Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys

Retention of crystals in the kidney is an essential early step in renal stone formation. Studies with renal tubular cells in culture indicate that hyaluronan (HA) and osteopontin (OPN) and their mutual cell surface receptor CD44 play an important role in calcium oxalate (CaOx) crystal binding during wound healing. This concept was investigated in vivo by treating rats for 1, 4, and 8 d with ethylene glycol (0.5 and 0.75%) in their drinking water to induce renal tubular cell damage and CaOx crystalluria. Tubular injury was morphologically scored on periodic acid-Schiff-stained renal tissue sections and tissue repair assessed by immunohistochemical staining for proliferating cell nuclear antigen. CaOx crystals were visualized in periodic acid-Schiff-stained sections by polarized light microscopy, and renal calcium deposits were quantified with von Kossa staining. HA was visualized with HA-binding protein and OPN and CD44 immunohistochemically with specific antibodies and quantified with an image analyzer system. Already after 1 d of treatment, both concentrations of ethylene glycol induced hyperoxaluria and CaOx crystalluria. At this point, there was neither tubular injury nor crystal retention in the kidney, and expression of HA, OPN, and CD44 was comparable to untreated controls. After 4 and 8 d of ethylene glycol, however, intratubular crystals were found adhered to injured/regenerating (proliferating cell nuclear antigen positive) tubular epithelial cells, expressing HA, OPN, and CD44 at their luminal membrane. In conclusion, the expression of HA, OPN, and CD44 by injured/regenerating tubular cells seems to play a role in retention of crystals in the rat kidney.     

Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis

BACKGROUND: Preterm neonates and renal transplant patients frequently develop nephrocalcinosis. Experimental studies revealed that crystal retention in the distal nephron, a process that may lead to nephrocalcinosis, is limited to proliferating/regenerating tubular cells expressing hyaluronan and osteopontin at their luminal surface. Fetal and transplant kidneys contain proliferating and/or regenerating cells since nephrogenesis is not completed until 36 weeks of gestation, while ischemia and nephrotoxic immunosuppressants may lead to injury and repair in renal transplants. This prompted us to investigate the expression of hyaluronan and osteopontin and to correlate this to the appearance of tubular calcifications both in fetal/preterm and transplanted kidneys. METHODS: Sections of fetal/preterm kidneys and protocol biopsies of transplanted kidneys (12 and 24 weeks posttransplantation from the same patients) were stained for osteopontin, hyaluronan, and calcifications (von Kossa). RESULTS: Hyaluronan and osteopontin were expressed at the luminal surface of the epithelial cells lining the distal tubules of all fetal kidneys at birth and in all kidney graft protocol biopsies 12 and 24 weeks posttransplantation. In 7 out of 18 surviving (at least 4 days) preterm neonates crystal retention developed. In renal allografts a striking increase (from 2/10 to 6/10) in tubular crystal retention between 12 and 24 weeks posttransplantation was observed. In addition, crystals were selectively retained in distal renal tubules containing cells with hyaluronan and osteopontin at their luminal surface. CONCLUSION: The results of this study show that luminal expression of hyaluronan and osteopontin preceded renal distal tubular retention of crystals in preterm neonates and renal transplant patients. We propose that the presence of this crystal binding phenotype may play a general role in renal calcification processes.     

透明质酸合成酶2在肾癌中的表达及临床意义

目的：研究透明质酸合成酶2在肾癌中的表达,并探讨其潜在的临床意义。方法：运用实时定量聚合酶链反应（qPCR）方法检测五种肾癌细胞（ACHN、Caki-1、OS-RC-2、786-O、SN12PM6）透明质酸合成酶三种亚型（HAs1、HAS2、HAs3）mRNA的表达,运用Westernblot方法进一步检测mRNA表达含量高的HAS亚型在五种肾癌细胞系及肾透明细胞癌（ccRCC）组织中的蛋白表达。结果：在五种肾癌细胞系中HAS2mRNA表达水平均明显高于正常肾小管上皮细胞（HK-2）,其中在。肾癌SN12PM6细胞系中表达水平最高（均P〈0．05）,HAS1mRNA的表达水平均明显低于正常肾小管上皮细胞（均P〉0．05）,而HAS3mRNA表达水平除肾癌Caki-1细胞系外均低于正常肾小管上皮细胞（P〉0．05）。在五种肾癌细胞系中HAS2蛋白均明显表达,而正常‘肾小管上皮细胞无明显表达。在肾透明细胞癌组织中HAs2蛋白表达也明显高于相应癌旁组织。结论：透明质酸合成酶2在多种肾癌细胞系和肾透明细胞癌组织中均明显表达,提示透明质酸合成酶2可能在肾癌尤其在肾透明细胞癌发生发展过程中起着至关重要的某种作用,并可能成为新的肾透明细胞癌分子标志物。     

Signaling through tumor necrosis receptor 2 induces stem cell marker in CD133+ regenerating tubular epithelial cells in acute cell‐mediated rejection of human renal allografts

Tumor necrosis factor receptor 2 (TNFR2) is strongly upregulated on renal tubular epithelial cells by acute cell‐mediated rejection (ACR. In human kidney organ culture, TNFR2 signaling both upregulates TNFR2 expression and promotes cell cycle entry of tubular epithelial cells. We find significantly more cells express CD133 mRNA and protein, a putative stem cell marker, in allograft biopsy samples with ACR compared to acute tubular injury without rejection or pretransplant “normal kidney” biopsy samples. Of CD133⁺ cells, ~85% are within injured tubules and ~15% are interstitial. Both populations express stem cell marker TRA‐1‐60 and TNFR2, but only tubular CD133⁺ cells express proximal tubular markers megalin and aquaporin‐1. TNFR2⁺CD133⁺ cells in tubules express proliferation marker phospho‐histone H3^S10 (pH3^S10). Tubular epithelial cells in normal kidney organ cultures respond to TNFR2 signaling by expressing CD133 mRNA and protein, stem cell marker TRA‐1‐60, and pH3^S10 within 3 hours of treatment. This rapid response time suggests that CD133⁺ cells in regenerating tubules of kidneys undergoing ACR represent proliferating tubular epithelial cells with TNFR2‐induced stem cell markers rather than expansion of resident stem cells. Infiltrating host mononuclear cells are a likely source of TNF as these changes are absent in acute tubular injury .     

Different type and localization of CD44 on surface membrane of regenerative renal tubular epithelial cells in vivo

BACKGROUND: CD44 is a transmembrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Previous studies demonstrated that CD44 was generally restricted to lateral-basal plasma membrane (PM) of epithelial cells, whether it localized on apical PM in vivo has not been clarified. METHODS: In this study, we used a gentamicin-induced acute tubular necrosis (ATN) and spontaneous recovery model in rats and two distinct antibodies, an anti-rat distal extracellular domain (OX49) of standard CD44 (CD44-OX49) and an anti-rat CD44 cytoplasmic tail (CD44CPT), to survey the localization of CD44-OX49 and CD44CPT on the PM in renal tubular epithelial cells in different recovery stages after ATN with immunohistochemistry and immunoelectron-microscopic examinations. RESULTS: CD44-OX49 was localized not only on the lateral-basal PM in tubular epithelial cells, but also on the apical surface membrane in PCNA-positive newly regenerative tubular epithelial cells in early recovery stages after ATN. However, CD44CPT was only localized on the lateral-basal PM. The immunoelectron-microscopic results showed that CD44-OX49 localization was changed from the apical to lateral to basal surface membrane in renal tubular epithelial cells during the recovery process after ATN, finally disappearing from basal PM when normal polarized epithelial cells formed. CONCLUSIONS: These results suggest that there were two types of CD44 including CD44 without a cytoplasmic tail localizing on the apical surface membrane related to newly regenerative epithelial cells, and CD44 with a cytoplasmic tail localizing on the lateral-basal PM related to establishment of tubular epithelial cell polarity after ATN in vivo.     

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.     

Osteopontin expression in acute renal allograft rejection

BACKGROUND: Osteopontin (OPN) is a potent chemoattractant for mononuclear cells that is up-regulated in various inflammatory states of the kidney. The role of OPN and its expression in human renal allograft rejection are unknown. METHODS: We examined by immunohistochemistry and in situ hybridization, renal biopsies from patients with acute rejection (N= 22), protocol biopsies without rejection (N= 9), and perioperative donor biopsies (N= 35) for intrarenal expression of OPN, and its correlation with clinical, laboratory, and histopathologic parameters. In the rejection biopsies, interstitial monocyte/macrophage infiltration, tubulointerstitial cell proliferation/regeneration and apoptosis were investigated. RESULTS: In the majority of rejection biopsies, OPN expression by proximal tubular epithelium was widespread, and tended to be enhanced in the tubules surrounded by numerous inflammatory cells. Conversely, in patients that did not experience episodes of rejection and in donor biopsies, OPN expression by proximal tubules was nil or weak. OPN mRNA was colocalized with its translated protein in the renal tubular epithelium. OPN expression positively correlated with the degree of interstitial inflammation (P < 0.05), CD68+ monocyte infiltration (P < 0.01), Ki-67+ regenerating tubular and interstitial cells (P < 0.05 and P < 0.005, respectively), but not with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive apoptotic tubular cells. CONCLUSION: These data suggest that inducible expression of OPN in the tubular epithelium may have a pathogenic role in acute renal allograft rejection by mediating interstitial monocyte infiltration and possibly tubular regeneration.     

Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells

Alteration in the glycosaminoglycan hyaluronan (HA) has been demonstrated in numerous renal diseases. We have demonstrated that renal proximal tubular epithelial cells (PTCs) surround themselves in vitro with HA in an organized pericellular matrix or 'coat', which is associated with cell migration, and also form pericellular HA cable-like structures which modulate PTC-mononuclear leukocytes interactions. The aim of this study was to characterize potential regulatory mechanism in the assembly of PTC-HA into pericellular cables. HA cables are generated by PTCs in the absence of serum. Immunohistochemical analysis demonstrates the incorporation of components of the inter-alpha-inhibitor (IalphaI) family of proteins and versican into HA cables. Addition of an antibody to IalphaI/PalphaI (pre-alpha-inhibitor) inhibits cable formation. In contrast, inhibition of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) has no effect on cable formation, suggesting that their generation is independent of the known heavy-chain transfer activity of TSG-6. Overexpression of HAS3 is associated with induction of HA cable formation, and also increased incorporation of HA into pericellular coats. Functionally, this resulted in enhanced HA-dependent monocyte binding and cell migration, respectively. Cell surface expression of CD44 and trypsin-released cell-associated HA were increased in HAS3-overexpressing cells. In addition, hyaluronidase (hyal1 and hyal2) and bikunin mRNA expression were increased, whereas PalphaI HC3 mRNA expression was unchanged in the transfected cells. The data demonstrate the importance of IalphaI/PalphaI in cable formation and suggest that expression of HAS3 may be critical for HA cable assembly.     

Expression of osteopontin in gentamicin-induced acute tubular necrosis and its recovery process

BACKGROUND: There is controversy regarding the exact localization and roles of osteopontin (OPN), a multipotential chemokine, in renal injury. There is little information on the expression and role of OPN in gentamicin-induced acute tubular necrosis (ATN) and its recovery process. METHODS: A severe ATN model was made using male Wistar rats by injecting gentamicin (150 mg/kg/day) for five days and limiting the provision of water. The expression and localization of OPN mRNA and protein, ED1 as a macrophage marker, proliferating cellular nuclear antigen (PCNA), CD44 as an OPN receptor, megalin as a proximal tubule marker, and their relationships to each other were examined from the early tubular necrotic period to the late recovery period by Northern blotting, in situ hybridization, and double immunohistochemical staining. RESULTS: In the gentamicin group, OPN mRNA and protein were expressed in only the PCNA-positive proliferating cortical distal tubules, not in the necrotic proximal tubules, until day 6 after the first administration, but were found markedly in PCNA-positive regenerative proximal and distal tubules on days 10, 15, and 30. The localization of PCNA-positive cells was almost always accompanied with the up-regulated expression of OPN using quantitative analysis (P < 0.01). CD44 expression was markedly up-regulated in the renal cortical tubular epithelium from days 6 to 30. In the control group, no expression of OPN and CD44 in the cortical area was found throughout the experimental period. CONCLUSIONS: These results suggested that OPN is related to the proliferation and regeneration of tubular epithelial cells after tubular damage.     

Molecular identification of canine podocalyxin-like protein 1 as a renal tubulogenic regulator

GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.     

Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells

Background. CD44 is the main receptor for the extracellular polysaccharide hyaluronan (HA). We have recently shown that CD44 is strongly induced on renal tubular epithelial cells (TEC) in autoimmune renal injury and that HA accumulates in the renal interstitium (Kidney Int 1996; 50: 156-163 and Nephrol Dial Transplant 1997; 12: 1344-1353). The functional significance of enhanced tubular CD44 expression and its interaction with HA are not known. The purpose of the present study was to characterize renal tubular CD44 expression and CD44-mediated HA binding in vitro and to investigate the growth modulating effects in response to HA binding by TEC. Methods. RT-PCR analysis, flow cytometry, confocal microscopy and Western blotting were used to examine cell surface and soluble CD44 expression by cultured TEC, using SV40-transformed mouse cortical tubular (MCT) cells. HA binding characteristics were examined by flow cytometry and effects of HA on TEC cell growth by [³H]thymidine incorporation. Results. By RT-PCR analysis MCT cells expressed predominantly the standard form of CD44 mRNA, whereas the expression of variant forms was very weak. Confocal microscopy showed that CD44 was expressed basolaterally and apically on MCT cells with strong staining on microvilli. Shedding of CD44 from MCT cells could be induced with crosslinking of anti-CD44 mAbs or with PMA stimulation. MCT cells constitutively bound HA and this binding could be modulated with anti-CD44 mAbs. Soluble and plate-bound HA markedly inhibited MCT cell growth. Conclusions. CD44 is a regulated HA receptor on MCT cells which can be shed into the cellular environment. Upon binding of HA, CD44 functions as a growth inhibitory cell surface protein in MCT cells. We speculate that the interaction of CD44 with HA may have important regulatory effects on cell proliferation in tubulointerstitial renal diseases.     

Hyaluronan and proximal tubular cell migration

BACKGROUND: The ubiquitous polysaccharide hyaluronan has been associated with both acute renal injury and progressive renal disease. The aim of this study was to examine the effect of hyaluronan on proximal tubular cell migration. METHODS: The proximal tubular cell line, HK-2 cells, were grown in monolayer culture, and cell migration following addition of hyaluronan characterized in an in vitro model of injury that we have previously developed and characterized. RESULTS: Addition of well-defined preparations of exogenous hyaluronan increased cell migration; however, optimum enhancement of migration was seen with hyaluronan of high molecular weight. Activation of the mitogen-activated protein kinase (MAPK) signaling cascade, as assessed by increased expression of the dually phosphorylated active form of MAPK, could be demonstrated following addition of hyaluronan. This was blocked by the addition of a specific antibody to the hyaluronan receptor, CD44. Hyaluronan-dependent enhanced migration was also abrogated by addition the CD44 blocking antibody, and by inhibition of MAPK kinase (MEK) activity. Generation of a denuded area also led to increased synthesis of endogenous hyaluronan and activation of MAPK, and blockage of either CD44 or MAPK activation inhibited proximal tubule cell (PTC) migration and re-epithelialization under nonstimulated conditions. CONCLUSION: We have demonstrated that hyaluronan activation of the MAPK pathway through binding to its receptor CD44, enhances proximal tubule cell (PTC) migration. In addition, the results suggest that mechanical injury of PTC stimulated hyaluronan generation. These observations may have implications for both recovery from acute tubular injury and progressive renal fibrosis.     

Proposed mechanisms in renal tubular crystal retention

The production of concentrated urine inevitably leads to the precipitation of poorly soluble waste salts in the renal tubular fluid. These crystallization processes are physiologic and without consequences as long as all crystals are excreted with the urine. The retention of crystals in the renal tubules, however, may lead to tubular nephrocalcinosis. Here, we present a brief survey of the possible mechanisms involved in this process, which seems to depend predominantly on the presence of regenerating/(re)differentiating cells in the renal tubules. Crystal binding to the surface of these cells can be mediated by a number of luminal membrane molecules, including acidic fragment of nucleolin-related protein, annexin-II, osteopontin, and hyaluronan.     

Spatial and temporal expression of CD44 isoforms in the developing and growing joints of the rat limb

Hyaluronan is an integral component of proteoglycan-rich extracellular matrices such as hyaline cartilage. Hyaluronan is commonly found in embryonic tissue and is important in the formation of hydrated matrices that allow cellular expansion and migration. Cell surface hyaluronan-binding proteins such as CD44 are presumed to be important in the cellular interactions with hyaluronan in both of these processes. The primary aim of this study was to document the spatial and temporal expressions of CD44 isoforms during the development and growth of the diarthrodial joints of rat limbs. With use of in situ hybridization and immunohistochemistry, the CD44s isoform is selectively identified as localized to a single cell layer on opposing sides of the joint at the first appearance of joint cavitation (on the 18th day of gestation). After joint formation in the neonate, the expression of the CD44s isoform in the cells at the joint surface is lost. These findings suggest that the CD44s isoform has a role in the development of the diarthrodial joint, presumably through interaction with hyaluronan.     

Renal osteopontin protein and mRNA upregulation during acute nephrotoxicity in the rat.

BACKGROUND: The effect of segment-specific proximal tubular injury on spatio-temporal osteopontin (OPN) distribution was determined in two different nephrotoxic rat models to evaluate its conceivability with a possible role for OPN in acute renal failure (ARF). OPN gene expression was further determined in proximal and distal tubular cells to investigate the origin of increased renal OPN. METHODS: Renal OPN protein and mRNA expression were compared in the rat during mercuric-chloride- vs gentamicin-induced ARF using immunohistochemistry and in situ hybridization. RESULTS: Mercuric chloride primarily induced tubular injury and subsequent cell proliferation in proximal straight tubules (PST), whereas gentamicin predominantly injured proximal convoluted tubules (PCT). In both models, the distribution of OPN protein was associated with increased OPN mRNA levels in proximal as well as distal tubular cells. However, upregulation was delayed in the proximal tubular segment suffering most from injury, i.e. PCT in gentamicin ARF vs PST in mercuric-chloride ARF. OPN immunostaining at the apical cell membrane from distal tubules was in contrast to perinuclear vesicular staining in proximal tubular cells. CONCLUSIONS: OPN gene and protein expression is induced in both proximal and distal tubular cells during rat toxic ARF. The distinct subcellular localization in proximal vs distal tubular cells indicates differences in OPN processing and/or handling. The spatio-temporal distribution is consistent with a possible role in renal injury and regeneration.     

肾小管上皮细胞程序性坏死对慢性肾脏病患者肾损伤的影响

目的:分析不同分期慢性肾脏病（chronic kidney disease,CKD）患者肾组织中发生程序性坏死的肾小管上皮细胞数量及其与临床病理指标的相关性,探讨肾小管上皮细胞程序性坏死在CKD患者肾小管上皮细胞过度死亡、慢性肾损伤进展中的作用。方法:收集2017年6月至2019年6月于海南医学院第一附属医院行肾活检且...