Skip to main content
NCBI home page
Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2014 May 15;25(10):2316–2326. doi: 10.1681/ASN.2013091001

Conformational Change in Transfer RNA Is an Early Indicator of Acute Cellular Damage

Eikan Mishima *, Chisako Inoue †, Daisuke Saigusa ‡, Ryusuke Inoue §, Koki Ito ‖, Yusuke Suzuki ‖, Daisuke Jinno ‡, Yuri Tsukui ‡, Yosuke Akamatsu , Masatake Araki **, Kimi Araki **, Ritsuko Shimizu ††, Haruka Shinke ‡‡, Takehiro Suzuki *, Yoichi Takeuchi *, Hisato Shima *, Yasutoshi Akiyama *, Takafumi Toyohara *, Chitose Suzuki *, Yoshikatu Saiki ‖, Teiji Tominaga , Shigehito Miyagi §§, Naoki Kawagisihi §§, Tomoyoshi Soga ‖‖, Takayoshi Ohkubo ¶¶, Kenichi Yamamura **, Yutaka Imai ***, Satohiro Masuda ‡‡, Venkata Sabbisetti †††, Takaharu Ichimura †††, David B Mount †††, Joseph V Bonventre †††, Sadayoshi Ito *, Yoshihisa Tomioka ‡, Kunihiko Itoh †, Takaaki Abe *,‡‡‡,§§§,✉
PMCID: PMC4178440  PMID: 24833129

Abstract

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Oxidative stress is considered to be one of the important pathogenic mechanisms in various acute and chronic diseases, such as AKI and CKD.1–3 Early detection of tissue damage by oxidative stress is clinically important and provides the potential opportunity of therapeutic intervention. However, because current available biomarkers are not sensitive enough to detect the appropriate early tissue damage, it is necessary to develop a much better marker for detecting oxidative stress and cellular damage based on the effective stress responses.

Recently, transfer RNA (tRNA) metabolism has been reported to have an important role in the cellular stress response.4–8 Under oxidative stress, tRNA is cleaved into half molecules (30–45 nucleotides in length) at the anticodon loop4,5 and referred to as tRNA-derived stress-induced fragments (tiRNAs).6 The resultant tiRNA halts global protein translation as a phosphor-eukaryotic initiation factor 2α–independent stress response.7 This cleaved tRNA also has a number of potential consequences, including depletion of the tRNA pool and modulation of cell viability.8 Although tRNA metabolism is reported to be prominently involved in the stress responses in vitro, the precise mechanism of the tRNA behavior under cellular damage as well as the relation to disease prognosis in humans remain to be elucidated.

We report here an approach to a method for the early detection of cellular damage using a tRNA-specific antibody. The approach is efficient and has the potential to be extended to determining all cellular and organ damage and estimating the relative risk for mortality in humans as well as facilitating the investigation into invisible oxidative mechanisms.

Results

An Anti–1-Methyladenosine Antibody Detects Both tRNA and tRNA-Derived Fragments

To examine the involvement of tRNA in the stress response, we established a monoclonal antibody against mononucleoside 1-methyladenosine (m1A), a key eukaryotic tRNA-specific modified nucleoside9 that is important for folding tRNA into a tertiary structure10 (Figure 1A). This antibody specifically recognizes m1A but no other nucleosides.11 To confirm that the anti-m1A antibody was able to detect mononucleoside m1A and also tRNA itself, we performed Northern blot analysis with the antibody. The antibody only detected mammalian and not bacterial tRNA, which has no m1A modification (Figure 1B). We next investigated whether the antibody would detect tRNA-derived fragments (e.g., tiRNA). It is well known that arsenite is a tRNA stress inducer,6 and our application of arsenite induced tiRNA (Figure 1C, left panel). Northern blot using the antibody detected the generation of tiRNA as well as tRNA itself (Figure 1C, right panel). In addition, incubation of tRNA with recombinant angiogenin, a member of the ribonuclease (RNase) A family, is thought to participate in stress-induced tRNA cleavage-generated tiRNA,5,6 and this cleavage step of tRNA into tiRNA was clearly identified (Figure 1D).

Figure 1.

Figure 1.

Open in a new tab

Anti-m1A antibody detects unfolded tRNA and tRNA-derived fragments. (A) Secondary and tertiary structure of tRNA and m1A. (B) Total RNA from mammal (Mam; rat kidney) or bacteria (Bac; Escherichia coli) was analyzed by SYBR staining or Northern blot (NB) using the anti-m1A antibody (Ab). (C) Human kidney HK-2 cells were treated with arsenite. Total RNA was analyzed by SYBR staining or NB. The levels of 5S rRNA are shown as loading controls. (D) In vitro RNA digestion by angiogenin. Total RNA isolated from normal rat kidney was incubated with recombinant human angiogenin. (E) Total RNA extracted from rat injured kidney was analyzed by NB. Kidney injury was induced by unilateral renal I/R or cisplatin injection. (F) Unfolded or refolded tRNA was immunoprecipitated by the anti-m1A Ab and analyzed by NB. Yeast tRNAphe and human tRNApro were used. IP, immunoprecipitation; nt, nucleotide.

Using this antibody, we next investigated whether this stress-induced tRNA cleavage occurs in vivo. Northern blot analysis showed that renal ischemia/reperfusion (I/R) and cisplatin nephrotoxic models, both of which induce tissue damage by oxidative stress,12,13 each generated stress-induced tRNA cleavage and tiRNA production in the damaged kidney (Figure 1E). These results showed that the cleavage of tRNA into tRNA-derived fragments is a phenomenon not only in vitro6 but also, in vivo under stress conditions.

The Anti-m1A Antibody Detects the Difference of tRNA Conformation

We next examined the relationship between the anti-m1A antibody epitope and tRNA conformation. It is well known that tRNA is folded and takes the form of a tertiary cloverleaf structure (Figure 1A).14 Because extracted tRNA from tissue is denatured and unfolded, we used an in vitro tRNA refolding technique14 to reconstitute tRNA tertiary structure. Subsequently, immunoprecipitation analysis was performed to assess antibody recognition to unfolded or refolded tRNA. When the anti-m1A antibody was incubated with unfolded or refolded tRNA, the antibody only immunoprecipitated unfolded tRNA (Figure 1F). These data suggest that the anti-m1A antibody only recognizes the unfolded tRNA form.

The Visualization and Quantification of tRNA Conformational Change

We next attempted to visualize the tRNA behavior in damaged tissues by immunohistochemistry (IHC). In the various organ I/R models, the anti-m1A antibody mainly stained the outer medullary tubules of the kidney, brain neuronal cells, and the pericentral area of hepatocytes corresponding to the regions vulnerable to ischemia15,16 (Figure 2A, Supplemental Figure 1). To further determine what kind of tRNA metabolism occurred in the damaged area, we next performed the following studies.

Figure 2.

Figure 2.

Open in a new tab

Anti-m1A antibody staining of damaged tissues. (A) IHC using the anti-m1A Ab. (Left panel) Rat kidney after unilateral renal I/R. (Right panel) Corticomedullary lesions (low magnification) and medullary tubules (high magnification). The stained tubular segments were mainly medullary thick ascending limbs and collecting ducts. (Upper panel) Rat brain after cerebral I/R. (Lower panel) Mouse liver after hepatic I/R. CV, central vein; PV, portal vein. Scale bar, 1 mm in low magnification; 50 μm. (B) Immunoelectron microscopy using the anti-m1A Ab. Outer medullary tubular cells of rat kidney exposed to renal I/R were analyzed. Mt, mitochondria. Scale bar, 500 nm. (C) Total amount of m1A-modified tRNA in tissues. The m1A levels in digested total RNA components were measured by LC-MS/MS. Total RNA was extracted from rat kidney with or without (sham) exposure to renal I/R. Relative values are shown (as sham=1.0). n=3. Data are the means±SEMs. n.s., not significant. (D and E) Wild-type and Ang1−/− mouse livers after hepatic I/R. (D) For NB, 1-hour ischemia and 2-hour reperfusion were performed. (E) For IHC, 30-minute ischemia and 1-hour reperfusion were performed. Scale bar, 100 μm. NB, northern blot; WT, wild-type.

First, we examined whether the anti-m1A antibody does recognize the tRNA present in the tissues. Treatment of osteosarcoma U2OS cells with RNase A canceled the immunopositive signals, suggesting that the antibody recognizes cellular tRNA (Supplemental Figure 2). In addition, electron microscopy revealed that, in the damaged tissue, enhanced immunopositive signals were distributed in the cytoplasmic compartment and endoplasmic reticulum where tRNA localizes (Figure 2B). These results suggested that the positive signals in the damaged tissues were derived from the tRNA components.

Second, we examined whether enhanced immunopositive signals reflected an increased amount of tRNA in the damaged tissues. Because m1A is only derived from tRNA, we measured the whole amount of m1A in the tissue by liquid chromatography–tandem mass spectrometry (LC-MS/MS). As a result, it was shown that the amount of m1A in the total RNA was not significantly different between the I/R and control kidneys (Figure 2C). These data suggest that the total amount of tRNA dose did not change in the damaged tissues.

Third, to exclude the possibility that the increased immunopositive signals in the damaged tissue reflect the amount of tiRNA, we generated knockout mice lacking angiogenin-1 (Ang1), a mouse homolog of human angiogenin that is responsible for stress-induced tRNA cleavage (Supplemental Figure 3, A and B). The ablation of Ang1 did not change the expression of the other Ang subtypes (Ang2, -3, -4, -5, and -6) (Supplemental Figure 3C). In Ang1 knockout mice, Northern blot analysis revealed that the production of tiRNA in the liver was not seen under the I/R condition (Figure 2D). Under similar conditions, the enhancement of the immunopositive signals detected by the antibody was still observed in the damaged regions in the Ang1 knockout mice (Figure 2E). These results suggest that the enhanced immunopositive signals in damaged tissues do not reflect the increased amount of tiRNA, suggesting the qualitative change of tRNA.

Fourth, we examined the possibility that a qualitative change in tRNA may account for the enhanced immunopositive signals in the damaged tissues. I/R caused strong oxidative stress. As shown in Figure 1E, the anti-m1A antibody recognizes the unfolded tRNA form but not folded tRNA. Therefore, we examined whether the folded tRNA structure was disrupted by oxidative stress using the immunoprecipitation system. After exposure of refolded tRNA to hydrogen peroxide (H2O2) or γ-radiation, the anti-m1A antibody strictly displayed the ability to immunoprecipitate tRNA, representing the stress-exposed unfolded form (Figure 3A). These results suggest that stress-induced conformational change of tRNA is generated by oxidative stress in damaged tissue and that such change may be visualized as enhanced immunopositive signals in IHC using the antibody.

Figure 3.

Figure 3.

Open in a new tab

Oxidative stress damage caused tRNA conformational change. (A) Refolded and unfolded yeast tRNAphe was treated with H2O2 or γ-irradiation and then immunoprecipitated by the anti-m1A antibody. (B) Upregulation of angiogenin did not induce tiRNA generation. (Left panel) Ang1 mRNA levels in mouse liver after the injection of thioglycollate (thio) medium. n=3. Data are the means±SEMs. *P<0.05; **P<0.01 versus the PBS-treated group. (Right panel) NB of liver total RNA. The generation of tiRNA was not detected in the thio-injected group. IP, immunoprecipitation; NB, northern blot.

To investigate whether the generation of tiRNA was affected only by the upregulation of angiogenin level, we injected thioglycollate, an angiogenin inducer,17 into mice and checked the induction of tiRNA. Although the angiogenin mRNA was upregulated by thioglycollate in the liver (Figure 3B, left panel), no generation of tiRNA was detected (Figure 3B, right panel). On the other hand, in vitro analysis showed that angiogenin preferentially cleaved unfolded-tRNA rather than refolded-tRNA (Supplemental Figure 4). These results suggest that tRNA conformational change is necessary prior to stress-induced tiRNA generation cleaved by angiogenin.

The Conformational Change of tRNA Is Much More Rapid than Apoptosis and DNA Damage

We next assessed the time course of tRNA conformational changes compared with the time course of DNA damage under tissue-damaging conditions. In the mouse hepatic I/R model, the enhancement of the immunopositive signals by the anti-m1A antibody occurred 30 minutes after reperfusion, which is much earlier than that of the DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL; 8 hours after reperfusion) (Figure 4A). Furthermore, the immunopositive signal by the anti-m1A antibody was also detected, even by much shorter ischemia exposure (Figure 4B). In a cisplatin kidney injury model, the enhanced 8-hydroxy-2′-deoxyguanosine (8-OHdG) signals, a direct DNA oxidation damage marker, were detected 72 hours after the injection. However, the enhanced immunopositive signals shown by the antibody were detected 24 hours after the injection (Figure 4C). These data indicate that stress-induced tRNA conformational changes occur much earlier than DNA damage and that this detection of tRNA changes is useful as a rapid stress marker.

Figure 4.

Figure 4.

Open in a new tab

The time course of the tRNA conformational change and DNA damages. The time course of IHC with anti-m1A Ab and TUNEL or 8-OHdG staining. (A and B) Hepatic I/R of mice. (A) Hepatic ischemia (1 hour) and reperfusion (at the indicated time). Black arrows denote TUNEL-positive cells. HE, hematoxylin and eosin staining. Scale bar, 100 μm. (B) Fifteen- or thirty-minute hepatic ischemia and 1-hour reperfusion. Scale bar, 100 μm. (C) Cisplatin-induced rat kidney damage (24 and 72 hours after cisplatin injection). The S3 segments in proximal tubules were observed. Scale bar, 50 μm.

The Levels of Circulating tRNA Derivatives Are Sensitive to Tissue Damage

Recently, it was reported that small RNA fragments (e.g., microRNA) are present in the circulation18 and can serve as useful biomarkers of various diseases.19 Therefore, we next measured the levels of circulating tRNA and its derivatives using an anti-m1A antibody-based ELISA.11 This ELISA assay detects tRNA (unfolded), tRNA-derived fragments (e.g., tiRNA), and mononucleoside free m1A (summarized in Figure 7). We collectively termed these three components tRNA derivatives. We measured free m1A by LC-MS/MS to assess the relative contribution of free m1A among the tRNA derivatives (Supplemental Figures 5–7). Exposure of cultured cells to H2O2 or arsenite increased the level of extracellular tRNA derivatives in the cell culture medium (Figure 5A). We also measured the circulating tRNA derivatives in I/R models. As shown in Figure 5B, the plasma tRNA derivatives measured by ELISA were also significantly increased in unilateral and bilateral renal I/R models (Figure 5B). In addition, cisplatin administration was also shown to increase the plasma level of tRNA derivatives (Figure 5B). Although the level of plasma free m1A measured by LC-MS/MS was increased significantly, the value was much lower than the value obtained by ELISA (Figure 5B, tRNA derivatives versus free m1A). The difference in the values between them obtained by ELISA and LC-MS/MS may contribute to the increase of tRNA-derived fragments other than free m1A in these models (Figure 7).

Figure 7.

Figure 7.

Open in a new tab

tRNA conformational change and degradation reflect cell damage. A proposed model of tRNA change in cell damage. Native tRNA is normally in a tertiary fold configuration. When cells are exposed to oxidative stress, tRNA is damaged, and the conformation is changed and unfolded. The unfolded tRNA is cleaved to tiRNA by angiogenin, and it suppresses global protein translation for the purpose of conserving energy and enhancing cell survival. The cleaved tRNA finally degrades to mononucleosides. Red circles denote m1A.

Figure 5.

Figure 5.

Open in a new tab

The circulating tRNA derivative level is a marker of tissue damage. The levels of tRNA derivatives and free m1A were measured by ELISA and LC-MS/MS, respectively. (A) Human hepatoma HepG2 or HK-2 cells were cultured with H2O2 or arsenite. The tRNA derivative level in the culture medium was measured. n=4. (B) The level of plasma tRNA derivatives and free m1A in rats after kidney injury. Rats were subjected to unilateral (uni) or bilateral (bil) renal I/R or cisplatin injection. Black bars, plasma tRNA derivatives; white bars, plasma free m1A. n=3–5 per group. (C) Plasma tRNA derivatives after tissue injury. Total RNA was isolated from rat plasma after bilateral renal I/R. The extracted RNA (from 70 μl plasma per lane) was analyzed by NB. (D) Samples from porcine renal I/R. n=4. (E) Samples from patients undergoing aortic arch replacement surgery. Urinary KIM-1 (u-Kim-1) and serum creatinine (s-Cr) were also measured. n=4. (F) The level of plasma tRNA derivatives in mice after γ-irradiation. (Left panel) Seven days after exposure to the indicated doses. (Right panel) At the indicated times after exposure to 10-Gy irradiation. n=4–5 per group. Data are the means±SEMs. *P<0.05; **P<0.01 versus control group (D) preischemia or (E) preoperation (pre-ope). NB, northern blot.

To further confirm the difference, we analyzed whole-plasma RNA by Northern blot using the anti-m1A antibody. tRNA and tiRNA were detected in the whole plasma, and the tRNA-derived fragments were increased after renal I/R (Figure 5C).

In addition, the plasma level of the tRNA derivatives measured by ELISA was rapidly increased after I/R in a porcine kidney (Figure 5D). The plasma free m1A measured by LC-MS/MS was also increased, but the level was lower than the level of tRNA derivatives (Figure 5D). Because the level of tRNA derivatives in renal venous blood was much higher than the level in peripheral blood, these data further suggest that the circulating tRNA derivatives originated from the damaged kidney (Figure 5D).

We also examined the plasma tRNA derivatives in patients who underwent total aortic arch replacement surgery. During this surgical procedure, ischemic tissue damage, including the ischemic tissue damage of the renal I/R, is inevitable. In the experiments, we simultaneously measured the urinary kidney injury marker-1 (KIM-1), a sensitive marker of AKI,20 to confirm and evaluate the renal damage. During the operation, the level of plasma tRNA derivatives was rapidly increased after the reperfusion procedure, whereas the level of serum creatinine did not change (Figure 5E). Although urinary KIM-1 was significantly and rapidly increased, the onset of the rise in the plasma tRNA derivatives shown by ELISA was much earlier than the onset of the rise of urinary KIM-1. These data strongly suggest that the circulating tRNA derivatives are a useful marker to predict I/R tissue damage and that they are much more sensitive than other tissue damage markers, including KIM-1 and creatinine.

We next examined the possibility of measuring the circulating tRNA derivatives in response to radiation, because ionizing radiation induces oxidative stress, leading to both cell and DNA damage.21 Exposure of mice to γ-radiation caused an elevation in the plasma tRNA derivatives (Figure 5F, left panel). The plasma tRNA derivatives determined by ELISA steadily increased for up to 7 days after irradiation (Figure 5F, right panel). These data also suggest the possibility that tRNA derivatives can serve as a marker of the invisible life-threatening event of radiation exposure.

tRNA Metabolism Is Linked to Mortality in Humans

It is well known that chronic oxidative stress has been shown to be a risk factor for mortality as well as morbidity in the general population.22 To evaluate the relationship, we examined the association between the circulating free m1A level and mortality in a general cohort study (n=1033)23 (Supplemental Table 1). During a mean follow-up of 6.7 years, 72 deaths were observed. The analysis showed that the circulating free m1A level was highly associated with whole death (Supplemental Figure 8), even after adjustment for age, sex, systolic BP, eGFR, smoking, use of antihypertensive medication, history of diabetes mellitus, hyperlipidemia, and cardiovascular disease (Figure 6A). The relative hazard risk of death was 2.99 (95% confidence interval [95% CI], 0.97 to 9.20) for the second quartile, 2.05 (95% CI, 0.63 to 6.69) for the third quartile, and 3.05 (95% CI, 1.02 to 9.39) for the fourth quartile compared with the relative hazard risk of death of the lowest m1A level group. These results suggest that the level of the circulating tRNA metabolites can serve as an indicator of prognosis.

Figure 6.

Figure 6.

Open in a new tab

Association of circulating free m1A, mortality, and chronic oxidative stress. (A) Association between the circulating free m1A level and mortality in the general population (n=1033) analyzed with a multivariate regression analysis. (B) Statin treatment reduces the circulating free m1A level. Pitavastatin (2 mg/d) was administered to CKD patients (n=29). The serum free m1A levels at pretreatment and after 3 months of statin treatment were measured by LC-MS/MS. Healthy controls (n=10). Data are means±SEMs. ***P<0.001 versus the control or pretreatment group.

In addition, CKD is also characterized by a state of increased oxidative stress, and this increased oxidative stress contributes to both a high rate of mortality and the rapid progression of kidney disease.2 Therefore, we next measured the free m1A in CKD patients. As a result, the circulating levels of free m1A were higher in the CKD group than a healthy control group (Figure 6B). Interestingly, treatment with a statin reduced the circulating free m1A level in CKD patients (n=29) independent of the change in renal function (Figure 6B, Supplemental Figure 9). These data also suggest that increased circulating free m1A, thus, reflects a disease prognosis that may be modified by drug intervention.

Discussion

Conformational Change of tRNA Is a Stress Sensor

It is now evident that RNA molecules are not only intermediates in the transfer of genetic information from DNA to proteins but also, key players in the control of cell metabolism.24 Cytochrome c is released from damaged mitochondria and promotes the apoptosome complex under cell stress condition.25 tRNA binds to cytochrome c and prevents the activation of downstream apoptotic process by serving as an effective scavenger of released cytochrome c.26 tiRNA-mediated halting of global protein translation through a phosphor-eukaryotic initiation factor 2α–independent stress response has also been suggested.7 Here, as shown in Figure 7, we have shown that loosening the tertiary conformation of tRNA in response to oxidative stress is the initial trigger of tRNA metabolism and that this tRNA conformational change is essential to the enzymatic cleavage of tRNA into tiRNA by angiogenin. Because RNA is mostly single-stranded and because its bases are not protected by hydrogen bonds, RNA is believed to be more susceptible to oxidative stress than DNA.27 tRNA has an L-shaped tertiary structure through coaxial stacking of the helices. To stabilize this structure, methylation at the N1 position of A58 introduces a positive charge into the elbow region of the tRNA tertiary configuration without disrupting any of the hydrogen bonds. Thus, we generated an anti-m1A antibody that recognizes 1-methylated adenosine, a specific nucleotide to tRNA, to identify this tRNA mobilization.

The secretion of angiogenin, which is responsible for stress-induced tRNA cleavage, is reported to be enhanced under stress conditions.28,29 However, the cleavage of intact tRNA by angiogenin does not result from a simple upregulation of angiogenin (Figure 3B). The knockout of Ang1 also did not affect the immunostaining by anti-m1A antibody in the damaged tissue (Figure 2E). Therefore, these data suggest that the disruption of the tertiary structure of tRNA enables angiogenin to gain access to tRNA and have an effect on the subsequent stress-induced tiRNA generation.

We were also able to visualize the immediate changes of tRNA in the damaged tissues, and the time course was more rapid than that of DNA damage (Figure 4, A and C). In cells damaged by various forms of stress (ischemia, toxic drug, radiation, etc.), mitochondria produce the superoxide radical, but this radical is produced in far greater quantities when mitochondrial respiration is compromised. Most of the oxidization by radicals occurs in the ribosomes around mitochondria under oxidative conditions.30 We identified a distribution of damaged unfolded tRNA around injured mitochondria (Figure 2B) and were able to visualize the immediate change in tRNA in many vulnerable tissues before the appearance of DNA damage (Figure 4, A and C). Thus, the detection of tRNA conformational changes using a tRNA-specific m1A antibody is much more useful for detecting early cell damage.

Although proximal tubules were also damaged in rat I/R models, the immunopositive signal by the anti-m1A antibody in IHC was relatively weak. In proximal tubules, ischemic damage causes the excretion of blebs to the lumen.31 As shown in Supplemental Figure 10, anti-m1A antibody stained such blebs by IHC. These results suggested that blebs contain damaged tRNA and that tRNAs damaged by oxidative stress were promptly excreted through the cytoplasmic blebbing from proximal tubule cell to lumen. Consequently, we consider that the immunopositive signal by the anti-m1A antibody, reflecting the amount of intracellular damaged tRNAs, was weak in proximal tubules.

Oxidative stress is produced in even normal physiologic conditions by abundant sodium transport activity and low blood supply in medullary tubular cells. In our data, IHC by anti-m1A antibody showed weak immunopositive signals in medullary tubules, even in normal condition (Figure 2A). The signals may reflect the tRNA conformational change by the baseline oxidative stress states. However, the reduction of oxidative stress through inhibition of the sodium transport activity by furosemide, a Na+-K+-2Cl− cotransporter antagonist, did not reduce the immunopositive signal levels by anti-m1A antibody (Supplemental Figure 11). We attributed this result to the limitation of the assay sensitivity or involvement of some stress factors (other than sodium transport–mediated oxidative stress) in tRNA damage.

Detection of tRNA Derivatives in the Circulation Is Sensitive

We have found that, under various stress conditions, tRNA derivatives (including unfolded tRNA, tiRNA, and degraded tRNA fragments) are released into the circulation. We also found that the determination of tRNA derivatives by an antibody-based ELISA is more sensitive than measuring the m1A nucleoside by LC-MS/MS, although the increase of m1A nucleoside was significant.

In the blood, naked RNA is degraded within seconds because of the high nuclease levels. However, it is well known that microRNA in the blood avoids degradation by binding to protective proteins, such as argonaute18 or being surrounded by exosomes.32 We confirmed that the circulating exosomes contain tRNA and tRNA-derived fragments (Supplemental Figure 12). Thus, tRNA and tRNA-derived fragments do seem to be prevented from degradation in the blood.

Urinary modified nucleosides have been reported to be excreted in abnormal amounts in patients with malignancies.33–35 An increased urinary level of oxidized nucleosides in patients with Alzheimer's disease was also reported.36 One possible cause of an increase in modified nucleosides has been ascribed to a close relationship between oxidative stress, carcinogenesis, and Alzheimer’s disease.37

The concentration of tRNA derivatives is also increased by γ-irradiation. Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through a direct interaction of the radiation with target macromolecules or through products of water radiolysis.38 In this setting, detection of tRNA conformational change would also be a potential biomarker for radiation tissue damage.

tRNA Degradation Affords a Prognosis That Allows Intervention

We have shown here that increased circulating free m1A is associated with mortality in the general population. We also reported that increased circulating m1A in CKD patients was decreased after statin treatment (Figure 6B). As discussed above, the high level of free m1A reflects the steady state level of oxidative stress. In addition, oxidative stress has been implicated in CKD patients.2 Therefore, we suggest that the cause of the increase of free m1A in such patients is the persistent RNA damage induced by oxidative stress. A recent meta-analysis reported that overall mortality was significantly lower in groups treated with a statin than placebo, independent of the statin lipid-lowering effect.39 Several reports have also indicated that statins possess antioxidant effects that are thought to help reduce overall mortality.40 We showed that statin treatment helped to decrease the free m1A level in CKD patients. As shown in Supplemental Figure 13, statin administration decreased the m1A-immunopositive signals in damaged tissues. In this regard, the level of tRNA derivatives can serve as a marker of oxidative stress and be used to predict the disease prognosis as well as serve as a marker of the efficacy of drug intervention.

In conclusion, stress-induced tRNA metabolism reflects cell damage, and monitoring the level as well as conformational change can serve as a novel tool for gauging the extent of cellular damage and forming more accurate prognoses and a intervene marker for CKD.

Concise Methods

RNA Isolation and Northern Blot Analysis

Total RNA was isolated using miRNeasy (Qiagen), separated by 12% polyacrylamide 8 M urea denaturing gel electrophoresis, and analyzed using SYBR Gold staining or Northern blot. The separated RNA was transferred onto nylon membranes. The membranes were ultraviolet-cross-linked, blocked, and incubated with the anti-m1A antibody (1:300). After washing, the membranes were incubated with horseradish peroxidase–conjugated goat anti-mouse IgG. ECL Plus (GE Healthcare) was used for visualization.

RNA Digestion

For in vitro angiogenin digestion assay, total RNA was incubated with recombinant human angiogenin (Sigma-Aldrich) in a buffer solution (30 mM NaCl and 30 mM Hepes [pH 6.8]) for 60 minutes at 37°C. The enzymatic digestion of RNA to the mononucleoside level was also performed with nuclease P1, phosphodiesterase I, and alkaline phosphatase as described.41

Immunoprecipitation of tRNA

Yeast tRNAphe (Sigma-Aldrich) or human tRNApro (Bio S&T) was refolded in 0.5×Tris-EDTA buffer (5 mM Tris⋅HCl [pH 7.5] and 0.5 mM EDTA) by heating at 80°C for 3 minutes and then cooling to 60°C for 2 minutes followed by the addition of 10 mM MgCl2 and gradual cooling to room temperature.42 Unfolded tRNA was prepared without the heating procedure. Refolded or unfolded tRNA was incubated with protein G agarose beads pretreated with anti-m1A antibody for 1 hour at room temperature in the immunoprecipitation buffer (50 mM Tris⋅HCl [pH 7.5], 130 mM NaCl, 10 mM MgCl2, and 0.05% NP-40). After washing with immunoprecipitation buffer three times, the precipitated RNA was extracted with Trizol and miRNeasy.

Refolded or unfolded tRNAphe were mixed with the indicated concentration of H2O2 in the reaction buffer (130 mM NaCl, 10 mM MgCl2, and 80 nM FeSO4). After 20 minutes of incubation at room temperature, two volumes of immunoprecipitation buffer were added to the reaction buffer containing tRNAphe and immunoprecipitated with the anti-m1A antibody as described above.

Refolded and unfolded tRNAphe were exposed to the indicated doses of γ-irradiation in a buffer solution (130 mM NaCl and 10 mM MgCl2). After irradiation, an equal volume of immunoprecipitation buffer was added to the buffer and immunoprecipitated.

Measurement of the Circulating tRNA Derivatives

Plasma tRNA derivatives were measured by ELISA using the anti-m1A antibody.11 Briefly, 96-microwell plates were coated with BSA-conjugated m1A in PBS. Aliquots of serially diluted m1A (Sigma-Aldrich) solution or measurement samples were applied to the wells. An equal volume of anti-m1A antibody solution was added to each well. After incubation, plates were washed, and alkaline phosphatase-conjugated anti-mouse IgG was added. After incubation, the plates were washed, and p-nitrophenyl phosphate in 1 M diethanolamine (pH 9.8) was added. The absorbance at 405 nm was measured.

Measurement of Circulating Free m1A

The free m1A levels were measured by LC-MS/MS. The sample preparation procedure and LC-MS/MS condition of m1A were described in a previous study.43–45 LC-MS/MS was comprised of a NANOSPACE SI-2 HPLC system and a TSQ Quantum Ultra (Thermo Fisher Scientific) triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. Samples were analyzed in selected reaction monitoring mode. The optimum value of selected reaction monitoring was determined by monitoring the following transitions: m/z 282.2>150.0 for m1A and m/z 267.1>135.0 for the internal standards. Liquid chromatographic separation was performed using an Xbridge C18 (150×2-mm inner diameter, 3.5-µm particle size; Waters) column with a gradient elution.

IHC

Tissues were fixed with 10% neutral buffered formalin. Paraffin-embedded sections were incubated with the anti-m1A antibody (1:100), blocked, and then incubated with horseradish peroxidase–labeled anti-mouse IgG; 3,3′-diaminobenzidine (DAB) was used for visualization.

The following antibodies were used: anti–Na+-K+-2Cl− cotransporter (Alpha Diagnostic) and anti–8-OHdG (JAICA). For 8-OHdG staining, Bouin's fixed tissues were used. For immunocytochemistry, human osteosarcoma U2OS cells were fixed in 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100 in PBS. Cells were incubated with anti-m1A antibody followed by incubation with the secondary antibody, CF488A anti-mouse IgG (H+L) F(ab)′2 fragments. For RNase A treatment, fixed and permeabilized cells were incubated with 1 mg/ml RNase A in PBS for 60 minutes at 37°C.

For immunoelectron microscopic analysis, frozen tissues fixed with 0.5% glutaraldehyde and 2% paraformaldehyde were incubated with an anti-m1A antibody, and then, DAB staining was performed using the appropriate secondary antibody and a DAB staining kit. After osmium staining, sections were embedded in resin. TUNEL staining was performed using a TUNEL Assay Kit (Wako).

Cell Experiments

Cells were cultured in DMEM supplemented with 10% FBS. For tiRNA induction, 200 μM sodium arsenite (Wako) was added to the medium. For measurements of tRNA derivatives in the culture medium, cells were plated in six-well dishes at 150,000 cells per well. After incubation for 12 hours, the cells were washed with PBS and treated with 1.5 ml medium containing H2O2 or sodium arsenite. After an additional incubation for 12 hours, the culture medium was collected and then centrifuged at 15,000×g for 15 minutes to discard the debris.

Animal Experiments

All animal experiments were approved by the Tohoku University Animal Care Committee. A segmental (70%) hepatic I/R model was performed in 8-week-old C57BL/6 male mice.46 For the renal I/R model, male Wister rats (weighing 250–350 g) were exposed to renal ischemia induced by clamping the unilateral or bilateral renal arteries for 60 minutes and then killed 60 minutes after removal of the clamp. For the cisplatin-induced kidney injury model, male Sprague–Dawley rats (weighing 300–350 g) received an intraperitoneal injection of cisplatin (8 mg/kg) and were killed 1 or 3 days after the injection. The rat focal cerebral I/R model was produced as described previously.47 For γ-irradiation, 9-week-old C57BL/6 male mice were exposed to the indicated dose of whole-body γ-irradiation in a Gammacell 40 exactor (Nordion). Plasma exosomes were collected from rat plasma using ExoQuick (SBI).

Ang1 Knockout Mice and Angiogenin Upregulation In Vivo

The Ang1+/− heterozygous mouse B6;CB-Rnase4 Gt(pU-21W)371Card was generated using the gene trap method as described.48 PCR primers used are summarized in Supplemental Table 2. To upregulate angiogenin expression, 1 ml solution of 3% thioglycollate medium (Difco Laboratories) was injected intraperitoneally into 8-week-old male C57BL/6 mice.17 Quantification of Ang1 mRNA was performed by real-time PCR analysis using the mouse Ang1 primer (Mm00833184_s1; Applied Biosystems). The gene expression levels were expressed relative to glyceraldehyde 3-phosphate dehydrogenase.

Human Experiment and Data Analyses

The review board of Tohoku University approved the study, and written informed consent was obtained from participants. Urinary KIM-1 was measured by ELISA.20 The subjects were patients undergoing aortic arch replacement surgery with the aid of cardiopulmonary bypass and hypothermic circulatory arrest for the treatment of aortic aneurysm. Blood and urine samples were collected both before and during surgery.

For the statin treatment study, pitavastatin (2 mg/d) was administered to CKD patients whose eGFR was below 60 ml/min per 1.73 m2 and who had not previously received statin treatment. eGFR was calculated using the abbreviated Modification of Diet in Renal Disease equation modified by the Japanese coefficient.49

For a general population-based study, the investigation was performed as a part of the Ohasama study,23,50 a longitudinal observation of subjects who had been participating in a BP measurement project in Ohasama, Iwate Prefecture, Japan since 1987. The socioeconomic and demographic characteristics of this region and full details of the project are described elsewhere.50 The serum free m1A levels were obtained from 1033 participants ≥35 years (mean=61.1 years). Cox proportional hazards models were used to estimate the relative hazard and 95% CIs adjusted for age, sex, systolic BP, eGFR, smoking, use of antihypertensive medication, history of diabetes mellitus, hyperlipidemia, and cardiovascular diseases.

Statistical Analyses

Data are the means±SEMs. Comparisons between groups were evaluated using a two-tailed paired or unpaired t test or ANOVA. P<0.05 was considered statistically significant.

Disclosures

None.

Supplementary Material

Supplemental Data
supp_25_10_2316__index.html (793B, html)

Acknowledgments

We thank Keiichi Shirasaki for technical assistance and Manabu Fukumoto, Hiroaki Shimizu, Tohru Onogawa, and Takuo Hirose for discussion.

This work was supported, in part, by National Grant-in-Aid 23390033 for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Japan Kidney Foundation, and the Miyagi Kidney Foundation.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, “t-RNA Fragmentation as an Early Biomarker of (Kidney) Injury,” on pages 2145–2147.

References

  • 1.Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39: 44–84, 2007 [DOI] [PubMed] [Google Scholar]
  • 2.Oberg BP, McMenamin E, Lucas FL, McMonagle E, Morrow J, Ikizler TA, Himmelfarb J: Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int 65: 1009–1016, 2004 [DOI] [PubMed] [Google Scholar]
  • 3.Sharfuddin AA, Molitoris BA: Pathophysiology of ischemic acute kidney injury. Nat Rev Nephrol 7: 189–200, 2011 [DOI] [PubMed] [Google Scholar]
  • 4.Thompson DM, Lu C, Green PJ, Parker R: tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA 14: 2095–2103, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Fu H, Feng J, Liu Q, Sun F, Tie Y, Zhu J, Xing R, Sun Z, Zheng X: Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett 583: 437–442, 2009 [DOI] [PubMed] [Google Scholar]
  • 6.Yamasaki S, Ivanov P, Hu GF, Anderson P: Angiogenin cleaves tRNA and promotes stress-induced translational repression. J Cell Biol 185: 35–42, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ivanov P, Emara MM, Villen J, Gygi SP, Anderson P: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 43: 613–623, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lee YS, Shibata Y, Malhotra A, Dutta A: A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 23: 2639–2649, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Saikia M, Fu Y, Pavon-Eternod M, He C, Pan T: Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs. RNA 16: 1317–1327, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Basavappa R, Sigler PB: The 3 A crystal structure of yeast initiator tRNA: Functional implications in initiator/elongator discrimination. EMBO J 10: 3105–3111, 1991 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Itoh K, Mizugaki M, Ishida N: Preparation of a monoclonal antibody specific for 1-methyladenosine and its application for the detection of elevated levels of 1-methyladenosine in urines from cancer patients. Jpn J Cancer Res 79: 1130–1138, 1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Granger DN, Korthuis RJ: Physiologic mechanisms of postischemic tissue injury. Annu Rev Physiol 57: 311–332, 1995 [DOI] [PubMed] [Google Scholar]
  • 13.dos Santos NA, Carvalho Rodrigues MA, Martins NM, dos Santos AC: Cisplatin-induced nephrotoxicity and targets of nephroprotection: An update. Arch Toxicol 86: 1233–1250, 2012 [DOI] [PubMed] [Google Scholar]
  • 14.Serebrov V, Clarke RJ, Gross HJ, Kisselev L: Mg2+-induced tRNA folding. Biochemistry 40: 6688–6698, 2001 [DOI] [PubMed] [Google Scholar]
  • 15.Brezis M, Rosen S: Hypoxia of the renal medulla—its implications for disease. N Engl J Med 332: 647–655, 1995 [DOI] [PubMed] [Google Scholar]
  • 16.Jaeschke H, Lemasters JJ: Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 125: 1246–1257, 2003 [DOI] [PubMed] [Google Scholar]
  • 17.Olson KA, Verselis SJ, Fett JW: Angiogenin is regulated in vivo as an acute phase protein. Biochem Biophys Res Commun 242: 480–483, 1998 [DOI] [PubMed] [Google Scholar]
  • 18.Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M: Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108: 5003–5008, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Etheridge A, Lee I, Hood L, Galas D, Wang K: Extracellular microRNA: A new source of biomarkers. Mutat Res 717: 85–90, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bonventre JV: Kidney injury molecule-1 (KIM-1): A urinary biomarker and much more. Nephrol Dial Transplant 24: 3265–3268, 2009 [DOI] [PubMed] [Google Scholar]
  • 21.Riley PA: Free radicals in biology: Oxidative stress and the effects of ionizing radiation. Int J Radiat Biol 65: 27–33, 1994 [DOI] [PubMed] [Google Scholar]
  • 22.Broedbaek K, Siersma V, Henriksen T, Weimann A, Petersen M, Andersen JT, Jimenez-Solem E, Hansen LJ, Henriksen JE, Bonnema SJ, de Fine Olivarius N, Poulsen HE: Association between urinary markers of nucleic acid oxidation and mortality in type 2 diabetes: A population-based cohort study. Diabetes Care 36: 669–676, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Boggia J, Li Y, Thijs L, Hansen TW, Kikuya M, Björklund-Bodegård K, Richart T, Ohkubo T, Kuznetsova T, Torp-Pedersen C, Lind L, Ibsen H, Imai Y, Wang J, Sandoya E, O’Brien E, Staessen JA, International Database on Ambulatory Blood Pressure Monitoring in Relation to Cardiovascular Outcomes (IDACO) Investigators : Prognostic accuracy of day versus night ambulatory blood pressure: A cohort study. Lancet 370: 1219–1229, 2007 [DOI] [PubMed] [Google Scholar]
  • 24.Qureshi IA, Mehler MF: Emerging roles of non-coding RNAs in brain evolution, development, plasticity and disease. Nat Rev Neurosci 13: 528–541, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Orrenius S, Gogvadze V, Zhivotovsky B: Mitochondrial oxidative stress: Implications for cell death. Annu Rev Pharmacol Toxicol 47: 143–183, 2007 [DOI] [PubMed] [Google Scholar]
  • 26.Mei Y, Yong J, Liu H, Shi Y, Meinkoth J, Dreyfuss G, Yang X: tRNA binds to cytochrome c and inhibits caspase activation. Mol Cell 37: 668–678, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Brégeon D, Sarasin A: Hypothetical role of RNA damage avoidance in preventing human disease. Mutat Res 577: 293–302, 2005 [DOI] [PubMed] [Google Scholar]
  • 28.Hartmann A, Kunz M, Köstlin S, Gillitzer R, Toksoy A, Bröcker EB, Klein CE: Hypoxia-induced up-regulation of angiogenin in human malignant melanoma. Cancer Res 59: 1578–1583, 1999 [PubMed] [Google Scholar]
  • 29.Nakamura M, Yamabe H, Osawa H, Nakamura N, Shimada M, Kumasaka R, Murakami R, Fujita T, Osanai T, Okumura K: Hypoxic conditions stimulate the production of angiogenin and vascular endothelial growth factor by human renal proximal tubular epithelial cells in culture. Nephrol Dial Transplant 21: 1489–1495, 2006 [DOI] [PubMed] [Google Scholar]
  • 30.Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA: Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 60: 759–767, 2001 [DOI] [PubMed] [Google Scholar]
  • 31.Donohoe JF, Venkatachalam MA, Bernard DB, Levinsky NG: Tubular leakage and obstruction after renal ischemia: Structural-functional correlations. Kidney Int 13: 208–222, 1978 [DOI] [PubMed] [Google Scholar]
  • 32.Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, Xiao T, Schafer J, Lee ML, Schmittgen TD, Nana-Sinkam SP, Jarjoura D, Marsh CB: Detection of microRNA expression in human peripheral blood microvesicles. PLoS ONE 3: e3694, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Nakano K, Nakao T, Schram KH, Hammargren WM, McClure TD, Katz M, Petersen E: Urinary excretion of modified nucleosides as biological marker of RNA turnover in patients with cancer and AIDS. Clin Chim Acta 218: 169–183, 1993 [DOI] [PubMed] [Google Scholar]
  • 34.Xu G, Schmid HR, Lu X, Liebich HM, Lu P: Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP-HPLC and factor analysis method. Biomed Chromatogr 14: 459–463, 2000 [DOI] [PubMed] [Google Scholar]
  • 35.Yang J, Xu G, Kong H, Zheng Y, Pang T, Yang Q: Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer. J Chromatogr B Analyt Technol Biomed Life Sci 780: 27–33, 2002 [DOI] [PubMed] [Google Scholar]
  • 36.Lee SH, Kim I, Chung BC: Increased urinary level of oxidized nucleosides in patients with mild-to-moderate Alzheimer’s disease. Clin Biochem 40: 936–938, 2007 [DOI] [PubMed] [Google Scholar]
  • 37.Sotgia F, Martinez-Outschoorn UE, Lisanti MP: Mitochondrial oxidative stress drives tumor progression and metastasis: Should we use antioxidants as a key component of cancer treatment and prevention? BMC Med 9: 62, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Azzam EI, Jay-Gerin JP, Pain D: Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett 327: 48–60, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Razzolini R, Tarantini G, Ossena G, Favaretto E, Bilato C, Manzato E, Dalla-Volta S, Iliceto S: Non-cardiovascular mortality, low-density lipoprotein cholesterol and statins: A meta-regression analysis. Cardiology 109: 110–116, 2008 [DOI] [PubMed] [Google Scholar]
  • 40.Davignon J: Pleiotropic effects of pitavastatin. Br J Clin Pharmacol 73: 518–535, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Crain PF: Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol 193: 782–790, 1990 [DOI] [PubMed] [Google Scholar]
  • 42.Hargittai MR, Musier-Forsyth K: Use of terbium as a probe of tRNA tertiary structure and folding. RNA 6: 1672–1680, 2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Saigusa D, Suzuki N, Takahashi M, Shiba K, Tanaka S, Abe T, Hishinuma T, Tomioka Y: Simultaneous determination of guanidinosuccinic acid and guanidinoacetic acid in urine using high performance liquid chromatography/tandem mass spectrometry. Anal Chim Acta 677: 169–175, 2010 [DOI] [PubMed] [Google Scholar]
  • 44.Djukovic D, Baniasadi HR, Kc R, Hammoud Z, Raftery D: Targeted serum metabolite profiling of nucleosides in esophageal adenocarcinoma. Rapid Commun Mass Spectrom 24: 3057–3062, 2010 [DOI] [PubMed] [Google Scholar]
  • 45.Woo HM, Kim KM, Choi MH, Jung BH, Lee J, Kong G, Nam SJ, Kim S, Bai SW, Chung BC: Mass spectrometry based metabolomic approaches in urinary biomarker study of women’s cancers. Clin Chim Acta 400: 63–69, 2009 [DOI] [PubMed] [Google Scholar]
  • 46.Selzner N, Selzner M, Jochum W, Clavien PA: Ischemic preconditioning protects the steatotic mouse liver against reperfusion injury: An ATP dependent mechanism. J Hepatol 39: 55–61, 2003 [DOI] [PubMed] [Google Scholar]
  • 47.Akamatsu Y, Saito A, Fujimura M, Shimizu H, Mekawy M, Hasumi K, Tominaga T: Stachybotrys microspora triprenyl phenol-7, a novel fibrinolytic agent, suppresses superoxide production, matrix metalloproteinase-9 expression, and thereby attenuates ischemia/reperfusion injury in rat brain. Neurosci Lett 503: 110–114, 2011 [DOI] [PubMed] [Google Scholar]
  • 48.Kim HR, Jeon BH, Lee HS, Im SH, Araki M, Araki K, Yamamura K, Choi SC, Park DS, Jun CD: IGSF4 is a novel TCR ζ-chain-interacting protein that enhances TCR-mediated signaling. J Exp Med 208: 2545–2560, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta K, Yamagata K, Tomino Y, Yokoyama H, Hishida A, Collaborators Developing the Japanese Equation for Estimated GFR : Revised equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis 53: 982–992, 2009 [DOI] [PubMed] [Google Scholar]
  • 50.Ohkubo T: Prognostic significance of variability in ambulatory and home blood pressure from the Ohasama study. J Epidemiol 17: 109–113, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_25_10_2316__index.html (793B, html)
ASN.2013091001_ASN2013091001SupplementaryData.pdf (1.2MB, pdf)

Articles from Journal of the American Society of Nephrology : JASN are provided here courtesy of American Society of Nephrology

RESOURCES