1The Renal Section, 4Renal Pathology Laboratory, Department of Pathology, Baylor College of Medicine, 3Department of Pathology, University of Texas Health Sciences Center, Houston, Texas 77030; and 2Kidney Laboratory, Austin Research Institute and Department of Nephrology, Austin Hospital, University of Melbourne, Australia 3084
Submitted 4 April 2003 ; accepted in final form 7 October 2003
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ABSTRACT |
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monocyte chemotactic protein-1; stromal cell-derived factor-1; ureteric obstruction; calcium homeostasis; kidney injury
Stanniocalcin-1 (STC1) is a calcium-regulating hormone in bony fish (8), where elevation of serum calcium triggers the release of STC1 from the corpuscles of Stannius (21), organs associated with the kidneys (27). Upon circulation in the gill and intestine, STC1 inhibits calcium flux from the aquatic environment through these organs, thus maintaining stable calcium concentrations in the blood (12, 22). In mammals, STC1 is expressed in multiple organs, including the brain, thyroid, spleen, thymus, parathyroid, lung, heart, skeletal muscle, kidney, pancreas, small intestine, colon, placenta, ovary, testes, and prostate (1, 2, 20). The wide expression of STC1 suggested that it might function in an autocrine and/or paracrine manner, while its localization to the thymus and spleen suggested a role in the immune/inflammatory response. Furthermore, through the evolutionary process from fish to mammals, STC1 appears to have maintained functional relevance to calcium regulation, as mammalian STC1 is involved in calcium homeostasis in the normal physiology of the gut (15) and in the adaptive response of brain cells to ischemic injury (30).
We hypothesized that, in macrophages, as in gut and brain cells, STC1 may affect calcium flux across cellular membranes, thereby leading to modulation of the inflammatory/immune response.
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METHODS |
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Unilateral ureteric obstruction in the mouse and rat. Under methoxyflurane inhalation anesthesia, unilateral ureteric obstruction (UUO) was created in 12-wk-old C57BL/6 mice by complete ligation of the left ureter at the ureteropelvic junction using double-silk sutures. A similar procedure was used to create UUO in the rat (male Sprague-Dawley rats; 200-g initial weight; Harlan Bioproducts for Science). Animals were placed on regular diet, allowed free access to tap water, and killed at varying time points (on days 4, 7, 15, 21, and 31; 3 animals per time point). These time points were chosen as they were shown in previous studies to reflect early and midpoint changes in the evolution of renal injury following ureteric obstruction (23, 24). Obstructed, nonligated-contralateral or sham-operated kidneys were processed for immunohistochemistry or snap-frozen in liquid nitrogen and stored at -70°C until used. The experimental protocols were performed in accordance with Baylor College of Medicine animal care committee.
Immunohistochemistry. Kidney tissue (mouse and rat) was fixed in 10% formaldehyde followed by dehydration in graded alcohols and embedded in paraffin blocks using standard techniques. Five-micrometer sections were cut, dried, and rehydrated for labeling with affinity-purified anti-hSTC1 antibodies, using peroxidase enzyme-based detection system (Vector Laboratories). Control for labeling was carried out in the presence of normal IgG and showed no staining. Colocalization of STC1 and macrophage markers (using anti-hSTC1 and mouse anti-rat monocyte/macrophage/dendritic cell clone ED1; Research Diagnostics, Flanders, NJ) was demonstrated using a dual-labeling kit (Zymed, San Francisco, CA). Photomicrographs were taken using Labophot-2 Nikon microscope with a MagnaFire Olympus digital camera.
Measurement of [Ca2+]i by fluorescence spectrophotometry. Measurements of fluorescence intensity of calcium fluoroprobe (Fluo3 at 3 µM) and sequential image recordings were made on a Wallac/Perkin Elmer (Gaithersburg, MD) Concord system incorporating a SpectraMaster multi-wavelength controller and temperature-controlled stage (Melville, NY). To detail events over time, sequential image captures spanning 2-5 s were selected, and video recordings of these events were made for a number of minutes (average of 25,000 image acquisitions) using an Olympix AstroCam CCD4100 Fast Scan (12 bit; 768 x 576: 1,000 frames/s; 9-µm resolution).
Chemotaxis and chemokinesis assays. Murine macrophage-like (RAW264.7) and human monoblast-type (U937) cells were used to assess the effects of STC1 on both random cell movement (chemokinesis) and chemokine-mediated chemotaxis. RAW264.7 cells were maintained in DMEM. U937 cells were maintained in RPMI. Both media were supplemented with 10% FBS. RAW264.7 cells are adherent cells, whereas U937 cells grow in suspension. Once confluent, RAW264.7 cells were passaged and resuspended at a concentration of 2 x 106 cells/ml in DMEM with 10% FBS. U937 cells in suspension were pelleted and resuspended in RPMI with 10% FBS at the same concentration (2 x 106 cells/ml). Both cell types were then incubated with STC1 (0, 10, 50, 100, 500 ng/ml) just before the assays. Denatured STC1 (boiled for 5 min) at the same concentration was used as a negative control.
Chemotaxis and chemokinesis were measured using either transfilter (cells trapped in pores-RAW264.7 cells) or transwell assays (cells reaching lower chamber-U937 cells) in 48-well chemotaxis chambers (Neuroprobe, Cabin John, MD), as previously described (3, 28). Media with or without recombinant rat MCP-1 (200 ng/ml; BD Pharmingen) or SDF-1 (150 ng/ml; BD Pharmingen) was placed in lower wells of chambers (30 µl) and separated from the cell suspension (50 µl) in the upper wells by polyvinylpyrrolidone-free polycarbonate filters (8- or 5-µm pore size). Chemotaxis chambers were incubated at 37°C in 5% CO2 for 90 min. In the transfilter assays (RAW264.7 cells), the filter membrane upper surface was subsequently washed three times with PBS and scraped to remove cells, which had settled on the membrane upper surface. Cells trapped in the filter pores or adherent to the undersurface were fixed in methanol, stained with Kwik-Diff (Thermo Shandon, Pittsburgh, PA), and counted. The total number of cells trapped in the pores was assessed for each well. In the transwell assays (U937 cells), the total number of cells reaching the lower well was counted using a hemacytometer. Counts were expressed as a percentage of the total number of cells added to the upper chamber. For both transfilter and transwell assays, each combination of upper and lower well configurations was performed in quadruplicate.
Ribonuclease protection assay. Total RNA was extracted using RNazol B (Tel-Test, Friendswood, TX) according to previously published protocols (4). Ten micrograms of total RNA were subjected to ribonuclease protection assay using RiboQuant kit and mCK-5 cDNA template set (Pharmingen). The cDNA template set used contained multiple probes, which allowed simultaneous detection of mRNAs corresponding to various inflammatory mediators (Ltn, RANTES, Eotaxin, MIP-1, MIP-1
, MIP-2, IP-10, MCP-1, TCA-3). cDNA probes for ribosomal protein L32 and glyceraldehyde-3-phosphate dehydrogenase were included as internal controls. In a separate ribonuclease protection assay, we examined the expression of STC1 and MCP-1 mRNAs in obstructed mouse kidneys, using murinespecific probes for STC1 (bp 1-200) and MCP-1 (bp 20-160). Both probes were sublconed into pGEM4Z with the 3' end of the cDNAs toward T7 promoter. Radiolabeled antisense RNA probes were synthesized using an in vitro transcription system (Promega, Madison, WI) and [
-32P]UTP (3,000 Ci/mmol; ICN). Ribonuclease protection assay was performed on 4 µg of total RNA using ribonuclease protection assay kit I (Torrey Pines Biolabs) according to the manufacturer's instructions. Antisense RNA probes were hybridized with the RNA samples at 90°C for 25 min. Unhybridized single-strand RNA was digested using Ribonuclease A/T1 (Sigma) for 30 min. Double-strand RNA was precipitated using stop solution at -80°C for 15-30 min and centrifuged at 15,000 g for 30 min. The samples were resolved on 6% sequencing gel. The gels were dried and exposed to X-ray films.
Statistical analysis. Results were expressed as means ± SD. Statistical significance was assessed using t-test or ANOVA and the Bonferroni multiple comparison test. P values <0.05 were considered statistically significant.
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RESULTS |
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STC1 attenuates MCP-1- and SDF-1-mediated chemotaxis. Because chemotaxis integrates multiple cellular functions that require calcium signaling, we examined the effect of STC1 on chemokine-mediated cell migration in both the macrophage-like RAW264.7 cells and U937 monoblasts. The effect on chemokinesis (random movement, no chemokine gradient) was also examined (Figs. 2 and 3). Treatment of both cell types with STC1 attenuated chemokinesis (Figs. 2D and 3C) as well as MCP-1- (Figs. 2, A and B, and 3A) and SDF-1
-mediated (Figs. 2C and 3B) chemotaxis. Denatured STC1 at the same concentrations (10, 50, 100, and 500 ng/ml) was used as a negative control in all assays and showed no effect on chemotaxis or chemokinesis. To avoid repetition, denatured STC1 results are indicated for the MCP-1-mediated chemotaxis assays only (Figs. 2B and 3A) and are not shown for the other assays.
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In RAW264.7 cells, the inhibitory effect of STC1 appeared particularly dose dependent (Fig. 2) with maximal effect generally achieved at a dose of 100 ng/ml. Figure 2A shows representative micropore membranes, which illustrate the effect of STC1 (100 ng/ml) on MCP-1-induced RAW264.7 cell migration. In U937 cells, we observed near-maximal inhibition of chemotaxis in the presence of MCP-1 (Fig. 3A) or SDF-1 (Fig. 3B) at a concentration of 10 ng/ml STC1, without an apparent dose-response curve. Furthermore, while chemokinesis was gradually inhibited by STC1 in RAW264.7 and U937 cells (Figs. 2D and 3C), and the half-maximal inhibitory effect was generally observed at/or near 50 ng/ml STC1 in both cell lines, the dose-response curve for inhibition of chemotaxis was different for each cell line. Whereas MCP-1- and SDF-1-mediated chemotaxis was gradually inhibited by STC1 in RAW264.7 cells (half-maximal inhibitory concentration of 50 ng/ml), it was abruptly and almost completely inhibited in U937 cells at the lowest concentration of STC1 (10 ng/ml) used (Fig. 3, A and B). Thus the effect of STC1 is not specific for one chemokine and does not appear to be limited to one cell line, suggesting broad effects for STC1 on the immune/inflammatory system.
Upregulation of MCP-1 and STC1 mRNAs in obstructive uropathy. Upregulation of MCP-1 and several chemokines has been shown to follow ureteric obstruction in humans (10) and experimental animals (5, 7, 11). In the following experiment, we used ribonuclease protection assay to determine mRNA levels of various inflammatory cytokines in obstructed mouse kidneys and to correlate these with STC1 mRNA levels. As previously reported (5, 7, 10, 11), ureteric obstruction induces the expression of MCP-1 and a number of chemokines, such as MIP-2, IP-10, and RANTES (Fig. 4A). Similarly, ureteric obstruction increases STC1 mRNA levels in a time-dependent manner with sustained expression through day 21 (Fig. 4, B and C). The induction of STC1 mRNA parallels that of MCP-1 in obstructed kidney suggesting a potential functional and regulatory linkage between the two molecules.
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STC1 is differentially expressed in obstructed rat kidney and localizes to macrophages. To further demonstrate relevance of STC1 to the immune/inflammatory response, it was necessary to show regulated expression of STC1 in a model of renal injury and localization of STC1 protein to inflammatory cells in vivo. Due to the suboptimal performance of available STC1 antibodies for immunohistochemical studies on mouse tissue, we examined rat kidneys for the expression of STC1 at various stages following UUO and sought to determine if STC1 colocalizes with specific macrophage markers. As previously reported, STC1 labeling in the normal kidney was found predominantly in distal nephron segments, whereas weak staining was observed in the proximal tubules (6, 17, 26). After ureteric obstruction, however, STC1 was strongly upregulated (through day 25 following ureteric obstruction) and was uniformly expressed in all tubular structures as well as in the glomeruli. In addition, STC1 was detected in interstitial cells that did not appear to have direct tubular association (Fig. 5). Dual staining for STC1 and macrophage markers using ED1 clone revealed expression of STC1 in macrophages. Thus contrary to the discrete expression of STC1 in some tubular structures in the normal kidney, STC1 was differentially expressed in obstructed kidney, where it was detected in all tubules, blood vessels, and glomeruli. In addition, STC1 was detected in macrophages, which appeared to have infiltrated the entire kidney. Of note, MCP-1 expression in obstructed kidneys was previously localized to the tubules and macrophages (25) and thus it parallels the expression of STC1 at these sites.
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DISCUSSION |
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The mechanism by which STC1 inhibits cell movement likely relates to its known effect to inhibit [Ca2+]i. Indeed, we were able to show a dose-response relationship between STC1 and [Ca2+]i in RAW264.7 cells. Similar effects have been shown by us and others (19, 30) in various cell types, including brain cells and cardiomyocytes. The specific mechanism by which [Ca2+]i is lowered is complex and may involve both alterations in calcium influx across the plasma membrane and effects on intracellular stores. Indeed, in cardiomyocytes we found that STC1 decreases calcium influx through inhibition of L-type channels (19) and possibly through modulation of IP3 channels as well (Sheikh-Hamad D, unpublished observations).
Our hypothesis is consistent with the known critical role for [Ca2+]i in monocyte and macrophage function. In macrophages, [Ca2+]i changes have been associated with activation of cellular kinases and phosphatases, degranulation, phagosome-lysosome fusion, cytoskeletal reorganization, transcriptional control, modulation of surface receptors, and regulation of cell adhesion, motility, and chemotaxis (16), processes that are important for the cellular response to antigenic stimuli. Occupancy of the chemokine receptor by its ligand results in the mobilization of [Ca2+]i through one of three possible pathways [IP3 receptor pathway, cyclic adenosine diphosphateribose (cADPR)/ryanodine receptor pathway, and S1P pathway]. In the IP3 receptor pathway, stimulation of G protein-coupled receptors by the chemokine (through Gq) leads to the activation of phospholipase C, which hydrolyzes inositol lipids in the plasma membranes, releasing IP3 into the cytosol and leaving DAG within the membrane. The primary function of IP3 is to mobilize calcium from IP3-responsive intracellular stores. In the cADPR/ryanodine receptor pathway, nicotin-amide adenine dinucleotide is converted to cADPR through the action of ADP-ribosyl cyclase. cADPR, in turn, stimulates ryanodine receptors in the sarcoplasmic reticulum and induces the release of calcium from this compartment (9, 13, 18). In the S1P pathway, sphingosine is phosphorylated by sphingosinekinase, generating S1P (31), which functions to release calcium from endoplasmic reticulum stores in a manner that is independent of IP3 and/or cADPR. Thus release of calcium from intracellular stores, irrespective of the mechanism employed for this release, plays a major role in macrophage chemotaxis.
The observation in our study that STC1 dose dependently reduced both [Ca2+]i and chemotaxis supports this pathway as the mechanism for the observed effect. Indeed, it is unlikely that STC1 acts by modulating cell membrane receptors for chemokines, as both MCP-1 and SDF act on different receptors (CCR2 and CXCR4, respectively). Furthermore, the effects on chemokinesis suggest a general mechanism, not simply a chemokine-dependent pathway. However, it is important to recognize that the concentration of STC1 needed to maximally inhibit chemokinesis and chemotaxis in RAW264.7 cells (100 ng/ml) was equivalent to 20% of the concentration needed for maximal attenuation of [Ca2+]i signals (500 ng/ml; see Fig. 1). Thus, while STC1-mediated alterations in [Ca2+]i signals may have an impact on chemokinesis and chemotaxis, we cannot rule out other mechanisms, such as activation/deactivation of intracellular signaling pathways.
Our observations in the two cell lines may be relevant in vivo. Here, we bring evidence for the involvement of STC1 in the response of the kidney to obstructive injury and possibly in the function of macrophages in vivo. In the normal kidney, STC1 is detected in epithelial cells of proximal S3-segments and distal nephrons (thick ascending limbs, distal convoluted tubules, and collecting ducts) and may have a role in regulating energy utilization in these metabolically active sites (17). In advanced stage renal injury that follows long-standing ureteric obstruction, however, in addition to its presence in tubular epithelium, STC1 strongly labels interstitial cells that are not associated with discernible tubular structures. A large number of these cells carry macrophage cell-surface markers, suggesting that STC1 is relevant to macrophage function in response to ureteric obstruction and possibly in other disease states. Furthermore, as the sites of MCP-1 and STC1 expression in obstructed kidneys appear to overlap, at least in macrophages and tubular cells (25), and show parallel induction at the mRNA level, it is possible that these proteins may function in concert to modulate the immune/inflammatory response in obstructive uropathy.
It is unclear at present whether staining for STC1 in macrophages represents de novo production of STC1 or adsorption of ambient STC1 to binding sites on macrophages. We propose that the role of STC1 in the inflammatory/immune response may relate to the timing and site of its production. Production of STC1 in the injured parenchyma would support a role for this molecule in attenuating the influx of inflammatory cells to the site of injury and thus protect the injured tissue from further influx of cytokines and toxic metabolites. De novo production of STC1 by the inflammatory cells, on the other hand, could potentially play a role in regulating the movement of inflammatory cells and their response to antigenic stimuli.
Through the evolutionary process from fish to mammals, STC1 appears to have maintained functional relevance to calcium homeostasis, as mammalian STC1 has been shown to regulate [Ca2+]i in the normal physiology of the gut (15), in the adaptive response of brain cells to ischemic injury (30) and in the regulation of cardiomyocyte L-type channel activity in heart failure (19). This paper provides another potential function for mammalian STC1; that is, modulation of the inflammatory response in a manner that involves alterations in calcium homeostasis. Indeed, the observation that STC1 inhibits spontaneous movement and the chemotactic response of two monocyte lines to two different chemokines in vitro and the demonstration that it is expressed at inflammatory sites in vivo both argue for a new and unrecognized function for STC1 in disease. Much emphasis has been placed in recent years on factors that drive the inflammatory response, while less is known about the endogenous factors that are involved in blocking or reversing inflammation. We propose that future studies are needed to determine if STC1 may be one of those critical endogenous factors that have intrinsic anti-inflammatory effects to modulate disease.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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