Miwako Suto

Interleukin-6 modulates oxidative stress produced during the development of cisplatin nephrotoxicity.

AIMS We reported that interleukin-6 (IL-6) plays a protective role in the development of cisplatin-induced acute renal failure (ARF) through upregulation of anti-oxidative stress factors. In this study, we examined the effects of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the development of cisplatin-induced ARF in wild-type (WT) and IL-6(-/-) mice to determine how IL-6 contributes to modulation of oxidative stress caused by cisplatin. MAIN METHODS WT and IL-6(-/-) male mice were given either cisplatin (30 mg/kg) or saline intraperitoneally. DMTU (100mg/kg) or saline was given 30 min before cisplatin or saline administration. Blood and kidney samples were collected on days 1 and 3 after cisplatin administration. KEY FINDINGS In WT mice, DMTU markedly improved cisplatin-induced renal dysfunction and survival rate. DMTU reduced the expression levels of TNF-α, Bax and c-fos and increased the expression levels of IL-6, Bcl-xL and Nrf2 in WT mice. Reduced reactive oxygen species (ROS) by DMTU resulted in increases of IL-6, anti-apoptosis and anti-oxidant gene expression levels. In IL-6(-/-) mice, DMTU also improved cisplatin-induced renal dysfunction and reduced expression levels of TNF-α, Bax and c-fos, but not Bcl-xL and Nrf2. Since Nrf2 induces IL-6 expression, IL-6 and Nrf2 may influence each other during anti-oxidant responses. The basal level of HO-1 in IL-6(-/-) mice was higher than that in WT mice. SIGNIFICANCE In IL-6(-/-) mice, overproduction of ROS by cisplatin results in upregulation of HO-1 expression in order to eliminate oxidative stress. IL-6 mediates the generation and elimination of ROS during cisplatin-induced ARF.

Interleukin-6 deficiency accelerates cisplatin-induced acute renal failure but not systemic injury

Cisplatin (CDDP), a major chemotherapeutic agent used to treat solid tumors, is known to induce acute renal failure (ARF). The progression of tissue injury involves the coordination of inflammatory and repair responses. Interleukin-6 (IL-6) has been suggested to modulate inflammatory and repair processes in various tissue injuries. In this study, we analyzed IL-6 regulation during CDDP-induced ARF in wild-type (WT) mice and determined the pathological role of IL-6 using IL-6 knockout ((-/-)) mice. A correlation between increase in serum IL-6 level and blood urea nitrogen level was found in WT mice. Renal IL-6 expression in most proximal tubular cells and suppressor of cytokine signaling 3 (SOCS3) gene expression significantly increased in WT mice after administration of CDDP, suggesting active IL-6 signaling during CDDP-induced ARF development. Interestingly, renal dysfunction occurred soon after administration of CDDP and became more severe in IL-6(-/-) mice than that in WT mice. In contrast, the survival rate of IL-6(-/-) mice (50% at 8 days) was better than that of WT mice (10%). Induction levels of proapoptotic Bcl-2 associated X protein (Bax) in renal proximal tubular cells was significantly higher in IL-6(-/-) mice than in WT mice at 24h after CDDP injection. Levels of antiapoptotic proteins, Bcl-2 and Bcl-extra large (Bcl-x(L)), in IL-6(-/-) groups were significantly higher than those in CDDP-treated WT groups throughout the experimental period. Bax might contribute to the development of CDDP-induced ARF at 24h; however, high expression levels of Bcl-x(L) and Bcl-2 might overcome the proapoptosis signaling at 72 h in IL-6(-/-) mice. These results indicated that local and systemic elevation of IL-6 contributes to the development of CDDP-induced ARF and that IL-6 produced in renal tubular cells prevents progression of ARF at the early stage. IL-6 deficiency accelerates CDDP-induced ARF but not development of systemic injury.

Phytoplankton gene detection in drowned rabbits

We have developed a sensitive and specific polymerase chain reaction (PCR) method for identifying phytoplankton in cases of death by drowning, and we have designed four primer pairs, EG1, EG2, SK1 and SK2, for chlorophyll-related genes of Euglena gracilis and Skeletonema costatum, which are commonly distributed in all types of water. In order to evaluate the usefulness of this method for diagnosis of drowning, we have used this method for detection of plankton genes in non-drowned rabbits submerged after death and in decomposed drowned rabbits. Plankton DNA was identified in lung samples obtained from the non-drowned rabbits because of postmortem plankton penetration into the respiratory system, and plankton DNA was identified in liver and kidney samples obtained from the decomposed drown rabbits. The results show that our new PCR method is a useful method for diagnosing drowning.

A novel PCR method for identifying plankton in cases of death by drowning.

We present a new PCR method for identifying plankton in cases of death by drowning. We designed four primer pairs for chlorophyll-related genes of Euglena gracilis (EG) and Skeletonema costatum (SK), which are commonly distributed in water. The primers were selected from sequences coding chloroplast/chlorophyll apoprotein of EG (EG1 and EG2) and fucoxanthin-chlorophyll a/c harvesting protein of SK (SK1 and SK2). With EG1 or EG2, up to 2 fg of EG-DNA was identified, and 0.2 pg of SK-DNA was detectable with SK1 or SK2. No PCR products were amplified from green vegetables (komatsuna, spinach, parsley) or human DNA with the four primer pairs. Regardless of the origin, seawater or fresh water, most diatoms were detectable with primer pairs of EG1 and EG2. With SK1, only Centrales diatoms were identified, and five diatom strains originating from seawater were detectable with SK2. EG1 and EG2 gave rise to PCR products from most water samples. By using Percoll®, plankton was easily isolated from human tissue or blood samples and good results of PCR analysis were obtained in cases of death by drowning.

Interleukin-6 plays a protective role in development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors

AIMS Cisplatin, a major chemotherapeutic agent, accumulates in proximal tubules of the kidneys and causes acute renal failure dose-dependently. We previously reported that cisplatin induced more severe renal dysfunction in interleukin-6 (IL-6) knockout (IL-6(-/-)) mice than in wild-type (WT) mice. Expression of a pro-apoptotic protein was significantly increased with cisplatin in IL-6(-/-) mice compared to that in WT mice. IL-6, locally expressed in renal tubular cells after cisplatin administration, prevents the development of renal dysfunction at an early stage. In the present study, we focused on downstream signals of IL-6 and oxidative stress induced by cisplatin in order to evaluate the protective role of IL-6 in the development of acute renal failure. MAIN METHODS WT and IL-6(-/-) mice were given either cisplatin (30 mg/kg) or saline intraperitoneally. Blood and kidney samples were collected at 24h and 72 h after cisplatin administration. The changes in expression of 4-hydroxy-2-nonenal protein (4-HNE, oxidative stress marker) and cyclooxygenase-2 (cox-2), activities of superoxide dismutases and caspase-3, and phosphorylation of extracellular signal-regulated kinase (ERK) were examined. KEY FINDINGS Cisplatin increased the expression of 4-HNE and cox-2, and phosphorylation of ERK in IL-6(-/-) mice than in WT mice. On the other hand, activity of superoxide dismutase, an anti-oxidative enzyme, was significantly decreased in the kidney obtained from IL-6(-/-) mice after cisplatin administration. SIGNIFICANCE Our findings suggest that IL-6 plays a protective role in the development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors.

Expression of cytokines, neurotrophins, neurotrophin receptors and NOS mRNA in dorsal root ganglion of a rat tourniquet model

We studied temporal changes in mRNA expression patterns for nitric oxide synthase (NOS), cytokines, neurotrophins and neurotrophin receptors in the dorsal root ganglion (DRG) of the rat, after application of a tourniquet to the hind limb. Collapsed myelin and degenerated axons were observed in the tourniquet segment of the sciatic nerve. Gene expression level of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) was significantly increased in ipsilateral DRG samples at 4h after application of the tourniquet but not in the contralateral or control DRG samples. Upregulation of tumor necrosis factor (TNF)-alpha, activating transcription factor (ATF)-3 and neurotrophin-3 (NT3) expressions began at 1h after application of the tourniquet in ipsilateral DRGs. It is likely that transient expression of these molecules triggers secondary events that may be beneficial to wound repair and regeneration.

PCR detection of bacterial genes provides evidence of death by drowning

We have developed a sensitive and specific PCR method for detecting plankton DNA in cases of death by drowning. However, this PCR method could not be used for cases of drowning in water containing no plankton. Bacteria species are normally localized in the throat and trachea and they may invade into blood through the respiratory tract in people who have drowned as well as species localized in water. The aim of this study was to establish a novel and expedient PCR method for detecting bacterial genes in samples from drowning cases. We designed primer pairs for Streptococcus salivarius (SL1) and Streptococcus sanguinis (SN1), which are common species in the throat, and for Aeromonas hydrophila (AH1), which has been found in various water samples. With SL1, SN1, and AH1, we detected 10, 0.1, and 1 pg of target DNA, respectively. Among 19 drowned cases within 3 days postmortem, SL-DNA was detected in all of the blood samples from hearts with SL1 and AH-DNA was detected in several samples with AH1. In a case of drowning in a bathtub, use of the conventional acid digestion method for diatom analyses and the PCR method for identifying plankton DNA revealed no plankton, but our PCR method for detecting bacterial DNA showed a positive result for SL-DNA in a blood sample from the heart. In conclusion, our novel PCR method is highly specific and sensitive for detecting bacterial DNA and is useful for cases of death by drowning in water containing no plankton.

Comparison of renal dysfunction in wild-type, IL-6 KO and iNOS KO mice hind limb tourniquet-reperfusion model.

The release of a tourniquet after hind limb ischemia results in vital organ injury, which progresses to multiple organ failure with a high mortality rate. Many events are involved in ischemia-reperfusion (I/R) injury. The purpose of this study was to determine how IL-6 or iNOS is involved in I/R injury using IL-6 knockout (KO) and iNOS KO mice. Male C57BL/6J wild-type (WT), IL-6 knockout (KO) and iNOS KO mice were anesthetized with pentobarbital, and rubber bands were fastened to the inguinal region of both hind limbs for 3h. Blood and kidney samples were obtained before reperfusion and at 1, 2, 3, and 12h after reperfusion. For the control group, mice were kept for 6h under an anesthetized condition without rubber bands. Blood gases and biochemical parameters were analyzed by i-STAT300F. Real-time PCR analyses were performed to examine the expression levels of IL-6 and iNOS mRNA in kidneys. Metabolic acidosis, hemoconcentration and renal dysfunction were significantly developed after reperfusion regardless of mouse genotype, and progression of this condition was earlier in IL-6 KO and iNOS KO mice than in WT mice. The expression level of kidney IL-6 mRNA increased and that of iNOS mRNA decreased after reperfusion. It is possible that late decrease recovery of iNOS mRNA expression in IL-6 KO mice and early progress of IL-6 mRNA expression in iNOS KO mice after reperfusion induce renal dysfunction.

Nitric oxide synthase expressions in mice skeletal muscle subjected to ischemia/reperfusion injury

Nitric oxide synthase (NOS) expressions in skeletal muscle subjected to ischemia/reperfusion (I/R) were studied using a hind limb tourniquet ischemia model in mice. A rubber band was applied to a hind limb for 3 h under isoflurane anesthesia followed by 1 or 4 h of reperfusion. Increased NADPH diaphorase activity and NOS immunoreactivity were histochemically detected in the cells of muscle that had been subjected to I/R. The results of RT-PCR of the muscle subjected to I/R showed that NOS mRNA expressions were not significantly increased until 4 h after the start of reperfusion. Since there was no significant difference between histochemical findings or between water contents of the hind limbs or organs in interleukin (IL)-6-deficient mice and the wild-type mice, IL-6 may not be involved in the early stage of I/R muscle injury such as that in this model. O(2)(-) production in the cells of muscle that had been subjected to I/R was observed using an in situ detection method with hydroethidine, and the O(2)(-) was inhibited by intravenous administration of L-NAME or L-NMMA, but not L-NIL, 30 min before tourniquet release. Further study is needed to evaluate the role of O(2)(-) produced by constitutive NOS in muscle subjected to I/R in the pathophysiology of tourniquet shock.

Changes in mRNA expression patterns for cytokines in blood leukocytes of a rat tourniquet model

We examined changes in mRNA expression patterns for proinflammatory cytokines and growth factors in blood samples after application of a tourniquet to the rat hind limb. Slight upregulations of interferon (IFN)-gamma, macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-beta1 mRNA began at 2h after tourniquet application and were short-lived. The levels of activating transcription factor (ATF)-3, a stress-inducible gene, had increased at 1h after tourniquet application. No significant expression of interleukin (IL)-6 mRNA was observed in most samples. There were no significant temporal changes in the levels of IL-1beta, cardiotrophin (CT)-1 mRNA compared to the control levels, but, downregulation of gp130, a receptor of the IL-6 family, began at 1h after tourniquet application. Nerve growth factor (NGF) mRNA gradually increased and reached a significantly high level at 4h after application of the tourniquet. Gene expression induction in blood leukocytes occurred soon after application of the tourniquet and was short-lived. The transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration.