European Review for Medical and Pharmacological Sciences 2019; 23: 3088-3095 The effects of TRPM2, TRPM6, TRPM7 and TRPM8 gene expression in hepatic ischemia reperfusion injury

T. BILECIK1, F. KARATEKE2, H. ELKAN3, H. GOKCE4

1Department of Surgery, Istinye University, Faculty of Medicine, VM Mersin Medical Park Hospital, Mersin, Turkey 2Department of Surgery, VM Mersin Medical Park Hospital, Mersin, Turkey 3Department of Surgery, Sanlıurfa Training and Research Hospital, Sanliurfa, Turkey 4Department of Pathology, Inonu University, Faculty of Medicine, Malatya, Turkey

Abstract. – OBJECTIVE: Mammalian transient Introduction receptor potential melastatin (TRPM) channels are a form of calcium channels and they trans- Ischemia is the lack of oxygen and nutrients port calcium and magnesium ions. TRPM has and the cause of mechanical obstruction in sev- eight subclasses including TRPM1-8. TRPM2, 1 TRPM6, TRPM7, TRPM8 are expressed especial- eral tissues . Hepatic ischemia and reperfusion is ly in the liver cell. Therefore, we aim to investi- a serious complication and cause of cell death in gate the effects of TRPM2, TRPM6, TRPM7, and liver tissue2. The resulting ischemic liver tissue TRPM8 gene expression and histopathologic injury increases free intracellular calcium. Intra- changes after treatment of verapamil in the he- cellular calcium has been defined as an important patic ischemia-reperfusion rat model. secondary molecular messenger ion, suggesting MATERIALS AND METHODS: Animals were randomly assigned to one or the other of the calcium’s effective role in cell injury during isch- following groups including sham (n=8) group, emia-reperfusion, when elevated from normal verapamil (calcium entry blocker) (n=8) group, concentrations. The high calcium concentration I/R group (n=8) and I/R- verapamil (n=8) group. leads to depressed compensative vasodilation TRPM 2, 6, 7, 8 gene expression level was as- following a period of vasoconstriction. All these sessed by Real Time-quantitative Polymerase reasons, including the lack of oxygen and nutri- Chain Reaction (RT-qPCR) and histopathologic ents start the pathway of apoptosis and necrosis3. changes were determined by the hematoxylin Verapamil, which is a member of the dihydropyri- and eosin (HE) examination. 4 RESULTS: The expression level of TRPM 2, 6, dine family, is a calcium entry blocker . Two stud- 7, and 8 genes was significantly higher in isch- ies have reported that the addition of verapamil emia-reperfusion (I/R), verapamil, IR-verapamil in perfusate and the iv/ip pretreatment showed groups compared to sham group. The p-values beneficial effects in ischemia-reperfusion inju- were 0.0024, < 0.0001, 0.0002, 0.006 for TRPM2, ry prevention on animal models. Moreover, ver- TRPM6, TRPM7, and TRPM8, respectively. Se- apamil has a vital effect on calcium homeostasis vere necrotic, degenerative differentiations and severe hemorrhagic areas were observed in he- and could reduce ischemia and reperfusion in the 5,6 patocytes from IR group. Also, moderate necrotic injured liver . The calcium channel blockers are and degenerative differentiations and moderate also used to improve the post ischemic myocar- hemorrhagic areas were observed in hepato- dial function7 and the survival of ischemic skin cytes from IR-verapamil group. flaps8. CONCLUSIONS: This is the first study report- The mammalian transient receptor potential ing an association between the expression level of TRPM 2, 6, 7, 8 in a hepatic ischemia-reperfu- (TRP) channels are a form of calcium channels sion rat model. Moreover, TRPM 2, 6, 7, 8 affect and they transport calcium, magnesium and trace hepatic ischemia-reperfusion. metal ions9. Therefore, TRPs cause changes in cal- cium, magnesium and trace metal ions concentra- Key Words Ischemia, Reperfusion, TRP, TRPM, Verapamil, Cal- tions. These contributions are pivotal for several cium entry blocker. physiological processes including pheromone sig- naling, temperature sensation, cell proliferation,

3088 Corresponding Author: Faruk Karateke, MD; e-mail: [email protected] TRPM calcium channels affect hepatic ischemia-reperfusion and muscle contraction10. Almost every cell type Ischemia-reperfusion group (I/R): none of the expresses TRPs which are localized in the plasma rats were treated with any substance. Ischemia membrane9. TRP superfamily includes TRPC (ca- was applied to the left hepatic artery and the por- nonical), TRPV (vanilloid), TRPM (melastatin), tal vein by a clamp for 60 min after the laparot- TRPML (mucolipin), TRPP (polycystin), TRPA omy. Within 60 min after reperfusion, relaparot- (ankyrin transmembrane protein) and TRPN (nom omy was performed on all group members, and PC-like)11. The family of TRPMs has eight sub- liver and blood of the subjects were isolated (n=8). classes including TRPM1-8 and they are expressed Ischemia-reperfusion/calcium entry blocker in different cells. TRPM2, TRPM6, TRPM7, (I/R-verapamil): as pretreatment, verapamil (1.25 TRPM8 are expressed especially in the liver cell12. mg/kg) was administered orally to the rats 30 min Moreover, TRPM7 has been found to be involved before anesthesia. Ischemia was applied to the left in delayed neuronal death after ischemia13. hepatic artery and the portal vein by a clamp for 60 There is no study about the association be- min after the laparotomy. Within 60 min after reper- tween the application of calcium entry blocker fusion, relaparotomy was performed on all group aiming at preventing ischemia-reperfusion liver members, and liver and blood were isolated (n=8). injury and TRPM calcium channels. To under- stand the roles of TRPM calcium channels in liver Surgical Procedures tissue, it is important to explain the pathogenesis Rats were anesthetized with intraperitoneal in- of ischemia-reperfusion. Therefore, we aim to in- jections of xylazine (12 mg/kg) and ketamine (80 vestigate the effects of TRPM2, TRPM6, TRPM7, mg/kg), and placed in a supine position on a tem- and TRPM8 gene expression and histopathologic perature-controlled heating table, maintaining the changes after treatment of the calcium entry block- body temperature in the range of 36.5-37.5°C. Rats er in the hepatic ischemia-reperfusion rat model. were allowed to breathe spontaneously during sur- gery. For the preparation of the liver, abdominal skin was shaved and sterilized with 70% of ethyl alcohol. Materials and Methods After midline laparotomy and subcostal incisions, the liver was carefully mobilized from all the liga- In vivo Experiment mentous attachments. The left hepatic artery and the The experiments were carried out on 32 male portal vein were clamped for 60 min using an atrau- Wistar rats (average body weight 225±25 g) matic vascular clamp for complete ischemia of the housed in an environmentally controlled room median and left hepatic lobes. Then, the edges of the (24°C to 26°C temperature, 50% to 60% moisture abdominal incision were approximated to each other rate) with a 12:12 h light: dark cycle, and kept on and covered by a piece of gauze soaked with warm commercial rat chow and tap water ad libitum. isotonic saline to prevent the undue loss of body flu- The experimental protocol was in accordance ids. After the removal of the clamp, the median and with EU directive 2010/63 for the protection of left hepatic lobes were removed and the abdomen animals used for scientific purposes. This investi- was properly irrigated with isotonic saline. During gation was also approved by the local Committee IR periods, the abdomen was covered with a plas- on the Ethics of Animal Experiments of the Mus- tic wrap to minimize fluid loss via evaporation. At tafa Kemal University, Hatay, Turkey (2014/5-9). the end of ischemia, the abdomen was closed with continuous stitches using Vicryl (Ethicon Endo-Sur- Experimental Design gery, Inc., Cincinnati, Ohio, USA). 3/0 sutures and The animals were randomly assigned to one the animals were returned to their cages. After 60 or other of the following groups including sham, min of reperfusion, animals were anesthetized with verapamil (calcium entry blocker), I/R group and intraperitoneal injection of ketamine and xylazine I/R- verapamil groups. (80 and 12 mg/kg, respectively) and were sacrificed Sham Group: rats were subjected to glucose by exsanguination via right ventricular puncture, pretreatment and surgical procedures, except for then blood and histological samples were taken. the induction of liver ischemia, but including liver resection (n=8). Hepatic Histopathologic Evaluation Verapamil (Calcium entry blocker) group: rats After ischemia-reperfusion, the liver specimens were treated with verapamil as a pretreatment were removed and placed in 10 % of formalin. Then, (1.25 mg/kg) and to none of them ischemia-reper- tissues were embedded in paraffin after alcohol and fusion (n=8) was applied. xylene according to the standard protocol. Five mi-

3089 T. Bilecik, F. Karateke, H. Elkan, H. Gokce

Table I. Primers used for TRPM gene expressions. Primers

β-Actin Left 5’-CCC GCG AGT ACA ACC TTC T-3’ β-Actin Right 5’-CGT CAT CCA TGG CGA ACT-3’ TRPM2 Left 5’-AAT TTG CTC ATC GCC ATG TT-3’ TRPM2 Right 5’-GAT CTG GTC TGT GTG CTC CTG-3’ TRPM6 Left 5’-GCA AGA ACT GGC TTT CCG TG-3’ TRPM6 Right 5’-ATC CGG GTC CTC TTG CAT CT-3’ TRPM7 Left 5’-AGA CGC TTT CCG ATA GAT GG-3’ TRPM7 Right 5’-CTA TCC AGG ATT TCT GGG ACA T-3’ TRPM8 Left 5’-GCC CAG TGA TGT GGA CAG TA-3’ TRPM8 Right 5’-GGA CTC ATT TCC CGA GAA GG-3’ crometer thick sections were stained with hematox- Statistical Analysis ylin and eosin (HE) for light microscopy studies in Data were analyzed by using GraphPad Prism a double-blind manner. The hepatic damage was 5 program (GraphPad Software Inc., La Jolla, evaluated with a histopathologic scoring system. CA, USA). Data were expressed as mean values The assessment was expressed as the sum of the in- ± standard error of the mean (SEM). For normally dividual score grades from 0 (no findings), 1 (mild), distributed data, the one-way analysis of variance 2 (moderate), to 3 (severe) for each of the following (ANOVA) with Bonferroni’s multiple comparison six parameters: cytoplasmic color fading, vacuoliza- post-hoc test was used to test significant differ- tion, nuclear condensation, nuclear fragmentation, ences. p-values < 0.05 were considered as statisti- nuclear fading and erythrocyte stasis. cally significant.

RNA Isolation and Quantitative Reverse Transcription Polymerase Results Chain Reaction (qRT-PCR) and Gene Expression Analysis Data of TRPM 2, 6, 7, 8 Gene Total RNA was isolated by using commer- Expressions cially available kits (RNeasy Mini Kit, Qiagen, Data were analyzed by using Rotor Gene Q Se- Hilden, Germany). cDNA was obtained through ries Software and the numbers of positive cham- the use of the reverse transcription assay kit (High bers were corrected to estimate the true number Capacity cDNA Reverse Transcription Kit, Ap- of copies. Detected numbers were used to deter- plied Biosystems, Foster City, CA, USA). Brief- mine the number of copies in the original sample. ly, 10X reverse transcriptase buffer (2 µl), dNTP Beta Actin was used as the housekeeping gene mix (0,8 µl), 10X RT random primers (2 µl) (Table for normalization of the expressions. The expres- I), AMV reverse transcriptase (1 µl), mRNA and sion levels of TRPM genes were compared in two RNase free water (4,2 µl) were mixed to obtain manners; the expression levels of TRPM genes cDNA. The reaction mixture was incubated at without treatment were named as “basal expres- 25°C for 10 min and 37°C for 120 min for reverse sion level.” The basal expression levels in sham transcription and heated at 85°C for 5 min to in- group, verapamil group, I/R group and I/R-ver- activate AMV reverse transcriptase. The cDNA apamil group were compared. Verapamil and obtained was stored at −20°C until tested. Then, sham groups were considered as control group. cDNA was denatured at 95°C for 10 min, at 95°C The expression levels of TRPM genes, with and for 15 s annealed at 60°C for 1 min (TRPM2, without treatment, in each liver tissue were com- TRPM6, TRPM7, TRPM8), and extended at 95°C pared within it. for 3 min and at 72°C for 30 s. The reaction mix- Data were shown to represent the Average ture was subjected to 40 cycles of PCR following ∆CT± SEM. TRPM2 expression was highest in an initial 15 s denaturation step at 95°C. the I/R group (Average ∆CT ±SEM: 1.190±0.04) The mRNA expressions of each of TRPM and and the values of Average ∆CT were 1.036±0.02, β-actin as housekeeping gene were analyzed by 0.983±0.05 and 1.048±0.02 for sham, verapamil quantitative reverse transcriptase PCR using the and I/R-verapamil groups respectively (Figure Rotor-Gene Q (Qiagen, Hilden, Germany). 1). The level of TRPM6 gene expressions was

3090 TRPM calcium channels affect hepatic ischemia-reperfusion

Figure 1. Level of TRPM2, TRPM6, TRPM7, TRPM8 gene expression in sham, ischemia-reperfusion (I/R), verapamil, ischemia-reperfusion with the treatment of verapamil (I/R-verapamil) from liver tissue in Wistar rats.

1.111±0.02, 1.219±0.02, 1.085±0.02, 1.121±0.02 for sham, I/R and I/R-verapamil groups (Fig- for sham, I/R, verapamil and I/R-verapamil ure 1). The p-values were 0.0024, < 0.0001, groups, respectively (Figure 1). For the TRPM7 0.0002, 0.0060 for TRPM2, TRPM6, TRPM7 gene, the value of expressions was 1.143±0.03, and TRPM8, respectively. Statistically signifi- 1.274±0.02, 1.121±0.03, 1.162±0.02 in hepato- cant differences in gene expression were deter- cytes from sham, I/R, verapamil, I/R-verapamil mined relative to control rats by an ANOVA test. groups, respectively (Figure 1). TRPM8 expres- According to Bonferroni’s multiple comparison sion was modest in verapamil group (Average post-test, the values of mean differences, t-value, ∆CT ±SEM: 1.178±0.04) and other expression p-value, confidence interval differences (95%) levels were 1.181±0.04, 1.312±0.02, 1.201±0.01 were assessed in Table II.

Table II. Comparison of the gene expressions in groups (∆CT ±SEM). Sham I/R Verapamil I/R-Verapamil p

TRPM2 1.036±0.02 1.190±0.04a,** 0.983±0.05 1.048±0.02b,** 0.0024a,b TRPM6 1.111±0.02 1.219±0.02a,*** 1.085±0.02 1.121±0.02b,*** <0.0001a,b TRPM7 1.143±0.03 1.274±0.02a,*** 1.121±0.03 1.162±0.02b,*** 0.0002a,b TRPM8 1.181±0.04 1.312±0.02a,* 1.178±0.04 1.201±0.01b,* 0.006a,b

∆CT±SEM; *: p<0.05; **: p<0.01; ***: p< 0.001; a: I/R vs. Sham, b: I/R-Verapamil vs. I/R.

3091 T. Bilecik, F. Karateke, H. Elkan, H. Gokce

Histopathologic Evaluation the reentering of oxygen and nutrients renovates Histopathologic evaluation was shown in Table the mitochondrial membrane potential and the so III. Furthermore, the findings of the histopatholog- rapid mitochondrial calcium uptake14. Therefore, ic analysis showed necrosis, hemorrhage and ne- calcium plays an important role in ischemia-reper- crosis in liver tissues. The changes became more fusion, homeostasis of cells and in calcium ion severe among shame, I/R, verapamil and I/R-ver- channels such as TRPM. apamil groups. Liver cells in sham and verapamil However, the mechanisms of ischemia-reperfu- groups were normal for the HE examination (0: no sion are totally unclear. The present study revealed findings). Severe necrotic, degenerative differenti- that the level of TRPM2, TRPM6, TRPM7, TRPM8 ations and severe hemorrhagic area were observed gene expressions was affected by ischemia-reper- in liver tissues from the IR group. Moderate ne- fusion and this effect can be related to the concen- crotic and degenerative differentiations and mod- tration of calcium ion changes during ischemia and erate hemorrhagic area were also observed in he- reperfusion. The second member of the TRPM fam- patocytes from IR-verapamil group (Figure 2). ily of cation channels is TRPM2. The activation of Severe necrotic, degenerative differentiations TRPM2 causes increases in intracellular calcium and severe hemorrhagic areas were observed in levels. The level of calcium concentration may serve hepatocytes from IR group. Also moderate ne- signaling roles in the secretory cells (e.g., hepatocyte) crotic and degenerative differentiations and mod- through the release of cytokines, neurotransmitters, erate hemorrhagic areas were observed in hepato- and insulin. Furthermore, this can induce apoptot- cytes from IR-verapamil group. ic and necrotic cell death under oxidative stress in extreme situations including ischemia-reperfusion16. Previous studies have shown that verapamil has a Discussion protective effect for hepatic, renal and cardiac isch- emia-reperfusion injuries17. Erdogan et al6 reported In ischemia, the restriction of nutrient and ox- that verapamil treats ischemia-reperfusion injury. ygen leads to changes in cell metabolism, includ- Unfortunately, the interaction among calcium chan- ing especially the efflux of calcium, which causes nel blockers, ischemia injury and reactive oxygen necrotic or apoptotic cell death. In reperfusion, species are not clear.

Figure 2. Photomi- crographs of liver tissue HE stain (magnifica- tion X100). Panel A rep- resents Sham group; Panel B represents verapamil group; Pan- el C represents severe necrotic, degenerative differentiations and se- vere hemorrhagic area in I/R group; Panel D represents moderate necrotic and degenera- tive differentiations and moderate hemorrhagic area in verapamil-treat- ed I/R group.

3092 TRPM calcium channels affect hepatic ischemia-reperfusion

Table III. Histopathologic scores in groups. Sham I/R Verapamil I/R-verapamil

1. sample 0 2 0 2 2. sample 0 3 0 2 3. sample 0 3 0 2 4. sample 0 3 0 1 5. sample 0 3 0 2 6. sample 0 3 0 2 7. sample 0 3 0 1 8. sample 0 3 0 2 0: no findings (normal), 1: mild, 2: moderate, 3: severe.

Numerous studies have demonstrated that functional in the reflow injury. Meng et al32 said brain, heart and kidney ischemia-reperfusions are that suppression of TRPM7 in renal tubules may associated with the level of calcium concentration reduce dysfunction in early renal ischemia-reper- and TRPM ion channels, especially TRPM218. fusion injury. Dusmez et al26 has demonstrated In in vitro and in vivo studies, TRPM2 is the that TRPM6 and TRPM7 genes were expressed cause of detrimental in brain ischemia19-21. There in the renal system. We show that TRPM 6 and are three studies on heart ischemia-reperfusion and TRPM7 are associated with liver ischemia and TRPM2. The first study22 reported that TRPM2 and the mentioned gene expression level may protect the activation of Poly (ADP ribose) polymerase 1 against liver ischemia (Figure 1). (PARP-1) were involved in oxidative stress-induced TRPM8 was first cloned from prostate tissue cardiomyocytes death. The second study23 reported and it is a calcium-permeable channel protein33. that TRPM2 is involved in Tumor Necrosis Fac- Nocchi et al34 demonstrated that TRPM8 shows tor-alpha (TNF-α) mediated cardiomyocytes death. the greatest expression level following hydro- The third study24 reported that TRPM2 protects gen peroxide (H2O2) treatment, and the oxida- cardiomyocytes from ischemia-reperfusion injury. tive stress can modulate TRPM8 expression. Our Scholars25,26 showed that TRPM2 cation channels work also demonstrates that the level of TRPM8 have a harmful function in ischemia-reperfusion expression is decreased by the treatment of ver- and thus the level of TRPM2 expression was in- apamil before ischemia-reperfusion. creased after 48 h of renal reperfusion in calcium Two studies35,36 have showed that the treatment channel blocker (verapamil). Our data is in agree- of carvacrol had no effects on hepatic histology ment with the mentioned literature findings and we in the hepatic tissue of ischemia-reperfusion. But, suggest that TRPM2 may be used as a therapeutic we found that the treatment of verapamil had pos- target for the ischemia-reperfusion treatment (Fig- itive effects, and the data of sham and verapamil ure 1). TRPM6 and TRPM7 are closely related, and groups were similar on hepatocytes. According have an intrinsic kinase domain. Both of them have to our histopathological evaluation, liver tissues the same electrophysiological properties, regulate were severe necrotic, apoptotic, degenerative dif- magnesium and calcium homeostasis and are ex- ferentiations and severe hemorrhagic in IR group, pressed by each mammalian cell27. Touyz 28 reported but ischemia-reperfusion with the treatment of that vascular TRPM6 or TRPM7 may contribute to verapamil liver tissues were moderate necrotic, the delivery of cellular magnesium which plays a apoptotic and degenerative differentiations and role in hypertension. TRPM7 has an effective role moderate hemorrhagic. in brain ischemic injury, as the seventh TRPM in cell and mitochondrial membranes29. Wey et al30 have shown that the concentration of extracellular Conclusions calcium and magnesium decreases during transient brain ischemia which is related with TRPM7 gene We first showed that the levels of TRPM2, expression. TRPM6, TRPM7, and TRPM8 expressions in- Furthermore, TRPM7 has been found to be in- crease in ischemia-reperfusion from hepatocytes volved in delayed neuronal death after ischemia13. of rats. Moreover, the mentioned genes expres- In the in vivo experiment31, the kidney was dys- sion levels close to normal levels in the treat-

3093 T. Bilecik, F. Karateke, H. Elkan, H. Gokce ment of calcium entry blocker (verapamil) before 10) Gees M, Colsoul B, Nilius B. The role of transient ischemia-reperfusion from rat hepatocytes. So, receptor potential cation channels in Ca2+ sig- we propose that verapamil can block TRPM2, naling. Cold Spring Harb Perspect Biol 2010; 2: a003962. TRPM6, TRPM7, and TRPM8 gene expression. 11) Pan Z, Yang H, Reinach PS. Transient receptor po- However, we need more information on which tential (TRP) gene superfamily encoding cation mechanism affects TRPM2, TRPM6, TRPM7 channels. Hum genomics 2011; 5: 108-116. and TRPM8 gene expression. 12) Nilius B, Owsianik G. The transient receptor poten- tial family of ion channels. 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