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Pyrophosphate, a ubiquitous small-molecule inhibitor of mineralization abundantly present in the extracellular environment, binds to calcium and mineral surfaces to inhibit crystal growth. O'Neill and colleagues show in uremic rats that systemic administration of pyrophosphate prevents or reduces uremia-related vascular calcification, without overt negative consequences for bone and without calcium pyrophosphate deposition disease. These findings prompt further research into the potential of pyrophosphate as treatment for vascular calcification in chronic kidney disease patients. [ABSTRACT FROM AUTHOR]

Background: Increased bone loss has been associated with the development of vascular calcification in patients with chronic renal failure (CRF). In this study, the effect of impaired bone metabolism on aortic calcifications was investigated in uremic rats with or without ovariectomy., Methods: CRF was induced by administration of a 0.75% adenine/2.5% protein diet for 4 weeks. In one group, osteoporosis was induced by ovariectomy (CRF-OVX), while the other group underwent a sham-operation instead (CRF). A third group consisted of ovariectomized rats with normal renal function (OVX). At regular time intervals throughout the study, bone status and aortic calcifications were evaluated by in vivo micro-CT. At sacrifice after 6 weeks of CRF, bone histomorphometry was performed and vascular calcification was assessed by bulk calcium analysis and Von Kossa staining., Results: Renal function was significantly impaired in the CRF-OVX and CRF groups. Trabecular bone loss was seen in all groups. In the CRF-OVX and CRF groups, trabecular bone density was restored after adenine withdrawal, which coincided with cortical bone loss and the development of medial calcifications in the aorta. No significant differences with regard to the degree of aortic calcifications were seen between the two CRF groups. Neither cortical bone loss nor calcifications were seen in the OVX group. Cortical bone loss significantly correlated with the severity of vascular calcification in the CRF-OVX and CRF groups, but no associations with trabecular bone changes were found., Conclusions: Cortical rather than trabecular bone loss is associated with the process of calcification in rats with adenine- induced CRF., (Copyright © 2011 S. Karger AG, Basel.)

In the present paper, vascular calcifications due to chronic renal failure in rats are studied by X-ray microtomography (micro-CT). Although micro-CT is traditionally used as an imaging technique, a quantitative analysis of data obtained by in vivo and ex vivo micro-CT is described and discussed. By comparison with traditional destructive methods, such as histomorphometry and atomic absorption, the detection limits for calcium were determined in living rats and in extracted aortas. micro-CT proved to be an effective non-invasive imaging technique allowing non-destructive quantification of ectopic calcifications.

Elevated serum phosphate levels as a consequence of chronic kidney disease (CKD) contribute to the increased cardiovascular risk observed in dialysis patients. Protein restriction and dialysis fail to adequately prevent hyperphosphatemia, and in general treatment with oral phosphate binding agents is necessary in patients with advanced CKD. Phosphate plays a pivotal role in the development of vascular calcification, one of the factors contributing to increased cardiovascular risk in CKD patients. Treatment of hyperphosphatemia with standard calcium-based phosphate binders and vitamin D compounds can induce hypercalcemic episodes, increase the Ca × PO(4) product and thus add to the risk of ectopic mineralization. In this review, recent clinical as well as experimental data on lanthanum carbonate, a novel, non-calcium, non-resin phosphate binding agent are summarized. Although lanthanum is a metal cation no aluminium-like toxicity is observed since the bioavailability of lanthanum is extremely low and its metabolism differs from that of aluminium. Clinical studies now document the absence of toxic effects of lanthanum for up to 6 years of follow-up. The effects of lanthanum on bone, vasculature and brain are discussed and put in perspective with lanthanum pharmacokinetics.

Accumulation of inorganic phosphate due to renal functional impairment contributes to the increased cardiovascular mortality observed in dialysis patients. Phosphate plays a causative role in the development of vascular calcification in renal failure; treatment with calcium-based phosphate binders and vitamin D can further increase the Ca x PO(4) product and add to the risk of ectopic mineralization. The new generation of calcium-free phosphate binders, sevelamer and lanthanum, can control hyperphosphatemia without adding to the patients calcium load. In this article, the metabolism of lanthanum carbonate and its effects in bone, liver and brain are discussed. Although lanthanum is a metal cation its effects are not comparable to those of aluminum. Indeed, in clinical studies no toxic effects of lanthanum have been reported after up to four years of follow-up. The bioavailability of lanthanum is extremely low. The effects observed in bone are due to phosphate depletion, with no signs of direct bone toxicity yet observed in rats or humans. The liver is the main route of excretion for lanthanum carbonate, which can be localized in the lysosomes of hepatocytes. No lanthanum could be detected in brain tissue.

Introduction: Beside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia-reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia-reperfusion. Therefore, a model of warm ischemia-reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage., Methods: Fifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1-4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of x500. Immunohistochemistry with antibody ED-1 (magnification x250) and antibody 1F4 (magnification x400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05., Results: Neutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion., Conclusions: After warm ischemia-reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion.

Inflammation has been established to contribute substantially to the pathogenesis of ischemia/reperfusion (I/R) with a central role for particular cells, adhesion molecules, and cytokines. Until recently, most of the research trying to unravel the pathogenesis of I/R injury has been focused on the role of neutrophils. However, recent studies have brought evidence that T cells and macrophages are also important leukocyte mediators of renal and extrarenal (liver) I/R injury. In vivo depletion of CD4+ cells but not CD8+ cells in wild-type mice was protective in I/R of the kidney. A marked preservation of liver function was also found after I/R in T-cell deficient athymic mice. Blocking the b130/CD28 costimulatory pathway by CTLA-4 Ig (recombinant fusion protein) ameliorated renal dysfunction and decreased mononuclear cell infiltration in I/R of the kidney. b130-1 expression was found limited to the membrane of the endothelial cells of the ascending vasa recta, resulting in trapping of CD28-expressing CD4 T cells. This trapping of leukocytes results in the upstream congestion in the ascending arterial vasa recta, generating the since more than 150 years described medullary vascular congestion of the kidney soon after ischemic injury. It seems worthwhile to study a combination therapy using anti-inflammatory/anti-adhesion molecules in the early phase of I/R.

Background: Osteopontin (OPN) is a phosphoprotein that is up-regulated in several experimental models of renal disease, including ischemia/reperfusion injury. OPN has been described as a macrophage chemoattractant, may serve as a survival factor for tubular cells, and is implicated in the development of tubulointerstitial fibrosis. However, the precise role of this protein in renal pathophysiology remains unclear., Methods: OPN knockout and wild-type mice were subjected to 30 minutes of warm renal ischemia combined with a contralateral nephrectomy, and sacrificed at six different time points, ranging from 12 hours to seven days after reperfusion. Besides functional and morphological parameters of postischemic acute renal failure (ARF), macrophage infiltration, apoptosis and expression of collagen types I and IV were investigated., Results: Postischemic ARF in OPN knockouts and wild-types showed a similar course and severity, without significant differences in either functional or morphological disease parameters. However, macrophage infiltration was significantly diminished in OPN knockouts after five and seven days, in cortex as well as in the outer stripe of the outer medulla (OSOM). Furthermore, OPN knockout mice showed significantly enhanced apoptosis in the injury phase and significantly less collagen I and IV expression in the regeneration phase of postischemic ARF., Conclusions: There was no influence of OPN protein on the severity or course of functional impairment or morphological injury in the first seven days after an ischemic insult to the kidney. However, our results demonstrate that OPN favors macrophage recruitment to the postischemic kidney, inhibits apoptosis, and stimulates the development of renal fibrosis after an acute ischemic insult.

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.

In normal human and rat kidneys, osteopontin (OPN) is present at the apical surface of cells in the distal nephron. After ischemic or toxic renal damage in rats, OPN is upregulated in distal tubular cells (DTC) and expressed de novo in perinuclear vesicles in proximal tubular cells (PTC). In the first phase of this study, OPN localization in ischemic human biopsies was compared with that in ischemic rat kidneys. In the second phase, cultures of PTC and DTC were used to investigate human renal OPN synthesis, secretion, and localization. OPN localization in human biopsies after renal ischemia was comparable to that in ischemic rat kidneys. Microscopic and flow cytometric detection of immunofluorescent OPN staining in tubular cell cultures demonstrated strong plasma membrane localization in DTC, whereas mainly perinuclear intracellular expression was observed in PTC. Northern blotting and reverse transcription-PCR demonstrated production of a single OPN mRNA in PTC and DTC. Detection of OPN by Western blotting and enzyme-linked immunosorbent assay demonstrated that PTC and DTC synthesized and secreted the same three molecular mass OPN forms, in comparable amounts. Finally, confocal microscopy demonstrated different staining patterns for endocytotic/lysosomal vesicles and perinuclear OPN; however, perinuclear OPN exhibited colocalization with the Golgi apparatus. In conclusion, human renal OPN localization in cell cultures demonstrated differences between PTC and DTC comparable to those observed after renal ischemia in vivo. Therefore, these cell cultures represented an excellent model for the study of human OPN synthesis, secretion, and localization in PTC versus DTC. It is reported for the first time that intracellular OPN is located in the Golgi apparatus of both PTC and DTC and that PTC and DTC are able to produce and secrete the same OPN isoforms, in comparable amounts.

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.

Background: Osteopontin (OPN) is a highly acidic phosphoprotein containing an arginine-glycine-aspartic acid (RGD) cell adhesion motif. High OPN expression has been found in tissues with high cell turnover, and OPN up-regulation has been demonstrated in several models of renal injury, suggesting a possible role in tissue remodeling and repair. However, its exact function in the kidney remains unknown. In this study, the possible contribution of OPN to regeneration and repair in the kidney was explored by studying the time course and subcellular localization of OPN up-regulation after renal ischemia/reperfusion injury in different nephron segments and by investigating its relationship with tubular morphology., Methods: Rats that underwent 60 minutes of left renal ischemia and a right nephrectomy sacrificed at 10 different time points (from 1 hr to 10 days after reperfusion) were compared with uninephrectomized rats at each time point. In renal tissue sections immunostained for OPN, proximal (PTs) and distal tubules (DTs) in both the renal cortex and outer stripe of the outer medulla (OSOM) were scored for the degree of OPN expression and tubular morphology., Results: Kidneys of uninephrectomized rats showed no injury, and the localization and intensity of their OPN expression remained unaltered compared with normal rats. After ischemia/reperfusion, morphological damage was most severe in PTs of the OSOM, but all examined nephron segments showed a significant increase in OPN expression. The time course of OPN up-regulation was different in PTs and DTs. DTs in both cortex and OSOM rapidly increased their OPN expression, with a maximum at 24 hours after reperfusion followed by a slow decrease. In contrast, PTs showed a delayed increase in OPN staining, with a maximum after five to seven days, higher in the OSOM than in the cortex. In OSOM PTs, OPN expression was predominantly associated with morphological regeneration, whereas DTs showed a substantial OPN up-regulation without major morphological damage. PTs and DTs displayed a different subcellular OPN staining pattern: OPN staining in DTs was located to the apical side of the cell; PTs, however, presented a vesicular, perinuclear staining pattern., Conclusions: Our study found a different pattern of OPN up-regulation after renal ischemia/reperfusion in PTs versus DTs, both with regard to time course and subcellular localization. DTs show an early and persistent increase in OPN staining in the absence of major morphological injury, whereas OPN staining in PTs is delayed and is mostly associated with morphological regeneration. PTs show a vesicular, perinuclear OPN staining pattern, whereas DTs show OPN staining at the apical cell side.