Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Retention behavior of short double-stranded oligonucleotide and its potential impurities by anion-exchange chromatography under non-denaturing conditions.

Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions.

Successful Use of Higher-Dose Etanercept for Multirefractory Systemic Flare of Adult-Onset Still's Disease with Liver Failure with No Response to Tocilizumab Therapy.

A 21-year-old woman with refractory systemic flare of adult-onset Still's disease with liver failure despite high-dose corticosteroids, cyclosporine, tacrolimus, and tocilizumab, was successfully treated with additional use of etanercept. Etanercept at a dose of 50 mg weekly was partially effective but could not reduce the dose of concomitant betamethasone from 5 mg/day. Etanercept at a dose of 75 mg weekly could lead her to clinical remission and enabled successful tapering off the corticosteroids and discontinuation of etanercept. Normalization of serum C-reactive protein and interleukin 6 and persistent elevation of serum tumor necrosis factor α under the treatment with high-dose corticosteroids and immunosuppressants suggest that tumor necrosis factor α was more deeply involved than at least interleukin 6 in the pathogenesis of refractoriness of the disease in this patient, and these findings might be indicative of potential efficacy for adjunctive use of a tumor necrosis factor inhibitor rather than an interleukin 6 inhibitor.

Circadian rhythm of urinary potassium excretion in patients with CKD.

AIMS: We previously reported in patients with chronic kidney disease (CKD) that the circadian rhythms of blood pressure (BP) and urinary sodium excretion were both impaired into non-dipper pattern as renal function deteriorated. However, the circadian rhythm of urinary potassium excretion has not been studied in relation to renal dysfunction. METHODS: BP and urinary excretion rates of sodium (UNaV) and potassium (UKV) were evaluated for daytime and nighttime to estimate their circadian rhythms in 83 subjects with CKD. RESULTS: As renal function deteriorated, night/day ratios of UNaV and UKV were both increased. Night/day ratio of UKV was positively correlated with night/day ratio of UNaV (r = 0.60, p < 0.0001). Multiple regression analysis (R2 = 0.37, p < 0.0001) revealed that night/day ratio of UKV was determined independently by the night/day ratio of UNaV (r = -0.55, p < 0.0001), rather than renal function or night/day ratio of BP. CONCLUSIONS: Circadian rhythm of natriuresis was regulated by renal function and night/day ratio of BP. On the other hand, the circadian rhythm of urinary potassium excretion was primarily determined by neither renal function nor BP, but was correlated with that of urinary sodium excretion.

Low birth weight and end-stage renal disease: demographic analysis by region in Japan.

UNLABELLED: BACKGROUND We have previously shown regional differences in the incidence of end-stage renal disease (ESRD)within Japan, which is ethnically homogenous, suggesting that non-genetic factors may contribute to the differences.We examined regional distribution in the incidence of low birth weight (LBW), a surrogate for low nephron number,in our search for an explanation. METHODS: Each year, the Ministry of Health, Labour and Welfare of Japan and the Japanese Society for Dialysis Therapy report the number of LBW babies and patients initiating maintenance dialysis in each prefecture of Japan,respectively. In this study, we calculated the annual incidences of LBW and ESRD in 11 regions of Japan over a 24-year period from 1984 to 2007. RESULTS: There were distinct regional differences in the annual incidences of both LBW and ESRD (p<0.0001).These regional distributions persisted despite consistent increases (p<0.0001) in incidences of both LBW and ESRD during the study period. Compared with the reference group consisting of 3 regions with the lowest LBW incidence, the odds ratios for ESRD (95% confidence interval) of the 5 regions with intermediate LBW incidence and the 3 regions with the highest LBW incidence are 1.09(1.05­1.14) and 1.29 (1.22­1.35), respectively. The annual incidence of LBW was positively correlated with annual incidence of ESRD in their regional distribution across 11 regions (r = 0.66, p = 0.03). CONCLUSIONS: The present study, relating regional distribution between LBW and ESRD dynamics in a nationwide population of Japan, revealed that the marked regional differences in the incidence of ESRD within Japan could be explained by a similar regional distribution in the incidence of LBW.

Predictors of proteinuria reduction by monotherapy with an angiotensin receptor blocker, olmesartan.

INTRODUCTION: It is known that reduced glomerular filtration rate (GFR) is a crucial factor to limit the blood pressure lowering effect of antihypertensives. In the present study, we tested whether the effects of monotherapy with an angiotensin receptor blocker (ARB) to lower proteinuria could be restricted by reduced GFR. MATERIALS AND METHODS: Thirty-five renal patients who had albuminuria more than 30 mg/day, but did not have diabetic nephropathy or nephrotic syndrome, were studied before and during eight weeks of monotherapy with ARB, olmesartan. RESULTS: Blood pressure was lowered from 129 ± 18/79 ± 12 to 116 ± 18/72 ± 12 mmHg (p < 0.0001), while albuminuria was reduced from 614±630 to 343±472 mg/day (p < 0.0001). Albuminuria was inversely correlated with GFR both before and during treatment. Albuminuria reduction was enhanced as plasma renin activity (p = 0.047) and dose of olmesartan were increased (p = 0.04). Although the absolute reduction in proteinuria was not correlated with GFR (p = 0.56), the % reduction was significantly proportional with GFR (p = 0.027). Multiple regression analysis demonstrated that 64% of proteinuria reduction could be explained by baseline levels of albuminuria, GFR and renin activity. CONCLUSIONS: The reduction in proteinuria by olmesartan may be roughly predicted using baseline GFR and other parameters. These findings clarify that the effect of ARB on proteinuria reduction is restricted by reduced GFR.

Morning hypertension in chronic kidney disease is sustained type, but not surge type.

OBJECTIVE: We have shown that as renal function deteriorates, the circadian blood pressure (BP) rhythm shifts to a nondipper pattern and the duration until nocturnal BP decline [dipping time (DT)] is prolonged. We investigated whether or not morning hypertension (BP 2 h after awakening >135/85 mmHg) in chronic kidney disease (CKD) was sustained type with a prolonged DT. MATERIALS AND METHODS: Twenty-four-hour BP was monitored in 104 patients with CKD. Fifty-one of 104 participants (group A) did not exhibit morning hypertension. The patients with morning hypertension (group B, n=53) were classified into three groups: group C (n=23), participants who exhibited morning hypertension but did not meet the criteria for the surge or sustained type; group D (n=29), the sustained type (with no night-time BP readings <120/70 mmHg); and group E (n=1), the surge type (systolic BP rises >25 mmHg after awakening). RESULTS: The night/day BP ratio and DT were compared among groups A, C, and D because there was only one participant in group E. Night/day ratio of BP and DT were both significantly higher in group D compared with groups A and C. The prevalence of nondippers tended to be higher in group D compared with the other groups (A, 65%; C, 57%; D, 86%, P=0.09). Creatinine clearance was significantly lower in group D compared with groups A and C. CONCLUSION: Sustained elevation of night-time BP until the early morning and high night/day ratio of BP may contribute to the high frequency of morning hypertension, which is generally the sustained rather than the surge type in CKD.

Geographic differences in the increasing ESRD rate have disappeared in Japan.

BACKGROUND: We previously showed that there are marked geographic differences in the incidence of end-stage renal disease (ESRD) within Japan. In addition, the use of renin-angiotensin system inhibitors was found to be inversely correlated with the increasing ESRD rate. It was recently demonstrated that the incidence of ESRD due to diabetic nephropathy is declining in both Europe and USA. Therefore, we investigated the increasing ESRD rate and its geographic difference in Japan. METHODS: Each year, the Japanese Society for Dialysis Therapy reports the numbers of patients initiating maintenance dialysis therapy in each prefecture of Japan. We used old (1984-1991) and recent (2001-2008) data to compare the increasing ESRD rate, which was estimated from the slope of the regression line of the annual incidence corrected for population, between the two periods in 11 regions of Japan. RESULTS: Increasing ESRD rate almost halved, from 11.1 ± 5.6 to 5.4 ± 0.7/million per year from the old to the recent period. Deceleration of the increasing ESRD rate from the old to the recent period was correlated with the incidence in the old period across 11 regions (r = 0.81, p < 0.003); i.e., the deceleration was greater in the regions where ESRD incidence had been higher. Whereas the increasing ESRD rate was significantly different among regions in the old period, this was not the case in the recent period, resulting in uniformity throughout Japan. CONCLUSIONS: The increasing ESRD rate is slowing in Japan, and its geographic differences, previously observed, have disappeared.

Hemosuccus pancreaticus associated with segmental arterial mediolysis successfully treated by transarterial embolization.

CONTEXT: Hemosuccus pancreaticus, a rare cause of intermittent upper gastrointestinal bleeding, is usually caused by the rupture of an aneurysm associated with chronic pancreatitis. Segmental arterial mediolysis is a rare non-arteriosclerotic, non-inflammatory vascular disease characterized by vacuolization and lysis of the arterial smooth muscle cells. Segmental arterial mediolysis often causes intra-abdominal hemorrhage through aneurysm rupture. To our knowledge, hemosuccus pancreaticus associated with segmental arterial mediolysis has not previously been reported. CASE REPORT: A 59-year-old man had suffered from recurrent episodes of epigastric pain and melena. Laboratory data showed mild anemia and an elevated serum amylase level. Upper gastrointestinal endoscopy revealed bloody pancreatic juice exuding from the papilla of Vater. There were no findings suggestive of chronic pancreatitis, such as pancreatic calcification, irregularity of the pancreatic duct and atrophy of the pancreas. Contrast-enhanced computed tomography revealed a large fusiform aneurysm of the middle-distal splenic artery, and dissection of the proximal splenic artery and celiac artery. Leakage of contrast medium from the aneurysm into the main pancreatic duct was also evident. Angiography clearly revealed a fusiform aneurysm of the splenic artery. This was managed successfully by transarterial coil embolization and the patient has subsequently shown no recurrence of epigastric pain or melena. CONCLUSION: Segmental arterial mediolysis is a very rare cause of hemosuccus pancreaticus not associated with chronic pancreatitis. Transcatheter arterial embolization is useful for treatment of ruptured aneurysm associated with segmental arterial mediolysis.