Thrombotic microangiopathy in a patient with anti-signal recognition particle antibody-positive immune-mediated necrotizing myopathy.

We describe the case of a 61-year-old woman with anti-signal recognition particle (SRP) antibody-positive immune-mediated necrotizing myopathy (IMNM) who exhibited biopsy-confirmed thrombotic microangiopathy (TMA). The patient developed proximal-dominant muscle weakness and was diagnosed with anti-SRP antibody-positive IMNM based on muscle biopsy results and serological examination. A high-dose corticosteroid prescription was initiated, followed by intravenous methylprednisolone and intravenous immunoglobulin therapy (IVIg). The patient showed IVIg-induced hemolytic anemia with preserved ADAMTS13 activity. Transient oral tacrolimus administration was initiated. Approximately 8 weeks after admission, the serum creatinine levels gradually increased. Renal histological examination revealed TMA, including ischemic changes in the renal tubules, stenosis, and occlusion of the interlobular arteries with fibrinoid necrosis of the afferent arteriolar walls. The arteriolar walls demonstrated an accumulation of C1q and C3c. Myofiber damage in patients with IMNM accounts for the activation of the classical pathway of the complement cascade in the sarcolemma due to antibody deposition. Additionally, a membrane attack complex is observed on capillaries in the muscle tissues of patients with anti-SRP antibody-positive IMNM. Although drug-induced pathomechanisms, such as IVIg and tacrolimus, can trigger the development of TMA, we suggest that the presence of serum anti-SRP antibodies would be implicated in complement-associated kidney vascular damage.

Oral Iron Absorption of Ferric Citrate Hydrate and Hepcidin-25 in Hemodialysis Patients: A Prospective, Multicenter, Observational Riona-Oral Iron Absorption Trial.

Oral ferric citrate hydrate (FCH) is effective for iron deficiencies in hemodialysis patients; however, how iron balance in the body affects iron absorption in the intestinal tract remains unclear. This prospective observational study (Riona-Oral Iron Absorption Trial, R-OIAT, UMIN 000031406) was conducted at 42 hemodialysis centers in Japan, wherein 268 hemodialysis patients without inflammation were enrolled and treated with a fixed amount of FCH for 6 months. We assessed the predictive value of hepcidin-25 for iron absorption and iron shift between ferritin (FTN) and red blood cells (RBCs) following FCH therapy. Serum iron changes at 2 h (ΔFe2h) after FCH ingestion were evaluated as iron absorption. The primary outcome was the quantitative delineation of iron variables with respect to ΔFe2h, and the secondary outcome was the description of the predictors of the body's iron balance. Generalized estimating equations (GEEs) were used to identify the determinants of iron absorption during each phase of FCH treatment. ΔFe2h increased when hepcidin-25 and TSAT decreased (-0.459, -0.643 to -0.276, p = 0.000; -0.648, -1.099 to -0.197, p = 0.005, respectively) in GEEs. FTN increased when RBCs decreased (-1.392, -1.749 to -1.035, p = 0.000) and hepcidin-25 increased (0.297, 0.239 to 0.355, p = 0.000). Limiting erythropoiesis to maintain hemoglobin levels induces RBC reduction in hemodialysis patients, resulting in increased hepcidin-25 and FTN levels. Hepcidin-25 production may prompt an iron shift from RBC iron to FTN iron, inhibiting iron absorption even with continued FCH intake.

Naked plasmid DNA-based alpha-galactosidase A gene transfer partially reduces systemic accumulation of globotriaosylceramide in Fabry mice.

Fabry disease is an X-linked recessive inborn metabolic disorder in which a deficiency in lysosomal enzyme alpha-galactosidase A (Gal A) causes the systemic accumulation of globotriaosylceramide (Gb3). Although many investigators have attempted to treat alpha-Gal A knock-out mice (Fabry mice) with gene therapy, no report has demonstrated therapeutic effects by the retrograde renal vein injection of naked DNA. We recently developed a naked plasmid vector-mediated kidney-targeted gene transfer technique. A solution containing naked plasmid DNA encoding human alpha-Gal A (pKSCX-alpha-Gal A) was rapidly injected into the left kidney of Fabry mice (pKSCX-alpha-Gal A mice). pKSCX was used for mock transfections (pKSCX mice). We confirmed that vector-derived human alpha-Gal A mRNA was present in the left kidney but not in other tissues, by reverse transcriptase polymerase chain reaction. Compared with the pKSCX mice, the pKSCX-alpha-Gal A mice showed partial therapeutic effects: increased alpha-Gal A activity in the injected kidney and in the liver, heart, and plasma, and decreased Gb3 in the injected kidney, contralateral kidney, liver, heart, and spleen. Our results demonstrated that, although further studies are needed to improve the outcome, this method has promise as a potential treatment option for Fabry disease.

Effects of viral interleukin 10 introduced by in vivo electroporation on arthrogen-induced arthritis in mice.

OBJECTIVE: Viral interleukin 10 (vIL-10) has a variety of immunomodulatory properties. We examined the applicability of vIL-10 gene transfer to the treatment of mice with arthrogen-collagen-induced arthritis (CIA), which is induced by anti-type II collagen antibodies. METHODS: One day after anti-type II collagen antibodies were injected into mice, 400 microg of plasmid DNA expressing vIL-10 (pCAGGS-vIL-10) was injected into the bilateral tibialis anterior muscles followed by in vivo electroporation consisting of four 50-ms electric pulses of 100 V (pCAGGS-vIL-10 mice). pCAGGS (400 microg) was similarly injected into control mice (pCAGGS mice). RESULTS: We observed high serum vIL-10 levels in the pCAGGS-vIL-10 mice, but no vIL-10 was detected in the serum of the pCAGGS mice. Using quantitative real-time polymerase chain reaction, we observed that the ratios of IL-6, tumor necrosis factor-a, and IL-1beta transcripts to those of G6PDH in the joints were significantly lower in the pCAGGS-vIL-10 mice than in the pCAGGS mice (p < 0.05). The pCAGGS-vIL-10 mice showed significant therapeutic effects: the severity of the macroscopic arthritis was significantly suppressed from Days 5 to 21 (p < 0.0001), and the histologically observable development of arthritis was also suppressed in these mice on Day 21 (p < 0.0001). CONCLUSION: These results demonstrated that pCAGGS-vIL-10 gene transfer by in vivo electroporation suppressed arthrogen-CIA.

Pretreatment plasma intact parathyroid hormone and serum calcium levels, but not serum phosphate levels, predict the response to maxacalcitol therapy in dialysis patients with secondary hyperparathyroidism.

BACKGROUND: The treatment strategy for secondary hyperparathyroidism is generally determined empirically with regards to present parathyroid function and serum calcium (Ca) and inorganic phosphate (Pi) levels. More evidence is needed to avoid the aimless continuation of active vitamin D therapy. METHODS: Nondiabetic dialysis patients whose plasma intact parathyroid hormone (iPTH) levels were greater than 300 pg/ml were included in the study. Maxacalcitol was intravenously injected three times a week. The treatment was continued for 48 weeks, unless the iPTH level was reduced to less than 300 pg/ml or unfavorable events occurred. The patients whose plasma iPTH levels were below 300 pg/ml within 48 weeks were defined as those who had been successfully treated. RESULTS: Findings for 146 patients were analyzed, and 96 patients were successfully treated. Serum Pi levels did not significantly increase during the therapy. The pretreatment plasma iPTH levels and serum Ca levels were lower in the patients who were successfully treated with maxacalcitol. A logistic regression study and classifying by stratum analyses revealed that the pretreatment serum Ca levels and plasma iPTH levels were significantly related to the result of maxacalcitol therapy, while the serum Pi levels were not. Analyses using a receiver-operating characteristic curve revealed that the areas under curves obtained for iPTH and Ca were significantly greater than those obtained for Pi (P < 0.0001). CONCLUSIONS: Serum Ca levels and parathyroid function were correlated with the results of maxacalcitol therapy. Pretreatment serum Pi levels could not predict the result.

Sustained transgene expression in rat kidney with naked plasmid DNA and PCR-amplified DNA fragments.

Recently, we developed a kidney-targeted gene transfer technique, in which naked DNA was injected into the renal vein while the renal vein and artery were clamped. Kidney-targeted DNA transfer with only the renal vein clamped is an important modification that may permit less invasive catheter-based gene transfer in future clinical applications. The preparation of PCR-amplified DNA fragments is less time-consuming than that of naked plasmid DNA. We examined rat erythropoietin (Epo) plasmid, pCAGGS-Epo, or PCR-amplified DNA fragment, fCAGGS-Epo, transfer into the rat kidney with only the renal vein clamped. The Epo level peaked at week 3 and then was sustained for 24 weeks, which resulted in significant erythropoiesis. This modified technique, allowing long-term expression of both PCR-amplified DNA fragments and naked plasmid DNA, could potentially be used for catheter-based gene transfer in humans, and could help determine the physiological functions of putative genes.

Direct comparison between two 1-84PTH assays in dialysis patients.

BACKGROUND/AIM: Today, two kinds of 1-84PTH assays are available in clinical practice. Few studies have directly compared the results of these assays in the same plasma. METHODS: Plasma samples were collected from 235 dialysis patients and analyzed by the 1-84PTH-IRMA, intact PTH-IRMA, 1-84PTH-CLIA, and intact PTH-CLIA assays simultaneously. RESULTS: The results obtained by the 1-84PTH-IRMA and 1-84PTH-CLIA were highly correlated to each other (r(2) = 0.971, p < 0.0001). In 90.2-92.3% of patients, the assays agreed when classifying them into three categories based on the K/DOQI guidelines. However, the 1-84PTH assays agreed in only 41.3-83.4% of patients when classifying them into two categories by calculating 1-84PTH/(intact PTH - 1-84PTH). CONCLUSION: The results obtained by the two assays could be regarded as comparable in clinical practice. However, the 1-84PTH/(intact PTH-1-84PTH) ratio has to be carefully applied since it amplified the error of these assays.

Rat kidney-targeted naked plasmid DNA transfer by retrograde injection into the renal vein.

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Recently, we demonstrated that naked plasmid deoxyribonucleic acid (DNA) can be transferred into renal interstitial fibroblasts near the peritubular capillaries (PTCs) in normal rats, by retrograde injection into the renal vein with the renal vein and artery clamped. The PTC network is a main target of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. We retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats using a 24-gage catheter. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTCs of the kidney by immunoelectron microscopy. Nephrotoxicity from the gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector pCAGGS-Epo in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 s in a volume of 1.0 mL. We detected transgene-derived Epo messenger ribonucleic acid by reverse transcriptase polymerase chain reaction only in the kidneys receiving pCAGGS-Epo. In this article, protocols for naked plasmid DNA transfer into rat kidney using this hydrodynamics-based transfection method and the immunoelectron microscopic technique to determine the lacZ gene transfer site are described in detail.

Rat liver-targeted naked plasmid DNA transfer by tail vein injection.

High levels of foreign gene expression in mouse hepatocytes can be achieved by "hydrodynamics-based transfection," the rapid injection of a large volume of a naked deoxyribonucleic acid (DNA) solution into the tail vein. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice and, thus, are more suitable for some biomedical research. Recently, we demonstrated that hydrodynamics-based transfection can also be used to deliver naked plasmid DNA into the normal rat, which is more than 10 times larger than the mouse. We performed the tail vein injection using a syringe with a winged needle equipped with an external tube. Injection of a lac Z expression plasmid, pCAGGS-lac Z by this technique resulted in the exclusive detection of beta-galactosidase in the liver. We also injected a rat erythropoietin (Epo) expression plasmid, pCAGGS-Epo (800 microg). Maximal Epo gene expression was achieved when a 25-mL injection volume (approx 100 mL/kg body wt) was transferred within 15 s.

Kidney-targeted naked DNA transfer by retrograde renal vein injection in rats.

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Previous gene transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have resulted in low-level expression for <1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity attributable to gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 microg. We detected the transgene-derived Epo mRNA by reverse transcription polymerase chain reaction only in the kidneys injected with pCAGGS-Epo. After an injection of 100 microg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 +/- 71.8 mU/ml at week 5, and gradually decreased to 116.2 +/- 38.7 mU/ml at week 24. A similar pattern was obtained using smaller doses of plasmid, 2 microg or 30 microg of pCAGGS-Epo. Transgene-derived Epo secretion resulted in significant erythropoiesis. This novel technique is simple and safe, allowing high-level and long-term stable gene expression specific to the fibroblasts near the PTC, and should have therapeutic value for future applications in humans.