Oxalate upregulates expression of IL-2Rβ and activates IL-2R signaling in HK-2 cells, a line of human renal epithelial cells

Abstract

The role of inflammation in oxalate-induced nephrolithiasis is debated. Our gene expression study indicated an increase in interleukin-2 receptor β (IL-2Rβ) mRNA in response to oxalate (Koul S, Khandrika L, Meacham RB, Koul HK. PLoS ONE 7: e43886, 2012). Herein, we evaluated IL-2Rβ expression and its downstream signaling pathway in HK-2 cells in an effort to understand the mechanisms of oxalate nephrotoxicity. HK-2 cells were exposed to oxalate for various time points in the presence or absence of SB203580, a specific p38 MAPK inhibitor. Gene expression data were analyzed by Ingenuity Pathway Analysis software. mRNA expression was quantitated via real-time PCR, and changes in protein expression/kinase activation were analyzed by Western blotting. Exposure of HK-2 cells to oxalate resulted in increased transcription of IL-2Rβ mRNA and increased protein levels. Oxalate treatment also activated the IL-2Rβ signaling pathway (JAK1/STAT5 phosphorylation). Moreover, the increase in IL-2Rβ protein was dependent upon p38 MAPK activity. These results suggest that oxalate-induced activation of the IL-2Rβ pathway may lead to a plethora of cellular changes, the most common of which is the induction of inflammation. These results suggest a central role for the p38 MAPK pathway in mediating the effects of oxalate in renal cells, and additional studies may provide the key to unlocking novel biochemical targets in stone disease.

kidney stones are a common source of urological morbidity. In fact, the treatment of kidney stone disease represents one of the most costly urological conditions in the United States (25). The formation of calcium oxalate stones in the kidney is the most common pathological condition involving oxalate. Oxalate is a metabolic end product, which is filtered at the glomerulus and undergoes bidirectional transport in the renal tubules before being excreted by the kidney (13, 14, 24). In addition to kidney stones, oxalate deposits are associated with renal cysts in acquired renal cystic disease, benign neoplasms of the breast, and hyperplastic thyroid glands, among others (7, 10, 28, 34). Many of these conditions are associated with tissue inflammation, atypical cell proliferation, and cell death. Unsurprisingly, the mechanism and modifications of calcium oxalate deposition and stone formation is more complex than the simple precipitation of calcium oxalate crystals (18).

Previous studies from our laboratory and those of others have demonstrated that the oxalate-renal cell interaction induces the initiation of DNA synthesis, cell growth, apoptosis, and changes in gene expression consistent with cellular stress (1, 12, 15, 17, 30). These data suggest that oxalate toxicity may result in tissue damage and/or inflammation. However, the cellular signaling pathways activated in renal cells following oxalate exposure are not well delineated and continue to be a large area of interest and study.

Previously, we have shown that exposure of LLC-PK₁ cells, a line of pig renal tubular epithelial cells, to oxalate or calcium oxalate monohydrate crystals results in selective activation of the p38 MAPK and JNK pathways (3, 19). The activation of these pathways and others is known to invoke changes in gene expression. Consistent with this notion, the expression of a number of stress- and inflammation-related genes has been shown to be increased in response to oxalate exposure (21). We recently reported preliminary results from a global (Affymetrix) gene expression analysis in the human proximal tubular epithelial cell line HK-2 in response to oxalate (22). These data demonstrated that oxalate induced the up- and downregulation of a large set of genes, including a 20-gene set that could potentially be used to monitor for oxalate-induced nephrotoxicity. One of these upregulated genes is interleukin-2 receptor β (IL-2Rβ).

IL-2R is a heterotrimeric protein consisting of three distinct membrane proteins: IL-2Rα (CD25), IL-2Rβ (CD122), and the common γ-chain (2). The IL-2R was initially thought to be primarily expressed on lymphoid cells where it is well known to activate cellular signaling programs in T cells and T_Reg cells; however, its expression has now been documented on human fibroblasts, endothelial cells, and intestinal and renal epithelial cells (2). IL-2Rβ is known to bind to IL-2, leading to ligand internalization and signal transduction. Consistent with our Affymetrix data, where the IL-2Rβ was differentially regulated, the IL-2R has been shown to be expressed on human renal proximal tubular epithelial cells (6). Therefore, in the present study, we investigated the role of oxalate on IL-2Rβ gene expression and its downstream signaling pathway, and the role of the p38 MAPK pathway, which may have a critical role in the progression oxalate-induced inflammation and urolithiasis.

MATERIALS AND METHODS

Cell culture.

HK-2 cells were obtained from ATCC (Manassas, VA), maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO₂. Cells were passaged upon reaching 80% confluence.

Reagents.

Sodium oxalate (20 mM; Sigma) was prepared in PBS, pH 7.4. Sodium oxalate as used at 1 mM in the experimental conditions as indicated in each experiment. SB203580 (Sigma) was resuspended in DMSO and used at either 10 or 20 μM as indicated. GAPDH, p38 MAPK, phospho p38 (Thr180/Tyr182), JAK1, phospho JAK1 (Tyr1022/1023), STAT5, and phospho STAT5 (Tyr694) antibodies were purchased from Cell Signaling Technology.

RNA preparation and real-time-PCR.

The total mRNA from the HK-2 cells was isolated (Qiagen), transcribed into cDNA (iScript; Bio-Rad), and real-time PCR was performed as described (22). Briefly, using a Light Cycler 480 (Roche Diagnostics), a multiwell 96-well plate containing 10 μM of each primer, 10 μl of 2× master mix, and 1 μl of cDNA template in a final reaction volume of 20 μl was used to amplify the specific mRNA sequences. IL-2Rβ primers were forward: 5′-GCTGATCAACTGCAGGAACA and reverse: 5′-TGTCCCTCTCCAGCACTTCT using the following cycle parameters: 95°C for 10 min; 45 cycles of 95°C for 10 s, 63°C for 10 s, and 72°C for 10 s. GAPDH primers (forward: 5′-AAGGTCGGAGTCAACGGATTTGGT; reverse: 5′-AGTGATGGCATGGACTGTGGTCAT) or 18S rRNA (forward: 5′-TCAGATACCGTCGTAGTTC; reverse: 5′-CCTTTAAGTTTCAGCTTTGC) were included to normalize variation from sample to sample. All experiments were repeated three times using three independent preparations of cDNA.

Western blot analysis.

Protein isolation and Western blot analysis were performed as previously described (20). Briefly, cells lysed in lysis buffer (20 mm Tris, pH 7.4, 1% Triton X-100, 1 mm sodium orthovanadate, 10 mm NaF, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin). Samples containing equal amounts of protein (50 μg) were separated on 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes using standard SDS-PAGE procedures and probed with various antibodies as indicated.

MAPKAPK2 immunocomplex kinase assays.

Nonradioactive MAPKAPK2 kinase was assayed using a MAPKAP Kinase 2 immunoprecipitation kinase assay kit (Upstate) according to the protocols of the manufacturer and as described previously (23).

Data analysis and bioinformatics.

Hybridization intensity quantification was performed using GeneChip Operating Software algorithms (GCOS1.2, Affymetrix) as previously described (22) and was visualized by STAGE, a software tool developed in house.

Cell Intensity files were processed into expression values for all the 55,000 probe sets (transcripts) on each array and following the respective normalization step. Differentially expressed genes were selected if they passed Welch's t-test and a parametric test (variance not assumed equal, P < 0.05) and showed at least twofold changes between control and oxalate-treated sets. Differentially expressed genes were classified according to the Gene Ontology functional category (GenMAPP v2), and the functional significance of differentially expressed genes was determined using Ingenuity Pathways Analysis software (Ingenuity Systems, www.ingenuity.com). This software takes the global gene expression array data and compares the global expression patterns with third-party biological databases to compile the complex pathways and interactions taking place (33). Cluster and Heatmap images were generated using BRB-Array tools (26).

RESULTS

Differential gene expression in genome-wide analysis of oxalate-treated HK-2 cells.

We recently published preliminary results from a genome-wide Affymetrix gene array of HK-2 cells in response to oxalate (22). These results demonstrated that oxalate modulated either the up- or downregulation of a large set of genes. In fact, the array revealed 20 oxalate-regulated genes that could potentially be used to monitor oxalate nephrotoxicity. Shown in Fig. 1A is a hierarchal cluster of a subset of the differentially regulated genes from the Affymetrix gene array at 0 (control), 4, and 24 h of oxalate exposure. As shown, many of these differentially regulated genes are involved in intracellular signaling, including components of the phosphatidylinositol 3-kinase (PI3K) and MAPK signaling pathways. Of these differentially regulated genes, the IL-2Rβ gene transcript was one of the highest overexpressed transcripts in the data set. Therefore, we initiated studies into the role of the IL-2Rβ signaling pathway in oxalate-exposed HK-2 cells.

Fig. 1.

Regulation of gene expression associated with the IL-2 receptor (IL-2R) pathway. Gene expression was analyzed from Affymetrix array data with respect to the IL-2R signaling pathway. Shown is a hierarchal cluster indicating the expression levels of those genes associated with the IL-2R signaling pathway in response to either 4 or 24 h of oxalate exposure. Green indicates downregulation, while red indicates upregulation.

IL-2Rβ mRNA and protein are upregulated in response to oxalate.

To confirm the Affymetrix data suggesting that IL-2Rβ was upregulated in response to oxalate, the human proximal tubular epithelial cell line HK-2 was treated with 1 mM sodium oxalate for various time points following serum starvation for 16–20 h. The upregulation of IL-2Rβ mRNA was confirmed by real-time PCR. As shown in Fig. 2A, IL-2Rβ mRNA is upregulated quickly within 15 min and remains elevated for over 1 h, indicating a time-dependent transcriptional upregulation of IL-2Rβ mRNA in response to oxalate.

Fig. 2.

Oxalate increases IL-2Rβ expression at the mRNA and protein level in a time-dependent manner. A: real-time PCR indicates the mRNA levels of IL-2Rβ were significantly upregulated within 1 h of oxalate exposure in HK-2 cells. B: Western blot analysis indicates that the protein levels of IL-2Rβ are upregulated in HK-2 cells at 4 h following oxalate exposure. The data from 3 independent experiments were analyzed statistically by 2-tailed Student's t-tests using SPSS software. *P < 0.05.

Since mRNA expression shows discordance with protein levels in many cases and because protein levels are necessary for biological activity, we also measured IL-2Rβ protein levels to determine whether increased IL-2Rβ mRNA expression resulted in increased IL-2Rβ protein levels. For these studies, HK-2 cells were exposed to oxalate for various time points and protein expression was evaluated by Western blot analysis using an anti-IL-2Rβ antibody. Results presented in Fig. 2B demonstrate that IL-2Rβ protein levels are also increased in response to oxalate exposure.

Taken together, our results show that oxalate exposure results in transcriptional upregulation of IL-2Rβ mRNA, resulting in an increase in IL-2Rβ protein levels.

IL-2Rβ Pathway Analysis.

Using Ingenuity Pathway Analysis software to mine the Affymetrix data set for signaling pathway involvement, we analyzed the effects of oxalate on the IL-2Rβ pathway. As shown in Fig. 3, IL-2Rβ can activate a number of downstream signaling pathways including the JAK/STAT pathway, the Ras/Raf/Erk MAPK pathway, and the PI3K pathway, among others. Ingenuity Analysis of our Affymetrix gene expression data with respect to the IL-2Rβ pathway indicated that the IL-2Rβ and members of its downstream signaling pathway were differentially regulated in response to oxalate. The results suggested differentially increased activity related to JAK/STAT components of the IL-2Rβ signaling cascade in response to oxalate.

Fig. 3.

Effects of oxalate: Ingenuity Pathway Analysis of the IL2-R pathway. For these studies, renal epithelial cells were exposed to oxalate and global gene expression changes were monitored using the Affymetrix platform. Ingenuity Pathway Analysis software was used to analyze gene expression data and model the IL2-R signaling pathway by comparing global expression patterns with third-party biological databases to compile the complex pathways and interactions taking place in the IL2-R pathway.

Oxalate activates the JAK/STAT arm of the IL-2Rβ pathway in a time-dependent manner.

The IL-2R pathway is a complex pathway, which can signal through a number of downstream signaling pathways, and since our gene expression analysis suggested activation of the JAK-STAT pathway (Fig. 3), we evaluated activity of JAK1 and STAT5 (downstream of the IL-2Rβ) in response to oxalate exposure. Results presented in Fig. 4, A and B, show that there is no change in total levels of JAK1 or STAT5 protein levels; however, oxalate exposure results in increased levels of phosphorylated JAK1 (Fig. 4A) and STAT5 (Fig. 4B) in a time-dependent manner. Since phosphorylation is a measure of kinase activation, these data indicate that the JAK1/STAT5 arm of the IL-2Rβ signaling pathway is activated in response to oxalate.

Fig. 4.

The IL-2Rβ pathway is activated in response to oxalate. Western blot analysis was performed on oxalate-treated HK-2 cells for the time points indicated. Increased levels of JAK1 and STAT5 phosphorylation were detected and were especially increased at the 4- and 18-h time points. Total protein levels of JAK1 and STAT5 were unchanged. The data from 3 independent experiments were analyzed statistically by 2-tailed Student's t-tests using SPSS software. *P < 0.05.

Oxalate exposure activates p38 MAPK in HK-2 cells.

Our laboratory has previously shown a central role for p38 MAPK activation in renal epithelial cells in response to oxalate in LLC-PK₁ cells (3). To determine whether oxalate induced phosphorylation of p38 in the human renal epithelial cells, HK-2 cells were treated with oxalate for up to 30 min. Within 5 min of oxalate exposure, we detected increased levels of p-p38, which was sustained for up to 30 min post-oxalate exposure (Fig. 5A). To further demonstrate that p38 was activated in this cell line, in situ activity of p38 MAPK, as determined by MAPKAPK2 activity, was measured in response to oxalate exposure in the absence and presence of a specific p38 MAPK inhibitor, SB203580. Figure 5B shows that MAPKAPK2 activity was increased fourfold in response to oxalate exposure and that increasing concentrations of SB203580 were able to diminish the activity of MAPKAPK2 in response to oxalate. These results demonstrate that like pig renal epithelial cells, oxalate exposure results in the phosphorylation and activation of the p38 MAPK signaling pathway in human renal epithelial cells as well.

Fig. 5.

Oxalate exposure results in activation of MAPK in HK-2 cells. A: representative Western blot showing time-dependent increase in p-p38 MAPK in HK-2 cells after oxalate treatment at multiple time points up to 30 min. B: in situ activity of p38 MAPK as determined by MAPKAPK2 activity in response to oxalate exposure in the absence and presence of the presence of p38 MAPK inhibitor SB203580. The data from 3 independent experiments were analyzed statistically by 2-tailed Student's t-tests using SPSS software. *P < 0.05 compared with control. **P < 0.05 compared with oxalate.

Oxalate-induced IL-2Rβ is dependent upon p38 MAPK activity.

To determine whether p38 MAPK activity was essential for the upregulation of IL-2Rβ expression, we treated HK-2 cells with oxalate in the presence or absence of SB203580, a select inhibitor of the p38 MAPK pathway. At indicated time points, the experiment was terminated and cells were processed for RNA or protein extraction. Results of the real-time PCR data presented in Fig. 6A reveal that pretreatment of the cells with SB203580 completely blocked the ability of oxalate to increase IL-2Rβ mRNA. Similarly, results of the Western blot analysis presented in Fig. 6B reveal that pretreatment of HK-2 cells with SB203580 resulted in abrogation of oxalate-induced IL-2Rβ protein levels. Taken together, these data demonstrate critical requirements of p38 MAPK activation in oxalate-induced upregulation of IL-2Rβ.

Fig. 6.

p38 MAPK inhibitor blocked upregulation of IL-2R induced by oxalate treatment in HK-2 cells. A: IL-2R mRNA level was assessed by qRT-PCR. HK-2 cells were treated with or without 1 mM sodium oxalate and 20 mM SB203580 for 2, 4, and 18 h (n = 3). 18S rRNA was used for normalization. B: Western blot analysis. The data from 3 independent experiments were analyzed statistically by 2-tailed Student's t-tests using SPSS software. *P < 0.05.

DISCUSSION

The present study confirms recently published Affymetrix array data (22) showing that IL-2Rβ mRNA is significantly upregulated by oxalate in the human renal epithelial cell line (HK-2) and that the IL-2Rβ downstream signaling pathway is also activated in response to oxalate exposure. Moreover, the p38 MAPK pathway is essential for the increased expression of IL-2Rβ as inhibition of the p38 pathway prevents oxalate-induced IL-2Rβ expression. These findings are significant, because the presence of oxalate in the kidneys is relatively common and is often associated with inflammation, tubulointerstitial damage, and kidney stone development (1, 18, 27). Moreover, the study of crystal deposition-induced inflammation goes beyond the kidney as the intra-articular deposition of octacalcium phosphate, calcium phosphate dihydrate, tricalcium phosphate, or hydroxyapatite causes inflammation and has been established as the cause of inflammatory arthritis (32).

Accumulating evidence over the past two decades suggests that proximal tubular epithelial cell injury and dysfunction play a central role in several disease states, including glomerular proteinuria and renal transplant rejection, and can result from significant ischemic insults. Following these various insults, the activation of signaling cascades can lead to the local production of cytokines, chemokines, complement, and matrix components (29). These mediators can then directly or indirectly enhance the influx of proinflammatory cells, including macrophages, dendritic cells, and T cells, leading to an increased proinflammatory environment and irreversible interstitial kidney injury and loss of renal function. The p38 MAPK pathway has been implicated as a central mediator of inflammation in the kidney (31). Oxalate has been previously implicated in p38 MAPK activation in LLC-PK₁ cells (3) and in this study in HK-2 cells. The activation of the p38 MAPK pathway is critical for the production of proinflammatory cytokines, including IL-1, IL-6, IL-8, and TNF-α, among others. Moreover, p38 MAPK induces key inflammatory enzymes such as inducible nitric oxide synthase and cyclooxygenase 2 (4). Thus our findings using the specific p38 inhibitor SB203580, demonstrating that the p38 MAPK pathway can also increase the expression of proinflammatory membrane receptors, not just proinflammatory ligands, represents yet another mechanism by which p38 MAPK can enhance inflammation and respond to proinflammatory stimuli in the tissue microenvironment.

A variety of crystals in the body have been shown to induce an inflammatory response by diverse cell types. There are several key chemotactic factors in the inflammatory reaction at various sites throughout the body, including the kidney, and it is recognized that cytokines perform ever-increasing roles in regulating immune homeostasis and activation in response to stimuli. Renal tubular cells have been shown to express several cytokines and other inflammatory mediators. IL-2 is a potent cytokine required for the generation and maintenance of T_effector and T_reg cells, and complicated mechanisms are responsible for keeping IL-2 levels in check and maintain a balanced proportion of these T cell subsets. The IL-2R is known to be expressed by activated infiltrating lymphocytes and is involved in inflammation during renal transplantation. In addition, the IL-2R is also expressed in kidney epithelial cells, namely, human proximal tubular epithelial cells (6). Therefore, our results suggest that activation of the IL-2R by IL-2 initiates a pleiotropic downstream signaling pathway in the kidney epithelium that may play a relevant role in inflammation, leading to kidney stone disease, which may be dependent and/or independent of inflammatory immune cells in the kidney.

It is interesting to point out here that elevated levels of IL-1β by calcium oxalate crystals have been shown to induce renal inflammation through IL-1R signaling in mice (27); however, this is the first study to our knowledge demonstrating increased expression and activation of IL-2Rβ pathway in response to oxalate in any cell type. The IL-2Rβ pathway is known to play a role in inflammation through immune cell activation in the kidney as well as other organs (2). However, it remains to be determined whether infiltrating inflammatory immune cells are responsible for IL-2 production leading to IL-2Rβ activation on the kidney cells (paracrine) or whether the kidney cells produce IL-2 in response to oxalate or other stimuli (autocrine). Either way, the IL-2/IL-2R pathway is activated in kidney cells in response to oxalate and may represent a major pathway in renal inflammation (29). The finding that IL-2Rβ expression is dependent upon an active p38 MAPK pathway is exciting in that the p38 MAPK pathway seems to play a central role in many aspects of oxalate-induced renal cell damage (3, 8, 16). It is necessary to point out here that increased binding of calcium oxalate monohydrate crystals has been shown in the damaged and proliferating renal epithelial cells (5, 30, 35), suggesting that renal epithelial cell damage or proliferation could lead to crystal retention. Moreover, oxalate exposure has been shown to increase renal epithelial cell dysfunction, cell death, and expression of proinflammatory cytokines (9, 11), as well as increased crystal binding to the renal epithelial cells (17). Taken together with findings presented in this paper, it is tempting to speculate that oxalate-induced renal epithelial cell dysfunction could promote a proinflammatory microenvironment in renal tubules and the resulting inflammation may promote crystal retention. Indeed, several lines of evidence point to association of renal crystal deposits and local inflammation (11). It is interesting to point out that, in preliminary studies, we have observed that activation of the p38 MAPK pathway precedes renal tubular damage and crystal retention in an ethylene glycol model of hyperoxaluria (16). Thus oxalate-induced p38 MAPK-mediated inflammation could promote inflammation and damage in the renal tubules, leading to areas of exposed basement membrane along the tubule, thereby providing for increased areas of crystal attachment and growth (29, 30). While many mechanistic insights remain unclear, the results of our study, by demonstrating a critical role of the p38 MAPK pathway in mediating the effects of oxalate on IL-2R β (Fig. 7), provide for a conceptual basis for the role of p38 MAPK pathway in oxalate-induced renal tubular inflammation. These considerations highlight the need for future studies 1) to understand the role of inflammation and the contribution of the IL-2R and p38 MAPK pathways in kidney stone formation in general and in subsets of patients with elevated urinary oxalate in particular and 2) to further test the utility of p38 MAPK inhibitors in preserving renal function by preventing renal damage and crystal retention.

Fig. 7.

Conceptual flow diagram (based on the data presented herein) depicting mechanistic link between p38 MAPK and IL-R signaling pathway activated by oxalate in HK-2 cells. Increased luminal oxalate levels may either lead to increased intracellular oxalate levels or may trigger p38 MAPK activation by acting on cell membranes; however, exact mechanisms have not been established. In any case, activated p38 MAPK may result in phosphorylation and activation of transcription factors that drive transcriptional activation of IL-2Rβ and consequent increase in IL-2Rβ protein levels.

GRANTS

This work was funded by National Institutes of Health Grant DK-RO1–54084 (H. Koul, PI) and the Department of Surgery, School of Medicine, University of Colorado (AEF funds). H. Koul is also supported by RO1-CA161880 and Veterans Affairs Merit Award 1BX001258.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: S.K. and H.K.K. provided conception and design of research; S.K., L.K., T.J.P., N.I., and M.P. performed experiments; S.K., L.K., N.I., M.P., J.J.S., and H.K.K. analyzed data; S.K. and H.K.K. interpreted results of experiments; S.K., L.K., T.J.P., N.I., M.P., J.J.S., and H.K.K. prepared figures; S.K., N.I., J.J.S., and H.K.K. drafted manuscript; S.K., M.P., J.J.S., and H.K.K. edited and revised manuscript; S.K., L.K., T.J.P., N.I., M.P., J.J.S., and H.K.K. approved final version of manuscript.