Next Article in Journal
Phosphinotripeptidic Inhibitors of Leucylaminopeptidases
Next Article in Special Issue
Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species
Previous Article in Journal
Initiation of Pancreatic Cancer: The Interplay of Hyperglycemia and Macrophages Promotes the Acquisition of Malignancy-Associated Properties in Pancreatic Ductal Epithelial Cells
Previous Article in Special Issue
Pleiotropic Effects of Sodium-Glucose Cotransporter-2 Inhibitors: Renoprotective Mechanisms beyond Glycemic Control
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells

Department of Internal Medicine IV–Nephrology and Hypertension, Medical University Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(10), 5089; https://doi.org/10.3390/ijms22105089
Submission received: 12 April 2021 / Revised: 4 May 2021 / Accepted: 10 May 2021 / Published: 11 May 2021
(This article belongs to the Special Issue SGLT2 Inhibitors: Emerging "Magic Bullets" beyond Glycemic Control)

Abstract

:
SGLT2 inhibitor-related nephroprotection is—at least partially—mediated by anti-inflammatory drug effects, as previously demonstrated in diabetic animal and human studies, as well as hyperglycemic cell culture models. We recently presented first evidence for anti-inflammatory potential of empagliflozin (Empa) under normoglycemic conditions in human proximal tubular cells (HPTC) by demonstrating Empa-mediated inhibition of IL-1β-induced MCP-1/CCL2 and ET-1 expression on the mRNA and protein level. We now add corroborating evidence on a genome-wide level by demonstrating that Empa attenuates the expression of several inflammatory response genes in IL-1β-induced (10 ng/mL) normoglycemic HPTCs. Using microarray-hybridization analysis, 19 inflammatory response genes out of >30.000 human genes presented a consistent expression pattern, that is, inhibition of IL-1β (10 ng/mL)-stimulated gene expression by Empa (500 nM), in both HK-2 and RPTEC/TERT1 cells. Pathway enrichment analysis demonstrated statistically significant clustering of annotated pathways (enrichment score 3.64). Our transcriptomic approach reveals novel genes such as CXCL8/IL8, LOX, NOV, PTX3, and SGK1 that might be causally involved in glycemia-independent nephroprotection by SGLT2i.

1. Introduction

SGLT2 inhibitors (SGLT2i) convey nephroprotection in type two diabetic patients with and without chronic kidney disease (CKD) [1,2,3,4]. Renal benefit has been primarily attributed to metabolic and hemodynamic drug effects, such as lowering of bodyweight, plasma volume, systemic blood pressure, and glomerular filtration pressure, as well as reducing renal hypoxia, rather than SGLT2i-mediated HbA1c-lowering [5]. In this regard, large clinical trials recently demonstrated renal benefit of SGLT2i irrespective of the presence or absence of diabetes mellitus in CKD [6] and heart failure [7] patients. Underlying molecular mechanisms for glycemia-independent protective effects of SGLT2i are still incompletely understood. Renal tissue inflammation, being directly involved in the pathogenesis of both diabetic and non-diabetic CKD, might play an important role in this context [8]. Indeed, anti-inflammatory potential of SGLT2i has been previously demonstrated in diabetic animal [9,10,11] and human studies [12,13], as well as hyperglycemic cell culture models [14]. In diabetic animal models, these SGLT2i-mediated anti-inflammatory effects were related to a decrease in glomerular and tubulo-interstitial damage [10,15]. This, however, has not been demonstrated under normoglycemic conditions until recently. In our own lab, we found an inhibitory effect of empagliflozin (Empa) on IL-1β-induced MCP-1/CCL2 and endothelin-1 expression on the mRNA and protein level under normoglycemic conditions in two independent human proximal tubular cells (HPTC) lines, namely HK-2 and RPTEC/TERT1. Both genes play an important role in early (inflammatory) pathogenesis of diabetic and non-diabetic kidney disease [16,17,18,19,20,21]. Genetic and pharmacologic inhibition of these two genes already demonstrated to convey beneficial renal effects in animal models [16,18,22,23,24,25,26] and humans [25,27,28,29,30,31] with diabetic and non-diabetic kidney disease. Most recently, Maayah and coworkers presented corroborating evidence for anti-inflammatory potential of Empa irrespective of glycemic effects by demonstrating a survival benefit among Empa-treated mice with LPS-induced septic shock. This was at least partially attributed to a suppression of renal and systemic inflammation, as well as a reduced rate of acute kidney injury [32]. We now aimed at evaluating the inhibitory effect of Empa on IL-1β-stimulated gene expression on the transcriptome level. Here we present novel evidence for anti-inflammatory potential of SGLT2i by demonstrating that Empa attenuates the expression of several inflammatory response genes (vide infra) in IL-1β-induced (10 ng/mL) normoglycemic HPTCs, namely HK-2 and RPTEC/TERT1 cells. Our transcriptomic approach presents novel genes such as CXCL8/IL8, LOX, NOV, PTX3, and SGK1, that might be causally involved in glycemia-independent nephroprotection by SGLT2i.

2. Results

While the effect of Empa on basal gene expression has been recently described in normoglycemic HPTCs by our lab [33], we present novel genome-wide evidence for Empa-mediated inhibition of inflammatory response in IL-1β-stimulated normoglycemic HPTCs. Microarray hybridization analysis identified 19 inflammatory response genes out of >30.000 human genes that present a uniform expression pattern, that is, inhibition of IL-1β (10 ng/mL)-stimulated gene expression by Empa (500 nM), in both HK-2 and RPTEC/TERT1 (Table 1AB) cells (n = 2, each) (Table 1). Additionally, we report only marginal effects of Empa (500 nM) on SGLT2 gene expression in IL-1β (10 ng/mL)-stimulated HK-2 and RPTEC/TERT1 cells (Table 2).
Based on these 19 genes-of-interest, pathway enrichment analysis (DAVID bioinformatics database) demonstrated statistically significant clustering of 30 annotated pathways (enrichment score 3.64) (Table 3).

3. Discussion

Inflammatory pathways play an important role during the development and progression of diabetic and non-diabetic CKD [8,34]. Circulating markers of systemic inflammation have been associated with renal damage [18]. Pro-inflammatory cytokines and reactive oxygen species are secreted by proliferating proximal tubular cells during initial CKD pathogenesis, thereby leading to inflammatory cell recruitment and activation within the glomerular and tubulo-interstitial compartment [35,36]. Local renal inflammation and thus kidney damage is further aggravated by the release of IL-1β from inflammatory cells upon inflammosome activation [37]. Interestingly, anti-IL-1β strategies already demonstrated beneficial renal effects in animal models of CKD [38,39]. Our own lab previously presented first evidence for anti-inflammatory effects of SGLT2i in normoglycemic HPTCs by demonstrating Empa-mediated inhibition of IL-1β-induced MCP-1/CCL2 and ET-1 expression at the mRNA and protein level [33]. Here we show novel evidence for anti-inflammatory potential of SGLT2i on the transcriptome level by demonstrating that Empa attenuates the expression of 19 inflammatory response genes in IL-1β-induced (10 ng/mL) normoglycemic HPTCs (Table 1). In line with recent findings in a murine animal model [32], pathway enrichment analysis of these genes-of-interest revealed that “cellular response to LPS” is among the Empa-regulated pathway cluster (Table 3). Based on the available evidence for involvement in kidney disease pathogenesis, five out of these 19 inflammatory response genes are discussed in more detail below:

3.1. Empa-Mediated Inhibition of IL-1β-Induced CXCL Isoform 1, 2, 5 and 8 (= IL8) Expression

Chemokines are chemotactic cytokines that have been demonstrated to play a fundamental role in orchestrating inflammation, and as such, are involved in experimental and human (inflammatory) kidney diseases. Chemokines are expressed by all types of renal cells, including HPTCs, upon pro-inflammatory cytokine stimulation (e.g., IL-1β or TNF-α). By regulating the process of leukocyte trafficking to the site of inflammation, chemokines control both acute and chronic inflammation that is crucial during development and progression of CKD and interstitial fibrosis [40,41]. For example, urinary and serum levels of CXCL8/IL8 are elevated in diabetic kidney disease [42], and CXCL1 has been described to be associated with interstitial fibrosis in progressive IgA nephropathy [43]. Chemokine antagonism has been shown to attenuate renal injury in various animal models [40,44,45,46]. Thus, it is tempting to speculate that Empa-mediated inhibition of tubular chemokine expression adds to the nephroprotective, anti-inflammatory potential of SGLT2i.

3.2. Empa-Mediated Inhibition of IL-1β-Induced LOX Expression

Lysyl oxidase (LOX) is a cellular amine oxidase facilitating cross-linking between collagen and elastin by catalyzing peptidyl lysine oxidation [47]. Excessive accumulation of these insoluble, cross-linked proteins leads to ECM fibrosis and remodeling. Both circulating and tissue levels of LOX are increased in patients with organ fibrosis, for example, in the heart, lung, and liver. Besides fibrotic diseases, altered LOX activity is related to inflammatory diseases, as well as cancer progression [48]. It has been previously speculated that LOX might be a potential therapeutic target in fibrotic diseases and cancer; however, specific treatment options are missing. Recently, elevated serum and tissue LOX levels have been described to strongly correlate with the presence and degree of kidney fibrosis in CKD patients. Serum LOX levels have been suggested to serve as non-invasive diagnostic biomarkers for renal fibrosis [49]. Further studies are mandatory to confirm this association and to investigate a potential causal relationship between LOX expression and kidney fibrosis. Based on these data and in line with our previous findings demonstrating a direct anti-fibrotic effect of SGLT2i in 2 independent HPTCs [50], it is tempting to speculate that Empa-mediated inhibition of IL-1β-mediated LOX expression might exert nephroprotective effects.

3.3. Empa-Mediated Inhibition of IL-1β-Induced NOV Expression

The nephroblastoma overexpressed (NOV/CCN3) gene is a member of the CCN family of matricellular proteins that play an important role during inflammation, tissue repair and cancer [51]. Genetic knockdown studies recently demonstrated that a reduced NOV expression in a murine model of progressive obstructive nephropathy limits inflammation as well as renal interstitial fibrosis compared to wild-type mice [52]. This animal model is corroborated by biopsy findings that demonstrate an increased tubulo-interstitial NOV expression in patients with tubulo-interstitial nephritis [52]. While this is the first report of a beneficial effect of NOV downregulation with respect to inflammation and renal fibrosis, previous studies demonstrated contradictory results by showing anti-inflammatory [53] and anti-fibrotic [54] NOV effects. The anti-inflammatory potential of NOV inhibition, however, is further corroborated by the fact that systemic overexpression of NOV increases MCP-1/CCL2 expression among nephritic rats [55]. It has been speculated that NOV might exert both pro- and anti-fibrotic effects depending on different cellular adaptive processes in experimental models [52]. It remains to be elucidated whether Empa-mediated downregulation of NOV mechanistically adds to the anti-inflammatory and anti-fibrotic potential of SGLT2i.

3.4. Empa-Mediated Inhibition of IL-1β-Induced PTX3 Expression

Pentraxins are multimeric pattern recognition proteins that can be divided into short (e.g., CRP and serum amyloid A) and long (e.g., pentraxin-3) isoforms. Pentraxin-3 (PTX3) is expressed in response to pro-inflammatory signals (e.g., IL-1, TNF-a, TLR-activation, LPS) by a variety of tissue and cell types, including innate immunity, as well as endothelial cells [56]. PTX3 is a multifunctional acute phase protein that is elevated in cardiovascular, autoimmune, and pulmonary disease [57,58,59], and has been described as independent marker of inflammatory activity [60,61]. PTX3 production has already been demonstrated in HPTCs [62], and elevated PTX3 expression levels have been documented in mesangial cells of patients with IgA nephropathy [63]. It has been speculated that PTX3 augments the renal inflammatory response as well as the atherogenic process during CKD [64]. PTX3 correlates negatively with GFR and is associated with cardiovascular disease, inflammation, and protein-energy wasting [64]. In CKD patients, PTX3 levels independently predict all-cause mortality [64]. It remains to be elucidated whether PTX3 is only a marker of renal inflammation or causally involved in CKD pathogenesis. Further mechanistic studies are mandatory to evaluate if an Empa-mediated inhibition of IL-1β-stimulated tubular PTX3 expression conveys nephroprotective effects or not. Interestingly, patients with IgA nephropathy (that have been demonstrated to present with high PTX3 levels) were highly represented among those non-diabetic CKD patients that showed a SGLT2i-mediated benefit [6].

3.5. Empa-Mediated Inhibition of IL-1β-Induced SGK1 Expression

Serum and glucocorticoid-induced kinase 1 (SGK1) is a ubiquitously expressed, cell-volume-regulated gene stimulated by various growth factors and hormones, including mineralo- and glucocorticoids. SGK1 regulates transepithelial ion transport by orchestrating trafficking of ion channels and transporters, thereby increasing both sodium reabsorption and potassium excretion [65,66]. It has been implicated in blood pressure control as well as the development of inflammatory and fibrotic processes [67]. Genetic or pharmacologic inhibition of SGK1 attenuates renal inflammation, proteinuria, and fibrosis in response to mineralocorticoids [68,69]. Its pleiotropic role in kidney disease is further supported by the fact that SGK1 is associated with aldosterone-induced oxidative stress and podocyte injury [70]. Being expressed in antigen-presenting T-cells, it has been implicated in renal inflammatory processes [71]. Increased SGK1 activity has been suggested to play a pathophysiologic role in kidney damage and CKD progression both in the presence and absence of hypertension [66]. It has been previously speculated that SGK1 might be a potential target to slow CKD progression [66]. By demonstrating an EMPA-mediated inhibition of IL-1β-induced SGK1 expression, this effect might be a potential explanation for the positive clinical effect seen with the use of SGLT2i in CKD patients irrespective of the presence and absence of diabetes.
In summary, our transcriptomic approach reveals 19 inflammatory response genes, such as CXCL8/IL8, LOX, NOV, PTX3, and SGK1, that might be causally involved in glycemia-independent nephroprotection by SGLT2i. Further molecular analyses on mRNA and protein level are mandatory to confirm our transcriptomic findings in HPTCs. Given the lack of data regarding SGLT2i effects at the human tissue level, it has been previously stated that kidney biopsy studies among SGLT2i-naive vs. SGLT2i-treated patients are inevitable to confirm a causal relationship between anti-inflammatory and nephroprotective potential of SGLT2i. In this regard, primary inflammatory kidney diseases, such as IgA nephropathy and acute interstitial nephritis, have been proposed to be most suitable for these future clinical trials [72].

4. Materials and Methods

4.1. Cell Culture

Human kidney 2 (HK-2) cells were purchased from American Type Culture Collection (Rockville, MD, USA) and RPTEC/TERT1 cells from Evercyte GmBH (Vienna, Austria). HPTCs were cultured under normoglycemic conditions as previously described in detail [73,74,75,76,77]. Cell culture media were obtained from Gibco Life Technologies (Paisley, UK) via Thermo Fisher Scientific (Waltham, MA, USA), and supplements and reagents from Sigma Aldrich Productions GmbH (Steinheim, Germany) and Roche (Basel, Switzerland). Empa was obtained from Selleckchem (Houston, TX, USA) and recombinant human IL‑1β from R&D Systems (Minneapolis, MN, USA) via Biomedica Medizinprodukte GmbH (Vienna, Austria). Empa was solubilized in dimethyl sulfoxide (DMSO) to a stock solution of 500 yM and diluted for further experiments to a final concentration of 500 nM (1:1000) containing <0.1% DMSO. IL-1β was solubilized in phosphate-buffered saline (PBS) to a stock solution of 10 yg/mL and diluted for further experiments to a final concentration of 10 ng/mL (1:1000). After 24 h serum and supplement starvation and an additional medium change, cells were either used for experiments or left untreated (control). Then, 24 h stimulation with IL‑1β (10 ng/mL) and the combination of IL-1β (10 ng/mL) and Empa (500 nM) was performed in the absence of any serum and growth supplements. As described previously [50], a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Cell Proliferation Kit I, Roche Diagnostics GmbH, Penzberg, Germany) was used to assess cell viability for each experimental condition according to manufacturer instructions. No significant difference in cell viability was observed between the experimental conditions.

4.2. Microarray Hybridization Analysis

Agilent oligonucleotide microarrays were used according to the Agilent One-Color Microarray protocol (Agilent Technologies, Santa Clara, CA, USA). A detailed description has been published previously [50]. Briefly, 200 ng of total RNA isolated from HPTCs by using the innuPREP RNA Mini Kit 2.0 (Analytic Jena AG, Jena, Germany) was used for hybridization. Microarray hybridization analysis was conducted in biological duplicates for each experimental condition and followed the Minimum Information About a Microarray Experiment (MIAME) guidelines [78]. The complete microarray data sets are available at Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) (accession numbers: GSE155867 and GSE169683).

4.3. Pathway Enrichment Analysis

Pathway enrichment analysis was conducted for differentially expressed genes in HPTCs (i.e., IL-1β-induced genes inhibited by Empa) by using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). This bioinformatics database provides functional gene annotation by utilizing a biological knowledgebase and analytic data-mining tools to systematically extract biological meaning from large gene lists [79].

4.4. Statistical Analysis

The expression analysis systematic explorer (EASE) score, a statistical test for over-representation of annotation classes, was calculated to statistically evaluate each annotation term. Statistically significant over-representation of annotation classes is reflected by an EASE score ≤0.05. The overall enrichment score, that is, the geometric mean of all annotation term p-values within an annotation cluster, was calculated to reflect biological significance [79].

Author Contributions

Concept and study design, M.P. and H.S.; data acquisition, P.S. and U.C.; data interpretation, M.P., S.S., J.L., and H.S.; figure preparation, M.P. and S.S.; resources, M.P. and G.M.; data curation, M.P.; writing—original draft preparation, M.P.; writing—review and editing, M.P., G.M., and H.S.; supervision, M.P.; project administration, M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete microarray data set has been submitted to the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) (accession number: GSE155867 and GSE169683).

Conflicts of Interest

M.P., S.S., P.S., U.C., J.L., and H.S. declare no conflict of interest; G.M. received travel support, consulting, and lecture fees from Boehringer Ingelheim and Astra Zeneca.

References

  1. Wanner, C.; Inzucchi, S.E.; Zinman-Parving, H.-H.; Lambers-Heerspink, H.; de Zeeuw, D. Empagliflozin and Progression of Kidney Disease in Type 2 Diabetes. N. Engl. J. Med. 2016, 375, 1801–1802. [Google Scholar] [CrossRef] [PubMed]
  2. Perkovic, V.; de Zeeuw, D.; Mahaffey, K.W.; Fulcher, G.; Erondu, N.; Shaw, W.; Barrett, T.D.; Weidner-Wells, M.; Deng, H.; Matthews, D.R.; et al. Canagliflozin and renal outcomes in type 2 diabetes: Results from the CANVAS Program randomised clinical trials. Lancet Diabetes Endocrinol. 2018, 6, 691–704. [Google Scholar] [CrossRef]
  3. Mosenzon, O.; Wiviott, S.D.; Cahn, A.; Rozenberg, A.; Yanuv, I.; Goodrich, E.L.; Murphy, S.A.; Heerspink, H.J.L.; Zelniker, T.A.; Dwyer, J.P.; et al. Effects of dapagliflozin on development and progression of kidney disease in patients with type 2 diabetes: An analysis from the DECLARE–TIMI 58 randomised trial. Lancet Diabetes Endocrinol. 2019, 7, 606–617. [Google Scholar] [CrossRef]
  4. Perkovic, V.; Jardine, M.J.; Neal, B.; Bompoint, S.; Heerspink, H.J.; Charytan, D.M.; Edwards, R.; Agarwal, R.; Bakris, G.; Bull, S.; et al. Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy. N. Engl. J. Med. 2019, 380, 2295–2306. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Heerspink, H.J.L.; Kosiborod, M.; Inzucchi, S.E.; Cherney, D.Z.I. Renoprotective effects of sodium-glucose cotransporter-2 inhibitors. Kidney Int. 2018, 94, 26–39. [Google Scholar] [CrossRef] [PubMed]
  6. Heerspink, H.J.L.; Stefánsson, B.V.; Correa-Rotter, R.; Chertow, G.M.; Greene, T.; Hou, F.-F.; Mann, J.F.E.; McMurray, J.J.V.; Lindberg, M.; Rossing, P.; et al. Dapagliflozin in Patients with Chronic Kidney Disease. N. Engl. J. Med. 2020, 383, 1436–1446. [Google Scholar] [CrossRef]
  7. Packer, M.; Anker, S.D.; Butler, J.; Filippatos, G.; Pocock, S.J.; Carson, P.; Januzzi, J.; Verma, S.; Tsutsui, H.; Brueckmann, M.; et al. Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure. N. Engl. J. Med. 2020, 383, 1413–1424. [Google Scholar] [CrossRef]
  8. Lv, W.; Booz, G.W.; Wang, Y.; Fan, F.; Roman, R.J. Inflammation and renal fibrosis: Recent developments on key signaling molecules as potential therapeutic targets. Eur. J. Pharmacol. 2018, 820, 65–76. [Google Scholar] [CrossRef]
  9. Kawanami, D.; Matoba, K.; Takeda, Y.; Nagai, Y.; Akamine, T.; Yokota, T.; Sango, K.; Utsunomiya, K. SGLT2 Inhibitors as a Therapeutic Option for Diabetic Nephropathy. Int. J. Mol. Sci. 2017, 18, 1083. [Google Scholar] [CrossRef]
  10. Wang, X.X.; Levi, J.; Luo, Y.; Myakala, K.; Herman-Edelstein, M.; Qiu, L.; Wang, D.; Peng, Y.; Grenz, A.; Lucia, S.; et al. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy: SGLT2 Protein Inhibition Decreases Renal Lipid Accumulation, Inflammation, and the Development of Nephropathy in Diabetic Mice. J. Biol. Chem. 2017, 292, 5335–5348. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. Ishibashi, Y.; Matsui, T.; Yamagishi, S. Tofogliflozin, A Highly Selective Inhibitor of SGLT2 Blocks Proinflammatory and Proapoptotic Effects of Glucose Overload on Proximal Tubular Cells Partly by Suppressing Oxidative Stress Generation. Horm. Metab. Res. 2015, 48, 191–195. [Google Scholar] [CrossRef] [PubMed]
  12. Heerspink, H.J.L.; Perco, P.; Mulder, S.; Leierer, J.; Hansen, M.K.; Heinzel, A.; Mayer, G. Canagliflozin reduces inflammation and fibrosis biomarkers: A potential mechanism of action for beneficial effects of SGLT2 inhibitors in diabetic kidney disease. Diabetologia 2019, 62, 1154–1166. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Dekkers, C.C.J.; Petrykiv, S.; Laverman, G.D.; Cherney, D.Z.; Gansevoort, R.T.; Heerspink, H.J.L. Effects of the SGLT-2 inhibitor dapagliflozin on glomerular and tubular injury markers. Diabetes Obes. Metab. 2018, 20, 1988–1993. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Panchapakesan, U.; Pegg, K.; Gross, S.; Komala, M.G.; Mudaliar, H.; Forbes, J.; Pollock, C.; Mather, A. Effects of SGLT2 Inhibition in Human Kidney Proximal Tubular Cells—Renoprotection in Diabetic Nephropathy? PLoS ONE 2013, 8, e54442. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Gembardt, F.; Bartaun, C.; Jarzebska, N.; Mayoux, E.; Todorov, V.T.; Hohenstein, B.; Hugo, C. The SGLT2 inhibitor empagliflozin ameliorates early features of diabetic nephropathy in BTBR ob/ob type 2 diabetic mice with and without hypertension. Am. J. Physiol. Renal. Physiol. 2014, 307, F317–F325. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Kitagawa, K.; Wada, T.; Furuichi, K.; Hashimoto, H.; Ishiwata, Y.; Asano, M.; Takeya, M.; Kuziel, W.A.; Matsushima, K.; Mukaida, N.; et al. Blockade of CCR2 Ameliorates Progressive Fibrosis in Kidney. Am. J. Pathol. 2004, 165, 237–246. [Google Scholar] [CrossRef] [Green Version]
  17. Sassy-Prigent, C.; Heudes, D.; Mandet, C.; Belair, M.F.; Michel, O.; Perdereau, B.; Bariety, J.; Bruneval, P. Early glomerular macrophage recruitment in streptozotocin-induced diabetic rats. Diabetes 2000, 49, 466–475. [Google Scholar] [CrossRef] [Green Version]
  18. Giunti, S.; Barutta, F.; Perin, P.C.; Gruden, G. Targeting the MCP-1/CCR2 System in diabetic kidney disease. Curr. Vasc. Pharmacol. 2010, 8, 849–860. [Google Scholar] [CrossRef] [PubMed]
  19. Tam, F.W.; Riser, B.L.; Meeran, K.; Rambow, J.; Pusey, C.D.; Frankel, A.H. Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy. Cytokine 2009, 47, 37–42. [Google Scholar] [CrossRef] [PubMed]
  20. Satirapoj, B.; Dispan, R.; Radinahamed, P.; Kitiyakara, C. Urinary epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors for rapid loss of renal function in type 2 diabetic patients with diabetic kidney disease. BMC Nephrol. 2018, 19, 246. [Google Scholar] [CrossRef] [Green Version]
  21. Kohan, D.E.; Pollock, D.M. Endothelin antagonists for diabetic and non-diabetic chronic kidney disease. Br. J. Clin. Pharmacol. 2012, 76, 573–579. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Boels, M.G.; Koudijs, A.; Avramut, M.C.; Sol, W.M.; Wang, G.; van Oeveren-Rietdijk, A.M.; van Zonneveld, A.J.; de Boer, H.C.; van der Vlag, J.; van Kooten, C.; et al. Systemic Monocyte Chemotactic Protein-1 Inhibition Modifies Renal Macrophages and Restores Glomerular Endothelial Glycocalyx and Barrier Function in Diabetic Nephropathy. Am. J. Pathol. 2017, 187, 2430–2440. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Chow, F.; Nikolic-Paterson, D.; Ozols, E.; Atkins, R.; Rollin, B.; Tesch, G. Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice. Kidney Int. 2006, 69, 73–80. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Tarabra, E.; Giunti, S.; Barutta, F.; Salvidio, G.; Burt, D.; Deferrari, G.; Gambino, R.; Vergola, D.; Pinach, S.; Perin, P.C.; et al. Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes. Diabetes 2009, 58, 2109–2118. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Dhaun, N.; Webb, D.J. Endothelins in cardiovascular biology and therapeutics. Nat. Rev. Cardiol. 2019, 16, 1–502. [Google Scholar] [CrossRef] [PubMed]
  26. Spires, D.; Poudel, B.; Shields, C.A.; Pennington, A.; Fizer, B.; Taylor, L.; McPherson, K.C.; Cornelius, D.C.; Williams, J.M. Prevention of the progression of renal injury in diabetic rodent models with preexisting renal disease with chronic endothelin A receptor blockade. Am. J. Physiol. Renal. Physiol. 2018, 315, F977–F985. [Google Scholar] [CrossRef] [PubMed]
  27. Menne, J.; Eulberg, D.; Beyer, D.; Baumann, M.; Saudek, F.; Valkusz, Z.; Więcek, A.; Haller, H. C-C motif-ligand 2 inhibition with emapticap pegol (NOX-E36) in type 2 diabetic patients with albuminuria. Nephrol. Dial. Transplant. 2016, 32, 307–315. [Google Scholar] [CrossRef] [Green Version]
  28. Wenzel, R.R.; Littke, T.; Kuranoff, S.; Jürgens, C.; Bruck, H.; Ritz, E.; Philipp, T.; Mitchell, A. Avosentan Reduces Albumin Excretion in Diabetics with Macroalbuminuria. J. Am. Soc. Nephrol. 2009, 20, 655–664. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Kohan, D.E.; Pritchett, Y.; Molitch, M.; Wen, S.; Garimella, T.; Audhya, P.; Andress, D.L. Addition of Atrasentan to Renin-Angiotensin System Blockade Reduces Albuminuria in Diabetic Nephropathy. J. Am. Soc. Nephrol. 2011, 22, 763–772. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. De Zeeuw, D.; Coll, B.; Andress, D.; Brennan, J.J.; Tang, H.; Houser, M.; Correa-Rotter, R.; Kohan, D.; Lambers, H.H.J.L.; Makino, H.; et al. The Endothelin Antagonist Atrasentan Lowers Residual Albuminuria in Patients with Type 2 Diabetic Nephropathy. J. Am. Soc. Nephrol. 2014, 25, 1083–1093. [Google Scholar] [CrossRef] [Green Version]
  31. Heerspink, H.J.L.; Parving, H.-H.; Andress, D.L.; Bakris, G.; Correa-Rotter, R.; Hou, F.-F.; Kitzman, D.W.; Kohan, D.; Makino, H.; McMurray, J.J.V.; et al. Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): A double-blind, randomised, placebo-controlled trial. Lancet 2019, 393, 1937–1947. [Google Scholar] [CrossRef]
  32. Maayah, Z.H.; Ferdaoussi, M.; Takahara, S.; Soni, S.; Dyck, J.R.B. Empagliflozin suppresses inflammation and protects against acute septic renal injury. Inflammopharmacology 2021, 29, 269–279. [Google Scholar] [CrossRef]
  33. Pirklbauer, M.; Bernd, M.; Fuchs, L.; Staudinger, P.; Corazza, U.; Leierer, J.; Mayer, G.; Schramek, H. Empagliflozin Inhibits Basal and IL-1β-Mediated MCP-1/CCL2 and Endothelin-1 Expression in Human Proximal Tubular Cells. Int. J. Mol. Sci. 2020, 21, 8189. [Google Scholar] [CrossRef] [PubMed]
  34. Tesch, G.H. Diabetic nephropathy—Is this an immune disorder? Clin. Sci. 2017, 131, 2183–2199. [Google Scholar] [CrossRef] [PubMed]
  35. Vallon, V.; Thomson, S.C. The tubular hypothesis of nephron filtration and diabetic kidney disease. Nat. Rev. Nephrol. 2020, 16, 317–336. [Google Scholar] [CrossRef] [PubMed]
  36. Chevalier, R.L. The proximal tubule is the primary target of injury and progression of kidney disease: Role of the glomerulotubular junction. Am. J. Physiol. Renal. Physiol. 2016, 311, F145–F161. [Google Scholar] [CrossRef]
  37. Anders, H.-J. Of Inflammasomes and Alarmins: IL-1β and IL-1α in Kidney Disease. J. Am. Soc. Nephrol. 2016, 27, 2564–2575. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Shahzad, K.; Bock, F.; Dong, W.; Wang, H.; Kopf, S.; Kohli, S.; Al-Dabet, M.M.; Ranjan, S.; Wolter, J.; Wacker, C.; et al. Nlrp3-inflammasome activation in non-myeloid-derived cells aggravates diabetic nephropathy. Kidney Int. 2015, 87, 74–84. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Lei, Y.; Devarapu, S.K.; Motrapu, M.; Cohen, C.D.; Lindenmeyer, M.T.; Moll, S.; Kumar, S.V.; Anders, H.-J. Interleukin-1β Inhibition for Chronic Kidney Disease in Obese Mice with Type 2 Diabetes. Front. Immunol. 2019, 10, 1223. [Google Scholar] [CrossRef]
  40. Wu, F.; Sun, C.; Lu, J. The Role of Chemokine Receptors in Renal Fibrosis. Rev. Physiol. Biochem. Pharmacol. 2020, 177, 1–24. [Google Scholar] [CrossRef] [PubMed]
  41. Segerer, S.; Nelson, P.J.; Schlöndorff, D. Chemokines, Chemokine Receptors, and Renal Disease: From Basic Science to Pathophysiologic and Therapeutic Studies. J. Am. Soc. Nephrol. 2000, 11, 152–176. [Google Scholar] [CrossRef] [PubMed]
  42. Wong, C.K.; Ho, A.W.Y.; Tong, P.C.Y.; Yeung, C.Y.; Kong, A.P.S.; Lun, S.W.M.; Chan, J.C.N.; Lam, C.W.K. Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy. Clin. Exp. Immunol. 2007, 149, 123–131. [Google Scholar] [CrossRef] [PubMed]
  43. Zhao, Y.; Zhu, L.; Zhou, T.; Zhang, Q.; Shi, S.; Liu, L.; Lv, J.; Zhang, H. Urinary CXCL1: A Novel Predictor of IgA Nephropathy Progression. PLoS ONE 2015, 10, e0119033. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Holdsworth, S.R.; Kitching, A.R.; Tipping, P.G. Chemokines as therapeutic targets in renal disease. Curr. Opin. Nephrol. Hypertens. 2000, 9, 505–511. [Google Scholar] [CrossRef]
  45. Svensson, M.; Yadav, M.; Holmqvist, B.; Lutay, N.; Svanborg, C.; Godaly, G. Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice. Kidney Int. 2011, 80, 1064–1072. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Cui, S.; Zhu, Y.; Du, J.; Khan, M.N.; Wang, B.; Wei, J.; Cheng, J.-W.; Gordon, J.R.; Mu, Y.; Li, F. CXCL8 Antagonist Improves Diabetic Nephropathy in Male Mice with Diabetes and Attenuates High Glucose–Induced Mesangial Injury. Endocrinology 2017, 158, 1671–1684. [Google Scholar] [CrossRef] [PubMed]
  47. Kagan, H.M.; Li, W. Lysyl oxidase: Properties, specificity, and biological roles inside and outside of the cell. J. Cell. Biochem. 2003, 88, 660–672. [Google Scholar] [CrossRef] [PubMed]
  48. Kumari, S.; Panda, T.K.; Pradhan, T. Lysyl Oxidase: Its Diversity in Health and Diseases. Indian J. Clin. Biochem. 2016, 32, 134–141. [Google Scholar] [CrossRef] [PubMed]
  49. Zhang, X.-Q.; Li, X.; Zhou, W.-Q.; Liu, X.; Huang, J.-L.; Zhang, Y.-Y.; Lindholm, B.; Yu, C. Serum Lysyl Oxidase Is a Potential Diagnostic Biomarker for Kidney Fibrosis. Am. J. Nephrol. 2020, 51, 907–918. [Google Scholar] [CrossRef]
  50. Pirklbauer, M.; Schupart, R.; Fuchs, L.C.; Staudinger, P.; Corazza, U.; Sallaberger, S.; Leierer, J.; Mayer, G.; Schramek, H. Unraveling reno-protective effects of SGLT2 inhibition in human proximal tubular cells. Am. J. Physiol. Renal. Physiol. 2019, 316, F449–F462. [Google Scholar] [CrossRef] [PubMed]
  51. Chen, C.-C.; Lau, L.F. Functions and mechanisms of action of CCN matricellular proteins. Int. J. Biochem. Cell Biol. 2009, 41, 771–783. [Google Scholar] [CrossRef] [Green Version]
  52. Marchal, P.-O.; Kavvadas, P.; Abed, A.; Kazazian, C.; Authier, F.; Koseki, H.; Hiraoka, S.; Boffa, J.-J.; Martinerie, C.; Chadjichristos, C.E. Reduced NOV/CCN3 Expression Limits Inflammation and Interstitial Renal Fibrosis after Obstructive Nephropathy in Mice. PLoS ONE 2015, 10, e0137876. [Google Scholar] [CrossRef]
  53. Lin, Z.; Natesan, V.; Shi, H.; Hamik, A.; Kawanami, D.; Hao, C.; Mahabaleshwar, G.H.; Wang, W.; Jin, Z.-G.; Atkins, G.B.; et al. A novel role of CCN3 in regulating endothelial inflammation. J. Cell Commun. Signal. 2010, 4, 141–153. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Kular, L.; Pakradouni, J.; Kitabgi, P.; Laurent, M.; Martinerie, C. The CCN family: A new class of inflammation modulators? Biochimie 2011, 93, 377–388. [Google Scholar] [CrossRef] [PubMed]
  55. Van Roeyen, C.R.; Boor, P.; Borkham-Kamphorst, E.; Rong, S.; Kunter, U.; Martin, I.V.; Kaitovic, A.; Fleckenstein, S.; Perbal, B.; Trautwein, C.; et al. A Novel, Dual Role of CCN3 in Experimental Glomerulonephritis: Pro-angiogenic and antimesangioproliferative effects. Am. J. Pathol. 2012, 180, 1979–1990. [Google Scholar] [CrossRef] [PubMed]
  56. Bottazzi, B.; Doni, A.; Garlanda, C.; Mantovani, A. An Integrated View of Humoral Innate Immunity: Pentraxins as a Paradigm. Annu. Rev. Immunol. 2010, 28, 157–183. [Google Scholar] [CrossRef] [PubMed]
  57. Kunes, P.; Holubcova, Z.; Kolackova, M.; Krejsek, J. Pentraxin 3(PTX 3): An Endogenous Modulator of the Inflammatory Response. Mediat. Inflamm. 2012, 2012, 920517. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. He, X.; Han, B.; Bai, X.; Zhang, Y.; Cypel, M.; Mura, M.; Keshavjee, S.; Liu, M. PTX3 as a potential biomarker of acute lung injury: Supporting evidence from animal experimentation. Intensiv. Care Med. 2010, 36, 356–364. [Google Scholar] [CrossRef]
  59. Van Rossum, A.P.; Pas, H.H.; Fazzini, F.; Huitema, M.G.; Limburg, P.C.; Jonkman, M.F.; Kallenberg, C.G.M. Abundance of the long pentraxin PTX3 at sites of leukocytoclastic lesions in patients with small-vessel vasculitis. Arthritis Rheum. 2006, 54, 986–991. [Google Scholar] [CrossRef]
  60. Fazzini, F.; Peri, G.; Doni, A.; Dell’Antonio, G.; Cin, E.D.; Bozzolo, E.; D’Auria, F.; Praderio, L.; Ciboddo, G.; Sabbadini, M.G.; et al. PTX3 in small-vessel vasculitides: An independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 2001, 44, 2841–2850. [Google Scholar] [CrossRef]
  61. Guo, T.; Huang, L.; Liu, C.; Shan, S.; Li, Q.; Ke, L.; Cheng, B. The clinical value of inflammatory biomarkers in coronary artery disease: PTX3 as a new inflammatory marker. Exp. Gerontol. 2017, 97, 64–67. [Google Scholar] [CrossRef] [PubMed]
  62. Nauta, A.J.; de Haij, S.; Bottazzi, B.; Mantovani, A.; Borrias, M.C.; Aten, J.; Rastaldi, M.P.; Daha, M.R.; Van Kooten, C.; Roos, A. Human renal epithelial cells produce the long pentraxin PTX3. Kidney Int. 2005, 67, 543–553. [Google Scholar] [CrossRef] [Green Version]
  63. Bussolati, B.; Peri, G.; Salvidio, G.; Verzola, D.; Mantovani, A.; Camussi, G. The Long Pentraxin Ptx3 Is Synthesized in IgA Glomerulonephritis and Activates Mesangial Cells. J. Immunol. 2003, 170, 1466–1472. [Google Scholar] [CrossRef] [Green Version]
  64. Tong, M.; Carrero, J.J.; Qureshi, A.R.; Anderstam, B.; Heimbürger, O.; Bárány, P.; Axelsson, J.; Alvestrand, A.; Stenvinkel, P.; Lindholm, B.; et al. Plasma Pentraxin 3 in Patients with Chronic Kidney Disease: Associations with Renal Function, Protein-Energy Wasting, Cardiovascular Disease, and Mortality. Clin. J. Am. Soc. Nephrol. 2007, 2, 889–897. [Google Scholar] [CrossRef] [Green Version]
  65. Lang, F.; Böhmer, C.; Palmada, M.; Seebohm, G.; Strutz-Seebohm, N.; Vallon, V. (Patho)physiological Significance of the Serum- and Glucocorticoid-Inducible Kinase Isoforms. Physiol. Rev. 2006, 86, 1151–1178. [Google Scholar] [CrossRef]
  66. Sierra-Ramos, C.; Velazquez-Garcia, S.; Keskus, A.G.; Vastola-Mascolo, A.; Rodríguez-Rodríguez, A.E.; Luis-Lima, S.; Hernández, G.; Navarro-González, J.F.; Porrini, E.; Konu, O.; et al. Increased SGK1 activity potentiates mineralocorticoid/NaCl-induced kidney injury. Am. J. Physiol. Renal. Physiol. 2021, 320, F628–F643. [Google Scholar] [CrossRef] [PubMed]
  67. Artunc, F.; Lang, F. Mineralocorticoid and SGK1-Sensitive Inflammation and Tissue Fibrosis. Nephron Physiol. 2014, 128, 35–39. [Google Scholar] [CrossRef]
  68. Artunc, F.; Amann, K.; Nasir, O.; Friedrich, B.; Sandulache, D.; Jahovic, N.; Risler, T.; Vallon, V.; Wulff, P.; Kuhl, D.; et al. Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1. J. Mol. Med. 2006, 84, 737–746. [Google Scholar] [CrossRef] [PubMed]
  69. Vallon, V.; Huang, D.Y.; Grahammer, F.; Wyatt, A.W.; Osswald, H.; Wulff, P.; Kuhl, D.; Lang, F. SGK1 as a determinant of kidney function and salt intake in response to mineralocorticoid excess. Am. J. Physiol. Integr. Comp. Physiol. 2005, 289, R395–R401. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Shibata, S.; Nagase, M.; Yoshida, S.; Kawachi, H.; Fujita, T. Podocyte as the Target for Aldosterone: Roles of oxidative stress and Sgk1. Hypertension 2007, 49, 355–364. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Norlander, A.E.; Saleh, M.A.; Pandey, A.K.; Itani, H.A.; Wu, J.; Xiao, L.; Kang, J.; Dale, B.L.; Goleva, S.B.; Laroumanie, F.; et al. A salt-sensing kinase in T lymphocytes, SGK1, drives hypertension and hypertensive end-organ damage. JCI Insight 2017, 2. [Google Scholar] [CrossRef]
  72. Pirklbauer, M. Anti-inflammatory potential of Empagliflozin. Inflammopharmacology 2021, 29, 573–576. [Google Scholar] [CrossRef]
  73. Pollack, V.; Sarközi, R.; Banki, Z.; Feifel, E.; Wehn, S.; Gstraunthaler, G.; Stoiber, H.; Mayer, G.; Montesano, R.; Strutz, F.; et al. Oncostatin M-induced effects on EMT in human proximal tubular cells: Differential role of ERK signaling. Am. J. Physiol. Renal. Physiol. 2007, 293, F1714–F1726. [Google Scholar] [CrossRef]
  74. Ryan, M.J.; Johnson, G.; Kirk, J.; Fuerstenberg, S.M.; Zager, R.A.; Torok-Storb, B. HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int. 1994, 45, 48–57. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Sarközi, R.; Flucher, K.; Haller, V.M.; Pirklbauer, M.; Mayer, G.; Schramek, H. Oncostatin M inhibits TGF-β1-induced CTGF expression via STAT3 in human proximal tubular cells. Biochem. Biophys. Res. Commun. 2012, 424, 801–806. [Google Scholar] [CrossRef] [PubMed]
  76. Sarközi, R.; Hauser, C.; Noppert, S.-J.; Kronbichler, A.; Pirklbauer, M.; Haller, V.M.; Grillari, J.; Grillari-Voglauer, R.; Mayer, G.; Schramek, H.; et al. Oncostatin M is a novel inhibitor of TGF-β1-induced matricellular protein expression. Am. J. Physiol. Renal. Physiol. 2011, 301, F1014–F1025. [Google Scholar] [CrossRef]
  77. Wieser, M.; Stadler, G.; Jennings, P.; Streubel, B.; Pfaller, W.; Ambros, P.; Riedl, C.; Katinger, H.; Grillari, J.; Grillari-Voglauer, R. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am. J. Physiol. Renal. Physiol. 2008, 295, F1365–F1375. [Google Scholar] [CrossRef] [Green Version]
  78. Brazma, A.; Hingamp, P.; Quackenbush, J.; Sherlock, G.; Spellman, P.; Stoeckert, C.; Aach, J.; Ansorge, W.; Ball, C.A.; Causton, H.C.; et al. Minimum information about a microarray experiment (MIAME)—toward standards for microarray data. Nat. Genet. 2001, 29, 365–371. [Google Scholar] [CrossRef] [PubMed]
  79. Huang da, W.; Sherman, B.T.; Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009, 4, 44–57. [Google Scholar] [CrossRef]
Table 1. Inflammatory response genes attenuated by empagliflozin in two independent human proximal tubular cell lines, namely HK-2 (A) and RPTEC/TERT1 (B).
Table 1. Inflammatory response genes attenuated by empagliflozin in two independent human proximal tubular cell lines, namely HK-2 (A) and RPTEC/TERT1 (B).
AHK-2 (Experiment 1)HK-2 (Experiment 2)
Gene NameFold Change to ControlFold Change to IL-1βFold Change to ControlFold Change to IL-1β
IL-1βIL-1β + EMPAIL-1β + EMPAIL-1βIL-1β + EMPAIL-1β + EMPA
ATP binding cassette subfamily A member 12 (ABCA12)2.231.83−0.402.391.28−1.11
Rho GTPase activating protein 22 (ARHGAP22)2.171.57−0.601.810.89−0.93
CD82 molecule (CD82)2.431.14−1.293.352.86−0.50
colony stimulating factor 2 (CSF2)4.523.43−1.094.133.53−0.60
C-X-C motif chemokine ligand 1 (CXCL1)6.896.81−0.083.953.34−0.61
C-X-C motif chemokine ligand 2 (CXCL2)7.176.92−0.254.654.13−0.52
C-X-C motif chemokine ligand 5 (CXCL5)5.305.29−0.022.982.63−0.35
C-X-C motif chemokine ligand 8 (CXCL8)6.296.21−0.084.093.71−0.38
insulin induced gene 1
(INSIG1)
3.973.87−0.094.523.88−0.65
lysyl oxidase (LOX)1.741.29−0.451.800.65−1.15
nephroblastoma overexpressed (NOV)1.900.62−1.272.370.75−1.63
prolyl 4-hydroxylase subunit alpha 2 (P4HA2)2.562.12−0.442.271.41−0.86
Pim-2 proto-oncogene, serine/threonine kinase (PIM2)2.972.92−0.053.092.89−0.19
pentraxin 3 (PTX3)3.542.43−1.112.872.17−0.70
serum/glucocorticoid regulated kinase 1 (SGK1)2.141.46−0.671.910.76−1.15
solute carrier organic anion transporter family member 2A1 (SLCO2A1)2.041.78−0.261.62−0.04−1.66
solute carrier organic anion transporter family member 4A1 (SLCO4A1)5.594.86−0.731.741.07−0.67
superoxide dismutase 2
(SOD2)
5.925.83−0.092.412.21−0.20
tissue factor pathway inhibitor 2 (TFPI2)3.703.22−0.472.251.69−0.57
BRPTEC/TERT1 (Experiment 1)RPTEC/TERT1 (Experiment 2)
Gene NameFold Change to ControlFold Change to IL-1βFold Change to ControlFold Change to IL-1β
IL-1βIL-1β + EMPAIL-1β + EMPAIL-1βIL-1β + EMPAIL-1β + EMPA
ATP binding cassette subfamily A member 12 (ABCA12)2.111.29−0.823.903.23−0.67
Rho GTPase activating protein 22 (ARHGAP22)1.930.79−1.142.942.27−0.68
CD82 molecule (CD82)3.702.99−0.712.651.42−1.24
colony stimulating factor 2 (CSF2)3.983.50−0.496.955.85−1.11
C-X-C motif chemokine ligand 1 (CXCL1)3.712.96−0.767.627.53−0.09
C-X-C motif chemokine ligand 2 (CXCL2)4.684.08−0.607.377.30−0.07
C-X-C motif chemokine ligand 5 (CXCL5)3.082.72−0.365.755.63−0.11
C-X-C motif chemokine ligand 8 (CXCL8)3.533.03−0.497.877.73−0.13
insulin induced gene 1
(INSIG1)
2.371.67−0.704.584.47−0.11
lysyl oxidase (LOX)1.530.27−1.263.582.97−0.61
nephroblastoma overexpressed (NOV)2.230.25−1.982.030.83−1.20
prolyl 4-hydroxylase subunit alpha 2 (P4HA2)2.291.88−0.402.401.85−0.55
Pim-2 proto-oncogene, serine/threonine kinase (PIM2)3.183.00−0.183.783.70−0.07
pentraxin 3 (PTX3)2.191.57−0.615.244.50−0.74
serum/glucocorticoid regulated kinase 1 (SGK1)1.640.38−1.262.181.91−0.27
solute carrier organic anion transporter family member 2A1 (SLCO2A1)1.940.13−1.812.952.38−0.57
solute carrier organic anion transporter family member 4A1 (SLCO4A1)2.081.51−0.575.895.23−0.67
superoxide dismutase 2
(SOD2)
2.622.46−0.155.805.73−0.07
tissue factor pathway inhibitor 2 (TFPI2)2.321.12−1.203.803.23−0.58
Inflammatory response genes were selected based on an IL-1β (10 ng/mL)-induced gene expression fold change >1.5 compared to control. Only genes with a uniform expression pattern in both HK-2 and RPTEC/TERT1 cells, that is, inhibition of IL-1β-induced gene expression by empagliflozin (500 nM) in two independent experiments per cell line (n = 2, each) are presented.
Table 2. Effect of empagliflozin (500 nM) on SGLT2 gene expression in IL-1β (10 ng/mL)-stimulated HK-2 (A) and RPTEC/TERT1 (B) cells.
Table 2. Effect of empagliflozin (500 nM) on SGLT2 gene expression in IL-1β (10 ng/mL)-stimulated HK-2 (A) and RPTEC/TERT1 (B) cells.
A HK-2 (Experiment 1)HK-2 (Experiment 2)
Gene NameFold Change to ControlFold Change to IL-1βFold Change to ControlFold Change to IL-1β
IL-1βIL-1β + EMPAIL-1β + EMPAIL-1βIL-1β + EMPAIL-1β + EMPA
Sodium glucose cotransporter, member 2 (SGLT2) (SLC5A2)−0.05−0.1−0.05−0.56−0.59−0.03
BRPTEC/TERT1 (Experiment 1)RPTEC/TERT1 (Experiment 2)
Gene NameFold Change to ControlFold Change to IL-1βFold Change to ControlFold Change to IL-1β
IL-1βIL-1β + EMPAIL-1β + EMPAIL-1βIL-1β + EMPAIL-1β + EMPA
Sodium glucose cotransporter, member 2 (SGLT2) (SLC5A2)−0.31−0.46−0.15−0.26−0.39−0.13
Table 3. Clustering of annotated pathways (enrichment score 3.64).
Table 3. Clustering of annotated pathways (enrichment score 3.64).
KnowledgebaseAnnotated Pathway/TermEASE Score (p Value)
INTERPROCXC chemokine2.2 × 10−7
INTERPROCXC chemokine, conserved site2.2 × 10−7
INTERPROchemokine interleukin-8-like domain1.1 × 10−5
SMARTSCY1.6 × 10−5
GOTERM_MF_DIRECTchemokine activity1.8 × 10−5
UP_KEYWORDScytokine1.9 × 10−5
UP_KEYWORDSdisulfide bond2.8 × 10−5
GOTERM_MF_DIRECTCXCR chemokine receptor binding3.8 × 10−5
GOTERM_BP_DIRECTcell chemotaxis4.3 × 10−5
GOTERM_BP_DIRECTchemokine-mediated signaling pathway5.6 × 10−5
UP_KEYWORDSsecreted1.2 × 10−4
KEGG_PATHWAYsalmonella infection1.9 × 10−4
UP_SEQ_FEATUREsignal peptide2.0 × 10−4
GOTERM_BP_DIRECTpositive regulation of neutrophil chemotaxis2.5 × 10−4
KEGG_PATHWAYcytokine-cytokine receptor interaction2.7 × 10−4
GOTERM_BP_DIRECTchemotaxis2.8 × 10−4
GOTERM_CC_DIRECTextracellular region5.5 × 10−4
GOTERM_BP_DIRECTinflammatory response6.1 × 10−4
GOTERM_BP_DIRECTresponse to lipopolysaccharide6.7 × 10−4
GOTERM_BP_DIRECTimmune response9.0 × 10−4
UP_KEYWORDSsignal9.9 × 10−4
GOTERM_CC_DIRECTextracellular space1.4 × 10−3
KEGG_PATHWAYchemokine signaling pathway2.0 × 10−3
KEGG_PATHWAYlegionellosis2.6 × 10−3
KEGG_PATHWAYNOD-like receptor signaling pathway2.8 × 10−3
UP_KEYWORDSchemotaxis3.2 × 10−3
KEGG_PATHWAYrheumatoid arthritis6.8 × 10−3
UP_KEYWORDSinflammatory response7.7 × 10−3
KEGG_PATHWAYTNF signaling pathway9.9 × 10−3
UP_SEQ_FEATUREdisulfide bond9.9 × 10−3
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Pirklbauer, M.; Sallaberger, S.; Staudinger, P.; Corazza, U.; Leierer, J.; Mayer, G.; Schramek, H. Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells. Int. J. Mol. Sci. 2021, 22, 5089. https://doi.org/10.3390/ijms22105089

AMA Style

Pirklbauer M, Sallaberger S, Staudinger P, Corazza U, Leierer J, Mayer G, Schramek H. Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells. International Journal of Molecular Sciences. 2021; 22(10):5089. https://doi.org/10.3390/ijms22105089

Chicago/Turabian Style

Pirklbauer, Markus, Sebastian Sallaberger, Petra Staudinger, Ulrike Corazza, Johannes Leierer, Gert Mayer, and Herbert Schramek. 2021. "Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells" International Journal of Molecular Sciences 22, no. 10: 5089. https://doi.org/10.3390/ijms22105089

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop