Even though CaOx is the majority crystal in most kidney stones, almost all stones contain some CaP, mostly in the form of hydroxyapatite (HA). These observations are not surprising since urine is often supersaturated with HA (10
). HA crystals are frequently encountered in the urine and are a good substrate for the nucleation of CaOx. Stone formation however, does not only require crystal formation but also their retention within the kidneys (11
). There are two major theories about crystal retention. According to one theory, stone formation begins with deposition of apatite crystals in the basement membrane of the loops of Henle (1
). The apatite crystal deposits then grow through the renal interstitium to subepithelial location in the renal papillae. These papillary subepithelial deposits called Type 1 Randall’s plaques become the nidi for the development of CaOx stones. Because the initial event is in the interstitium as opposed to moving urine in the renal tubules, attachment is not a factor for crystal retention. An alternate theory of stone formation considers stone formation as an intratubular event requiring crystal attachment to the cell surfaces and some form of cell membrane alteration or injury for the retention of crystals (5
A variety of renal epithelial cells including LLC-PK1
, MDCK, HK-2 (human kidney), NRK (normal rat kidney) and RPTEC (human renal proximal tubular epithelial cells), when exposed to high levels of oxalate, or brushite or CaOx crystals, show signs of membrane damage, and release LDH and enzymes such as γ-glutamyl trasnspeptidase, and N-acetyl-β-glucoseaminidase (4
It has also been proposed that exposure of renal epithelial cells to higher than normal levels of calcium and oxalate can perturb the plasma membrane, causing lateral and trans-membrane migration of phospholipids, sequestering them in specific domains. Migration of the acidic phospholipids such as phosphatidylserine from the inner leaflet of the plasma membrane to the outside (14
) promotes adhesion of CaOx crystals to the epithelial cells. Crystal attachment to the inner medullary collecting duct cells has also been correlated with membrane fluidity (14
Recent studies have provided evidence that both Ox and CaOx crystals selectively activate p38 MAPK signal transduction pathways (15
). In addition p38 MAPK is essential for re-initiation of the induced DNA synthesis. Ox exposure also causes modest activation of JNK as determined by c-Jun phosphorylation. Apparently the renal epithelial response to oxalate involves signal transduction via MAP kinases, similar to the cellular responses to many other challenges. Cytosolic phospholipase A2
) is released upon the activation of MAP kinases and translocated to the cell membrane. cPLA2
preferentially hydrolyses arachidonoyl phospholipids generating a number of byproducts including arachidonic acid and lysophospolipids. Exposure of MDCK cells to oxalate produces a time and concentration dependent increase in cPLA2
). Inhibition of cPLA2
activity blocks the oxalate-induced upregulation of Egr-1, c-jun and c-myc genes.
A limited analysis of renal epithelial cell response to HA exposure has also been carried out. The HA has been shown to adhere to clumps of cells in primary cultures of rat renal papillary collecting tubule (RPTC) rather than the cells in the monolayer (14
). It was concluded that a sub-lethal injury caused a loss of tight junction integrity and separation of cells exposing binding sites on cell surfaces. Results also indicate that CaOx and apatite crystals share the same binding sites. Monkey kidney epithelial cells of non-transformed BSC-1 cell line endocytose the CaOx, apatite or brushite crystals (17
). The reaction of non-renal cell types to HA has also been investigated because the deposition of calcium pyrophosphate dihydrate (CPPD) and a variety of other calcium phosphates including hydroxyapatite, carbonate apatite, octacalcium phosphate and tricalcium phosphate collectively termed basic calcium phosphate (BCP), causes many diseases of the joints (18
). BCP and to some extent CPPD crystals induce mitogenesis, stimulate production of prostaglandin E2 (PGE2), activate phospholipase C, promote the synthesis of metalloproteinases (MMP’s) and induce proto-oncogenes c-fos and c-myc.
The results of the current study show that exposure to apatite crystals, the most common crystals found in the human urine, stones and the Randall’s plaques injures the renal epithelial cells in culture as demonstrated by the release of LDH into the medium from the NRK52E cells. There were signs of apoptotic cell death. On apical exposure cells endocytosed the crystals present on the surface. Thus the cellular response to HA crystals was similar to that of the COM crystals (6
), which have been shown to be endocytosed, induce apoptosis and cause cell injury. Exposure to HA on both the apical and basal surfaces resulted in significantly increased release of LDH. However the release was markedly higher when cells were exposed to the HA and COM crystals from the apical side than when exposed on the basal side. More LDH was released when exposed to a crystal concentration of 133μg/cm2
for longer duration of 6 hours.
Results also show the generation of free radicals during the interaction between HA crystals and epithelial cells as evidenced by the production of H2
. The amount of H2
produced was significantly higher when cells were exposed to higher amounts of HA or COM crystals for a longer duration. H2
has been shown to cause lipid peroxidation, DNA damage and in the end, cell death. 8-Isoprostane is produced by the random oxidation of tissue phospholipids by reactive oxygen species. Our data show that 8-IP was elevated in cells exposed to 67μg/cm2
HA or COM. With respect to the production of 8-IP, there were similar cell responses to both HA and COM crystals. However basal exposure appeared to be more effective than the apical exposure except in the case of 133μg/cm2
HA crystals when significantly less 8-1P was produced after basal exposure than after the apical exposure. Exposure to CaP crystals also resulted in the production of MCP-1 and PGE2, two important mediators of inflammation. In this respect too exposure to HA crystals was similar to the brushite and CaOx crystals. Reactive oxygen species are also shown to be involved in the CaOx and brushite crystal induced production of MCP-1. MCP-1 is a major mediator of monocyte/macrophage movement to the site of inflammation. CaOx crystal deposits in both the human and rat kidneys have been shown to be surrounded by the macrophages and giant cells (5
It is apparent that both apatite and CaOx crystals are injurious to renal epithelial cells, whether supplied from the apical or basal side. In vivo animal model studies as well as human studies have also shown CaOx to be injurious to the renal epithelium. Urinary excretion of many enzymes regarded as markers of renal tubular injury is increased by rats with CaOx nephrolithiasis (8
) as well as human CaOx stone formers (19
). In addition CaOx nephrolithiais in both rats and humans is associated with increased urinary excretion of lipid peroxides (8
). Administration of antioxidants to hyperoxaluric rats leads to a decrease in renal injury, the production of lipid peroxides and CaOx crystal deposition in the kidneys indicating the involvement of reactive oxygen species in the crystal induced renal injury.
Recent morphological studies of the papillae obtained from human kidney stone patients showed that human kidneys respond differently to different type of stones (2
). No morphological renal injury was detected in kidneys of idiopathic stone formers even though apatite crystals were present in their interstitium and stones were seen attached to the apatitic subepithelial Randall’s plaques. On the other hand, kidneys of brushite stone formers showed interstitial apatite deposits without any signs of cell injury while crystal filled medullary collecting ducts had extensive cell injury and interstitial fibrosis. The kidneys of intestinal bypass patients with calcium oxalate stones, on the other hand, showed only apatite crystals in their inner medullary collecting ducts and the ducts of Bellini. Tubules filled with apatite crystals showed extensive cell injury and death. Thus human renal data from stone formers indicate that Randall’s plaques in the form of interstitial apatite crystals with basal cell exposure are not noticeably injurious and inflammatory however injury and inflammation occurs when there are tubular deposits with apical cell exposure. What are the reasons for reported cellular injury in the presence of intratubular apatite crystals and the absence of injury and inflammation when apatite crystals are present as Randall’s plaque in the renal interstitium? We can only speculate at this time. Individual apatite crystals are extremely small and like all the other crystals produced in the biological systems are always coated with organic molecules. The presence of this organic coat around the crystals may interfere with the crystal cell interaction. Tubular crystals formed in the flowing urine may have a thinner coat which may allow a better cell crystal contact. In addition the intratubular crystals completely filled the inner medullary ducts and blocked the urinary movement, which may be partially responsible for the observed injury and inflammation. The crystals produced in the stationary milieu of the interstitium are likely to be slow growing and thus contain a thicker organic coating and a better barrier between the crystals and the cell membrane. We have recently shown that biological crystals obtained from the kidney stones are less injurious to the renal epithelial cells than the inorganic crystals (20
). Alternatively, the interstitial crystals may provoke the production of pro-inflammatory molecules by the renal epithelium without leading to migration of inflammatory cells, the histological marker of inflammation. Thus inflammation and injury associated with sub-epithelial apatite of Randall’s plaque may not be morphologically visible. Our results presented here and elsewhere clearly show that an exposure of renal epithelial cells to CaP as well as CaOx crystals leads to the production of inflammatory molecules. This apatite induced injury and inflammation may be involved in the eruption of subepithelial plaques to the papillary surface, establishing a substrate for the deposition of CaOx and formation of kidney stones.