Article pubs.acs.org/jpr
Patterns of Gene and Metabolite Define the Effects of Extracellular Osmolality on Kidney Collecting Duct Hyo-Jung Choi,†,# Yu-Jeong Yoon,‡,# Yong-Kook Kwon,‡,∥ Yu-Jung Lee,† Sehyun Chae,§ Daehee Hwang,§ Geum-Sook Hwang,*,‡,∥ and Tae-Hwan Kwon*,† †
Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea Integrated Metabolomics Research Group, Seoul Center, Korea Basic Science Institute, Seoul, Korea § Department of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea ∥ Graduate School of Analytic Science & Technology, Chungnam National University, Daejeon, Korea ‡
S Supporting Information *
ABSTRACT: To investigate the effects of changes in extracellular osmolality on the function of kidney collecting duct cells, particularly on water and sodium reabsorption in the conditions of diuresis and antidiuresis, we generated transcriptome and metabolome profiles of primary cultured inner medullary collecting duct (IMCD) cells. They were grown in hyperosmolar culture medium (640 mOsm) for 4 days and then exposed to either reduced (300 mOsm) or same osmolality for 1 or 2 days more. Integrated analysis of the transcriptome and metabolome revealed that decreased extracellular osmolality was associated with decreased levels of organic osmolytes, glucose, intermediates of citric acid cycle, and branchedchain amino acids (BCAA) in IMCD cells, along with significantly decreased gene expression and protein abundance of P-type transporters (ATP1B1), ABC transporters (ABCC5 and ABCG1), and insulin signaling pathways (IRS2). Quantitative real-time RT-PCR and semiquantitative immunoblotting confirmed the changes of transcript levels of differentially expressed genes and protein levels. Taken together, integrated analysis of omics data demonstrated that water and sodium reabsorption could be reduced by decreased extracellular osmolality per se, through decreased levels of ABC transporters and IRS2, which play a potential role in the transport of organic osmolytes, BCAA, glucose, and trafficking of epithelial sodium channel. KEYWORDS: kidney collecting duct, organic osmolyte, osmolality, metabolomics, transcriptomics
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fluid shear stress affects the structure and function of the collecting duct cells, as we demonstrated in the “collecting ducton-a-chip” analysis in the kidney inner medullary collecting duct (IMCD) cells.12 In addition, an in vitro study demonstrated that hypoosmotic cell swelling counteracts protein catabolism, whereas hyperosmotic cell shrinkage promotes protein breakdown.13 In the present study, we hypothesized that altered extracellular osmolality per se could change a broad spectrum of cellular processes by affecting transcriptomic and metabolomic profiles in IMCD cells irrespective of hormonal stimulation, and hence, it could produce the changes in renal tubular function, particularly on water and sodium reabsorption. This was directly investigated by exploiting an integrated omics analysis of the primary cultured IMCD cells in rat kidney. First, we generated the metabolomic profiles through 1H NMR-based metabolomic analysis and identified the differentially expressed metabolites (DEMs) by the changes of extracellular osmolality. Second, gene expression profiles were generated via transcriptomic analysis and identified
INTRODUCTION A major function of the kidney is to regulate body water and electrolyte balance. This function is achieved and finely regulated by a number of cellular and molecular processes in the tubular epithelial cells. This includes the tubular reabsorption of water and electrolytes through water channels (aquaporins: AQPs) and ion transporters expressed along the renal tubule.1−3 Consequently, a variety of different extracellular microenvironments are formed in the tubular lumen and interstitium, to which renal tubular epithelial cells and interstitial cells are directly exposed. In particular, collecting duct cells are confronted by a wide range of urine flow rate, luminal fluid pH, and extracellular osmolality, which are produced by vasopressin- and aldosterone-mediated actions.1,4−6 For instance, hyperosmolality in the medulla is established to concentrate urine,2,7 and renal medullary cells adapt to the hyperosmolality by accumulating organic osmolytes,8 which is provided by activation of enzymes and transporters induced by tonicity-responsive enhancer-binding protein (TonEBP).9,10 This was also demonstrated by our previous1H NMR-based metabolomic analysis of the kidneys from rats with lithiuminduced nephrogenic diabetes insipidus.11 Moreover, luminal © 2012 American Chemical Society
Received: March 30, 2012 Published: June 12, 2012 3816
dx.doi.org/10.1021/pr300309d | J. Proteome Res. 2012, 11, 3816−3828
Journal of Proteome Research
Article
Figure 1. Representative 600-MHz 1H NMR spectra. Inner medullary collecting duct (IMCD) cell extracts of 24 h-640 (A), 24 h-300 (B), 48 h-640 (C), and 48 h-300 (D) were examined. Key: 1, valine; 2, isoleucine; 3, leucine; 4, lactate; 5, alanine; 6, acetate; 7, arginine; 8, glutamate 9, glutamine; 10, 4-aminobutyrate; 11, succinate; 12, citrate; 13, asparate; 14, creatine; 15, creatinine; 16, GPC; 17, betaine; 18, taurine; 19, glucose; 20, myo-inositol ; 21, glycine; 22, choline; 23, pyruvate; 24, sorbitol.
Figure 2. PCA score plots derived from the 1H NMR spectra. Inner medullary collecting duct (IMCD) cell extracts of 24 h-640 (open triangles) and 24 h-300 (closed triangles) (A), and 48 h-640 (open diamonds) and 48 h-300 (closed diamonds) (B) were examined. Metabolic differences between the groups were clearly seen. (A) 24 h640 vs 24 h-300 (R2X = 0.679 and Q2 = 0.574); (B) 48 h-640 vs 48 h-300 (R2X = 0.635 and Q2 = 0.407). The R2X value represented the goodness of fit of the models and Q2 value represented the predictability of the models.
differentially expressed genes (DEGs) by the changes of extracellular osmolality. Third, cellular processes affected by the changes of extracellular osmolality were then identified through functional enrichment analysis of the DEGs. This integrated analysis revealed that several important metabolic processes were affected, where the alterations were collectively indicated by both DEMs and DEGs. Lastly, quantitative real-time RT-PCR analysis and immunoblotting analysis were performed to confirm the changes of mRNA expression of genes representing the cellular processes and their protein abundance in the IMCD cells.
After isolating IMCD cell suspension, cells were seeded in type 1 collagen-coated 100 mm dish (Iwaki Science products Department of Asahi Glass Co. LTD, Japan, catalog no. 4020-010). IMCD cells were fed every 48 h and grown in culture medium (osmolality: 640 mOsm/kg H2O) supplemented with 10% FBS at 37 °C in 5% CO2−95% air for 4 days. Then, the cells were grown either in the culture medium having the same osmolality (640 mOsm/kg H2O) or the decreased osmolality (300 mOsm/ kg H2O) for an additional 1 or 2 days of the experiments. The osmolality of culture medium (Dulbecco’s modified Eagle’s medium/F12 without phenol red, 80 mM urea, 130 mM NaCl, 10 mM HEPES, 2 mM L-glutamine, penicillin/streptomycin 10 000 units/mL, 50 nM hydrocortisone, 5 pM 3,3,5-triiodothyronine, 1 nM sodium selenate, 5 mg/L transferrin, 10% fetal bovine serum, pH 7.4, 640 mOsm/kg H2O) was adjusted by changing NaCl and urea concentration and was determined by freezing-point depression (Osmomat 030-D, Gonotec, Berlin, Germany).
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MATERIALS AND METHODS
Primary Culture of IMCD Cells of Rat Kidney
The animal protocols were approved by the Animal Care and Use Committee of the Kyungpook National University, Korea (KNU 2012-10). Primary cultures enriched in IMCD cells were prepared from pathogen-free male Sprague−Dawley rats [200− 250 g, Charles River (Orient Bio, Seongnam, Korea)], as we described previously in detail.14 Briefly, rats were anesthetized under enflurane inhalation, and kidneys were rapidly removed.
Sample Preparation for Metabolomic Analysis
After harvesting ∼1.5 × 106 IMCD cells/sample, the extraction of intracellular metabolites was performed using a mixture of 3817
dx.doi.org/10.1021/pr300309d | J. Proteome Res. 2012, 11, 3816−3828
Journal of Proteome Research
Article
Table 1. 1H-Chemical Shift Assignment of the Major Metabolites in the IMCD Cell Extracts and Their Relative Concentrations in Each Group 24 h metabolite
chemical shift (ppm) and multiplicitya
relative concentration (300/640)b
Valine Isoleucine Leucine Lactate Alanine Acetate Arginine Glutamate Glutamine Succinate Citrate Creatine Creatinine GPCd Betaine Taurine Glucose Myo-inositol Glycine Choline Pyruvate
3.59 (d), 2.25 (m), 0.98 (d), 1.03 (d) 0.93 (t), 1.00 (d), 1.28 (m),1.47 (m), 1.96 (m), 3.68 (d) 3.72 (t), 1.69 (m), 0.97 (d), 0.94 (d) 4.12 (q), 1.33 (d) 3.81 (q), 1.48 (d) 1.91 (s) 1.68 (m), 1.90 (m), 3.23 (t), 3.76 (t) 3.76 (m), 2.06 (m), 2.36 (m) 3.76 (m), 2.15 (m), 2.46 (m) 2.41 (s) 2.69 (d), 2.54 (d) 3.04 (s), 3.93 (s) 3.05 (s), 4.06 (s) 3.24 (s), 3.67 (dd), 3.78 (m), 4.36 (m) 3.26 (s), 3.89 (s) 3.27 (t), 3.43 (t) 5.22 (d) 3.53 (dd), 4.06 (dd), 3.28 (t), 3.63 (t) 3.55 (s) 3.19 (s), 3.50 (m), 4.07 (m) 2.37 (s)
0.817 0.826 0.750 1.197 0.778 1.273 1.580 0.808 0.968 1.448 1.195 0.879 1.211 0.425 0.801 0.472 1.247 0.371 0.792 1.244 1.137
48 h P valuec
relative concentration (300/640)b
P valuec
0.0450 0.0458 0.0004 0.0115e 0.0003 0.0109 0.0062 0.0038 ns 0.0015e ns ns 0.0033
