Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.

BACKGROUND: The interaction between renal tubular cells and crystals generated in the tubular fluid could play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. Crystals are expected to form in the renal collecting ducts, but not in the proximal tubule. In the present investigation, we studied the damaging effect of calcium oxalate crystals on renal proximal and collecting tubule cells in culture. METHODS: Studies were performed with the renal proximal tubular cell lines, porcine proximal tubular cells (LLC-PK(1)) and Madin-Darby canine kidney II (MDCK-II) and the renal collecting duct cell lines, RCCD(1) and MDCK-I. Confluent monolayers cultured on permeable growth substrates in a two-compartment culture system were apically exposed to calcium oxalate monohydrate crystals, after which several cellular responses were studied, including monolayer morphology (confocal microscopy), transepithelial electrical resistances (TER), prostaglandin E(2) (PGE(2)) secretion, DNA synthesis ([(3)H]-thymidine), total cell numbers, reactive oxygen species [hydrogen peroxide (H(2)O(2))] generation, apoptotic (annexin V and DNA fragmentation), and necrotic (propidium iodide influx) cell death. RESULTS: Crystals were rapidly taken up by proximal tubular cells and induced a biphasic response. Within 24 hours approximately half of the cell-associated crystals were released back into the apical fluid (early response). Over the next 2 weeks half of the remaining internalized crystals were eliminated (late response). The early response was characterized by morphologic disorder, increased synthesis of PGE(2), H(2)O(2), and DNA and the release of crystal-containing cells from the monolayers. These released cells appeared to be necrotic, but not apoptotic cells. Scrape-injured monolayers generated even higher levels of H(2)O(2) than those generated in response to crystals. During the late response, crystals were gradually removed from the monolayers without inflammation-mediated cell death. Crystals did not bind to, were not taken up by, and did not cause marked responses in collecting tubule cells. CONCLUSION: This study shows that calcium oxalate crystals cause acute inflammation-mediated necrotic cell death in renal proximal tubular cells, but not in collecting tubule cells. The crystal-induced generation of reactive oxygen species by renal tubular cells is a general response to tissue damage and the increased levels of DNA synthesis seem to reflect regeneration rather than growth stimulation. As long as the renal collecting ducts are not obstructed with crystals, these results do not support an important role for crystal-induced tissue injury in the pathophysiology of calcium oxalate nephrolithiasis.

Oxalate is toxic to renal tubular cells only at supraphysiologic concentrations.

BACKGROUND: Oxalate-induced tissue damage may play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. The concentration of oxalate is higher in the renal collecting ducts ( approximately 0.1 to 0.5 mmol/L) than in the proximal tubule ( approximately 0.002 to 0.1 mmol/L). In the present investigation, we studied the damaging effect of oxalate to renal proximal and collecting tubule cells in culture. METHODS: Studies were performed with the renal proximal tubular cell lines, LLC-PK1 and Madin Darby canine kidney II (MDCK-II), and the renal collecting duct cell lines, rat renal cortical collecting duct (RCCD1) and MDCK-I. Confluent monolayers cultured on permeable growth substrates in a two-compartment culture system were apically exposed for 24 hours to relatively low (0.2, 0.5, and 1.0 mmol/L) and high (5 and 10 mmol/L) oxalate concentrations, after which several cellular responses were studied, including monolayer morphology (confocal microscopy), transepithelial electrical resistances (TER), prostaglandin E(2) (PGE(2)) secretion, lactate dehydrogenase (LDH) release, DNA synthesis ([(3)H]-thymidine incorporation), total cell numbers, reactive oxygen species (H(2)O(2)) generation, apoptotic (annexin V and DNA fragmentation), and necrotic (propidium iodide influx) cell death. RESULTS: Visible morphologic alterations were observed only at high oxalate concentrations. TER was concentration-dependently decreased by high, but not by low, oxalate. Elevated levels of PGE(2), LDH, and H(2)O(2) were measured in both cell types after exposure to high, but not to low oxalate. Exposure to high oxalate resulted in elevated levels of DNA synthesis with decreasing total cell numbers. High, but not low, oxalate induced necrotic cell death without signs of programmed cell death. CONCLUSION: This study shows that oxalate is toxic to renal tubular cells, but only at supraphysiologic concentrations.

Pericellular matrix formation by renal tubule epithelial cells in relation to crystal binding.

BACKGROUND/AIM: Retention of crystals in the kidney ultimately leads to renal stone formation. Hyaluronan (HA) has been identified as binding molecule for calcium oxalate monohydrate crystals. The association of high molecular mass (M(r)) HA with cell surface receptors such as CD44 gives rise to pericellular matrix (PCM) formation by many eukaryotic cells in culture. Here, we study the ability of several renal tubular cell lines to assemble PCMs and to synthesize high-M(r) HA during proliferation in relation to crystal retention. METHODS: PCM assembly by MDCK-I, MDCK-II, and LLC-PK1 cells was visualized by particle exclusion assay. Metabolic labeling studies were performed to estimate the cellular production of HA. The expression of CD44 and HA was studied using fluorescent probes, and crystal binding was quantified with radiolabeled calcium oxalate monohydrate. RESULTS: PCMs were formed, and HA was expressed by most MDCK-I and some MDCK-II, but not by LLC-PK1 cells. All cell types expressed CD44 at their apical surface. MDCK-I and MDCK-II cells secreted, respectively, 14.7 +/- 1.6 and 0.5 +/- 0.2 pmol [3H]glucosamine incorporated in high-M(r) HA, whereas LLC-PK1 cells did not secrete HA. Streptomyces hyaluronidase treatment significantly decreased crystal binding (microg/cm2) to MDCK-I cells (from 8.6 +/- 0.4 to 3.9 +/- 0.9), but hardly to MDCK-II cells (from 10.2 +/- 0.2 to 9.6 +/- 0.1) or LLC-PK1 cells (from 10.2 +/- 0.8 to 9.9 +/- 0.3). CONCLUSIONS: There are various forms of crystal binding to renal tubular cells in culture. Crystal attachment to MDCK-I and some MDCK-II cells involves PCM assembly that requires high-M(r) HA synthesis. HA production and PCM formation do not play a role in crystal binding to LLC-PK1 and the majority of MDCK-II cells. It remains to be determined which form of binding is involved in renal stone disease.

Internalization of calcium oxalate crystals by renal tubular cells: a nephron segment-specific process?

BACKGROUND: Crystal retention in the kidney is caused by the interaction between crystals and the cells lining the renal tubules. These interactions involve crystal attachment, followed by internalization or not. Here, we studied the ability of various renal tubular cell lines to internalize calcium oxalate monohydrate (COM) crystals. METHODS: Crystal-cell interactions are studied by light-, electron-, and confocal microscopy with cells resembling the renal proximal tubule [porcine kidney (LLC-PK1)], proximal/distal tubule [Madin-Darby canine kidney II (MDCK-II)], and distal tubule and/or collecting ducts [(Madin-Darby canine kidney I (MDCK-I), rat cortical collecting duct 1 (RCCD1)]. Crystal-binding strength and internalization are characterized and quantified with radiolabeled COM. RESULTS: Microscopy studies showed that crystals were firmly embedded in the membranes of LLC-PK1 and MDCK-II cells to be subsequently internalized. On the other hand, crystals bound only loosely to MDCK-I and RCCD1 and were not taken up by these cells. Crystal uptake by LLC-PK1 and MDCK-II, expressed in microg/10(6) cells, is temperature-dependent and gradually increases from 0.88 and 0.15 in 30 minutes, respectively, to 4.70 and 3.85, respectively, after five hours, whereas these values never exceeded background levels in MDCK-I and RCCD1 cells. CONCLUSION: The adherence of COM crystals to renal cells with properties of the proximal tubule is inevitable and actively followed by their uptake, whereas crystals attached to cells resembling the distal tubule and/or collecting duct are not internalized. Since crystal formation usually occurs in segments beyond the renal proximal tubule, crystal uptake may be of less importance in the etiology of idiopathic calcium oxalate stone disease.

Crystal retention capacity of cells in the human nephron: involvement of CD44 and its ligands hyaluronic acid and osteopontin in the transition of a crystal binding- into a nonadherent epithelium.

Nephrolithiasis requires formation of crystals followed by their retention and accumulation in the kidney. Crystal retention can be caused by the association of crystals with the epithelial cells lining the renal tubules. The present study investigated the interaction between calcium oxalate monohydrate (COM) crystals and primary cultures of human proximal (PTC) and distal tubular/collecting duct cells (DTC). Both PTC and DTC were susceptible to crystal binding during the first days post-seeding (4.9 +/- 0.8 micro g COM/cm2), but DTC lost this affinity when the cultures developed into confluent monolayers with functional tight junctions (0.05 +/- 0.02 micro g COM/cm2). Confocal microscopy demonstrated the expression of the transmembrane receptor protein CD44 and its ligands osteopontin (OPN) and hyaluronic acid (HA) at the apical membrane of proliferating tubular cells; at confluence, CD44 was expressed at the basolateral membrane and OPN and HA were no longer detectable. In addition, a particle exclusion technique revealed that proliferating cells were surrounded by HA-rich pericellular matrices or "cell coats" extending several microns from the cell surface. Disintegration of these coats with hyaluronidase significantly decreased the cell surface affinity for crystals. Furthermore, CD44, OPN, and HA were also expressed in vivo at the luminal side of tubular cells in damaged kidneys. These results suggest (1) that the intact distal tubular epithelium of the human kidney does not bind crystals, and (2) that crystal retention in the human kidney may depend on the expression of CD44-, OPN-, and-HA rich cell coats by damaged distal tubular epithelium.

Urinary crystallization inhibitors do not prevent crystal binding.

PURPOSE: Renal stone formation requires the persistent retention of crystals in the kidney. Calcium oxalate monohydrate (COM) crystal binding to Madin Darby canine kidney strain I (MDCK-I), a cell line that resembles the epithelium in the renal distal tubule/collecting duct, is developmentally regulated, while LLC-PK1 cells (American Type Tissue Collection), which are widely used as a model of the renal proximal tubule, bind crystals irrespective of their stage of epithelial development. Whereas to our knowledge the binding molecules for COM at the surface of LLC-PK1 cells are still unknown, crystals adhere to the hyaluronan (HA) rich pericellular matrix transiently expressed by mobile MDCK-I cells. In the current study we investigated whether crystal binding to either cell type is influenced by urinary substances, including glycoprotein inhibitors of crystallization MATERIALS AND METHODS: We studied crystal binding to MDCK-I cells during wound repair, to confluent LLC-PK1 cells and to HA immobilized on a solid surface using [14C] COM pretreated or not pretreated with urine from healthy male volunteers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed to assess whether the crystals became coated with urine derived proteins RESULTS: Western blot analysis demonstrated that pretreated COM crystals were covered with protein inhibitors of crystallization. However, this protein coat had no significant effect on the level of crystal binding to either cell type. In contrast, the adherence of urine treated crystals to immobilized HA was significantly reduced CONCLUSIONS: The adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide HA. Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may predominantly depend on the surface properties of the renal tubular epithelium.