitors show mitochondria to be significant site of CaP crystal induced superoxide production and glutathione depletion in both LLC-PK1 and MDCK cells [52]. Exposure of LLC-PK1 cells to oxalate significantly increased cellular ceramides, however, pretreatment with glutathione precursor N-acetylcysteine (NAC) blocked this increase. Isolated mitochondria responded to oxalate exposure by the accumulation of ROS, lipid peroxides and oxidized thiol proteins [53]. Citrate is also involved in maintaining endogenous antioxidant defenses. Administration of exogenous citrate to LLC-PK1 and MDCK cells bolstered these defenses and diminished the cellular injury inflicted by exposure to increased Ox and CaP crystals [54]. The presence of citrate in the culture medium was associated with a significant increase in GSH peroxidase and a drop in the production of H2O2 and 8-isoprostane (8-IP), which is an end product of lipid breakdown. There was a significant improvement in cell viability as demonstrated by decreased LDH release and increased trypan blue exclusion.
Mitochondrial damage is suggested to be induced by the opening of mitochondrial permeability transition pore (mPTP). mPTP opening depends upon the activation of cyclophilin D in the mitochondrial matrix by ROS produced by NADPH oxidase and is inhibited by cyclosporine A (CSA) [55]. CSA prevented the depolarization of mitochondrial membrane, decrease in SOD expression, increase in 4-hydroxy-2-nonenal (4HNE) and release of cytochrome-c into the cytosol in NR52E renal epithelial cells exposed to CaP monohydrate crystals in vitro. CSA treatment of hyperoxaluric rats resulted in reduced mitochondrial damage, OS and CaP crystal deposition in the kidneys.
2.9 RELATIONSHIP BETWEEN ROS AND CRYSTALLIZATION
ROS have many important regulatory roles, they normally occur at a steady state level, & are generated as required by the system, and are then removed by the activity of antioxidants and scavengers present in the system. They have very short life and have tightly controlled actions and are localized to sites it is needed. They do not travel too far because of their potential that can harm the cells. ROS can produce chemical modifications & damage to proteins, carbohydrates, lipids and nucleotides, and modulate renal and cardiovascular systems through redox dependent signaling pathways. Uncontrolled generation of reactive oxygen creates oxidative stress (OS) by boosting levels of intracellular antioxidants. Pathological changes may result from the damaging effects of ROS and from changes in gene expression and signal transduction.
Since most ROS are short lived & do not travel through a long distance, the presence of oxidative stress is generally recognized by the abundance of byproducts of ROS interaction with cell constituents.
2.10 CELL LINES INVOLVED IN STUDY OF KIDNEY STONE FORMATION
It is very difficult to study the kidney stone in situ, some models like animal model, the membrane model, and the cell model are used to explore the pathological mechanism of kidney stones. In cell model renal epithelial cell line of Madin-Darby canine kidney cell line (MDCK),Hampshire swine (LLC-PK) , the, the African green monkey renal epithelial cell line (Vero).
2.10.1 Vero cell line
Vero cells are derived from the kidney of a normal, adult green African monkey ( Cercophithecus aethiops) and are one of the commonly used mammalial cell lines. These are anchorage-dependent, having morphology similar to epithelial cells i.e. appear flattened and polygonal in shape [56- 58]. Under proper conditions, cells attach and spread onto the substrate and gradually grow out to a confluent monolayer. One of the most important advantage of choosing Vero cell lines lies in the fact that the ape is evolutionary close to humans.
2.11 INHIBITORS OF STONE FORMATION
Inhibitors of crystallization can force crystal-crystal interaction. These are compounds that can interfere with nucleation, growth, and retention of crystals, so diminishing the risk of actual stone formation. During urine small precipitated crystals can be harmlessly excreted with an ease. Urinary macromolecules, like glycosaminoglycans and proteins can affect the aggregation and growth of these crystals, acting as either inhibitors or promoters [58]. Among those are prothrombin fragment 1, inter--trypsin inhibitor, osteopontin, albumin, and 1-microglobulin. Small organic or inorganic compounds, like magnesium, citrate, & pyrophosphate also act as inhibitors.
2.12 CRYSTAL-CELL INTERACTION
The mechanisms of crystal-cell interaction is very complex process. Crystallization is caused by the condition of urinary supersaturation. The crystals that have formed bind to renal epithelial cells and are taken into them. The process of attachment of crystals to renal cells is crystal-cell interactions. These functional & structural studies of crystal-cell interactions in culture indicate that CaP crystals immediately adhere to microvilli on the cell surface and are later internalized. Khan et al. concluded that crystal-cell interaction is an one of the important element in the development of urinary stone disease [59]. Kohjimoto et al. reported that crystal-cell interactions is one of the earliest processes in the formation of kidney stones. Finlayson and Reid hypothesized that it was unlikely that Cap crystals could grow large enough to be retained within the renal tubules, and that attachment of crystals was necessary for stone formation initiation. Animal model and tissue culture studies have provided evidence for crystal retention within the kidneys via attachment to renal epithelial cells. Some urinary macromolecules have an inhibitory effect on Cap crystal attachment. Lieske et al. reported that diverse polyanionic molecules in urine, such as specific GAGs, glycoproteins, and citrate, block the binding of CaP crystals to the cell membrane.
RELATIONSHIP BETWEEN RENAL CELL INJURY AND CRYSTAL-CELL INTERACTION
Calcium phosphate & oxalate crystals are injurious to renal epithelial cells. Cultured renal epithelial cells exhibited evidence of damage after exposure to CaP crystals. Addition of Cap crystals to monolayers of Vero cells led to a marked increase in the release of malonaldehyde content [60]. In animal models of renal stone produced by the administration of high oxalate concentration, the presence of CaP crystals inside the renal cell is associated with renal cell damage, as evidenced by enzymuria and the presence of membranous debris within the epithelial lumina [61]. Animal model of stone disease, high concentrations of oxalate ion and CaP crystals the in proximal tubular fluid appear to be toxic for renal epithelial cells.
Many reports have suggested the involvement of renal epithelial cell injury in the crystal-cell interaction process.
Injury to the renal epithelial cells results in the production of membranous vesicles and cellular degradation. The crystals are passed as endocytosed by the epithelial cells to be refined by their lysosomal system or crystalluria particles. Deposition of CaP crystal in the kidneys upregulates the expression and synthesis of macromolecules that promote inflammation to membrane and ultimately leads to fibrosis. Hyperoxaluriais is a risk factor for CaP urolithiasis [62]. Hyperoxaluria promotes increased production of crystallization modulators, such a bikunin, Sosteopontin. These macromolecules are involved in controlling crystal nucleation, growth, and aggregation, but also crystal-cell interaction and crystal retention within the kidney.