Diagnosis of Developmental Dysplasia of the Hip by Ultrasound Imaging Using Deep Learning.

BACKGROUND: A timely diagnosis of developmental dysplasia of the hip (DDH) is important for satisfactory clinical outcomes. Ultrasonography is a useful tool for DDH screening; however, it is technically demanding. We hypothesized that deep learning could assist in the diagnosis of DDH. In this study, several deep-learning models were assessed to diagnose DDH on ultrasonograms. This study aimed to evaluate the accuracy of diagnoses made by artificial intelligence (AI) using deep learning on ultrasound images of DDH. METHODS: Infants who were up to 6 months old with suspected DDH were included. DDH diagnosis using ultrasonography was performed according to the Graf classification. Data on 60 infants (64 hips) with DDH and 131 healthy infants (262 hips) obtained from 2016 to 2021 were retrospectively reviewed. For deep learning, a MATLAB deep learning toolbox (MathWorks, Natick, MA, US) was used, and 80% of the images were used as training data, with the rest as validation data. Training images were augmented to increase data variation. In addition, 214 ultrasound images were used as test data to evaluate the AI's accuracy. Pre-trained models (SqueezeNet, MobileNet_v2, and EfficientNet) were used for transfer learning. Model accuracy was evaluated using a confusion matrix. The region of interest of each model was visualized using gradient-weighted class activation mapping (Grad-CAM), occlusion sensitivity, and image LIME. RESULTS: The best scores for accuracy, precision, recall, and F-measure were all 1.0 in each model. In DDH hips, the region of interest for deep learning models was the area lateral to the femoral head, including the labrum and joint capsule. However, for normal hips, the models highlighted the medial and proximal areas where the lower margin of the os ilium and the normal femoral head exist. CONCLUSIONS: Ultrasound imaging with deep learning can assess DDH with high accuracy. This system could be refined for a convenient and accurate diagnosis of DDH. LEVEL OF EVIDENCE: Level-Ⅳ.

Multicenter study on atypical femoral fractures in patients with bone metastases taking bone- modifying agents.

Bone-modifying agents (BMAs), with bone-resorptive inhibitory effects, such as zoledronic acid and denosumab, are widely used at higher doses for bone-related events caused by bone metastasis of malignant tumors. These drugs have been suggested to be associated with atypical femoral fractures (AFFs), and the relationship between BMAs and AFFs has attracted attention. To investigate the clinical features including bone union time of AFFs in patients administered BMA for bone metastasis, we conducted a retrospective multicenter study. Thirty AFFs from 19 patients were enrolled in this study. Thirteen patients had bilateral AFFs, and nineteen AFFs had prodromal symptoms. Eighteen AFFs underwent surgery after complete fracture, three failed to achieve bone union and required nonunion surgery, and 11 AFFs that achieved bone union had an average period until bone union of 16.2 months, which was much longer than that previously reported for ordinary AFFs. Seven patients discontinued the BMAs, but not due to AFFs. Stopping BMAs in patients with bone metastasis would make it difficult to secure their performance of activities of daily living, and AFF with BMA administration might require a longer time for union. Therefore, it would be important to prevent incomplete AFF from becoming complete AFF via prophylactic internal fixation.

Clinical Outcome of Patients with Pelvic and Retroperitoneal Bone and Soft Tissue Sarcoma: A Retrospective Multicenter Study in Japan.

This study aimed to retrospectively analyze the clinical outcomes of patients with pelvic and retroperitoneal bone and soft tissue sarcoma (BSTS). Overall, 187 patients with BSTS in the pelvis and retroperitoneal region treated at 19 specialized sarcoma centers in Japan were included. The prognostic factors related to overall survival (OS), local control (LC), and progression-free survival (PFS) were evaluated. The 3-year OS and LC rates in the 187 patients were 71.7% and 79.1%, respectively. The 3-year PFS in 166 patients without any distant metastases at the time of primary tumor diagnosis was 48.6%. Osteosarcoma showed significantly worse OS and PFS than other sarcomas of the pelvis and retroperitoneum. In the univariate analyses, larger primary tumor size, soft tissue tumor, distant metastasis at the time of primary tumor diagnosis, P2 location, chemotherapy, and osteosarcoma were poor prognostic factors correlated with OS. Larger primary tumor size, higher age, soft tissue tumor, chemotherapy, and osteosarcoma were poor prognostic factors correlated with PFS in patients without any metastasis at the initial presentation. Larger primary tumor size was the only poor prognostic factor correlation with LC. This study has clarified the epidemiology and prognosis of patients with pelvic and retroperitoneal BSTS in Japan.

Proximal-type epithelioid sarcoma in pubic region expressing L-type amino acid transporter 1: A case report.

Proximal-type epithelioid sarcoma is an aggressive malignant soft-tissue neoplasm, a "proximal" variant of epithelioid sarcoma, resistant to multimodal therapy and involved in early tumor-related death. Pertinent treatments are, therefore, continually being explored. A 24-year-old woman with nonmetastatic proximal-type epithelioid sarcoma, originating subcutaneously on the right side of the vulva, underwent surgical resection; the lesion recurred, however, leading to death 3 months after the second surgery. Here described is a case of proximal-type epithelioid sarcoma expressing L-type amino acid transporter 1 (LAT1) that transports essential amino acids and p-borono-L-phenylalanine (BPA)-the chemical compound used in boron neutron capture therapy (BNCT)-and is highly expressed in many malignant tumors. Recently, LAT1 has drawn attention, and relevant treatments have been studied-LAT1 inhibitor and BNCT. LAT1 expression in proximal-type epithelioid sarcoma may lead to cogent treatments for the disease.

BNCT for primary synovial sarcoma.

Synovial sarcoma is a rare tumor requiring new treatment methods. A 46-year-old woman with primary monophasic synovial sarcoma in the left thigh involving the sciatic nerve, declining surgery because of potential dysfunction of the affected limbs, received two courses of BNCT. The tumor thus reduced was completely resected with no subsequent recurrence. The patient is now able to walk unassisted, and no local recurrence has been observed, demonstrating the applicability of BNCT as adjuvant therapy for synovial sarcoma. Further study and analysis with more experience accumulation are needed to confirm the real impact of BNCT efficacy for its application to synovial sarcoma.

Clinical Outcome of the Patients With Brain Metastasis from Soft Tissue Sarcomas.

BACKGROUND/AIM: This study aimed to evaluate the association of clinical characteristics with treatment outcomes to ascertain the appropriate treatment options for soft tissue sarcomas (STS) patients with brain metastasis (BM). PATIENTS AND METHODS: Medical records of STS patients with BM who were treated in our institutions were retrospectively reviewed, and analyzed to identify the factors associated with post-BM survival. RESULTS: Among the 509 STS patients, BM occurred in five patients (0.98%). The median survival after BM was 1.5 months. Histological subtypes of the primary lesions in the five BM patients were: two synovial sarcomas, one myxoid liposarcoma, one alveolar soft part sarcoma, and one rhabdomyosarcoma. Among the five BM patients, the post-BM survival of two patients, who underwent surgery and postoperative radiotherapy, was longer than that of the other patients (p<0.01). CONCLUSION: Combined surgery and postoperative radiotherapy effectively managed symptoms and prolonged survival in STS patients with BM.

Chondrolipoma of the finger in a child: A case study.

Chondrolipoma is, based on the limited case reports available, an extremely rare histological variant of lipoma with the proliferation of mature adipocytes containing an area of true hyaline cartilage. Chondrolipoma is characterized by adult onset and is often identified in the breast, pharynx and tongue. The current study presents a case of chondrolipoma of the finger in an 11 year-old girl. Physical examination indicated a well-defined elastic soft mass, measuring 2.5x2 cm, on the dorsal aspect of the proximal phalanx of the left middle finger. Magnetic resonance imaging (MRI) revealed a well-circumscribed lesion with heterogeneous signal intensity. On T1- and T2-weighted images, the lesion indicated a predominantly marked hyperintense signal containing linear hypointense regions, and on fat-suppressed short-tau inversion recovery sequences, the lesion indicated a predominant hypointensity, with linear regions displaying hyperintensity. Marginal excision of the tumor was performed. Histologically, the major component of the tumor was mature adipose tissue containing a limited area of mature hyaline cartilage matrix, without lipoblasts or malignancy. The postoperative course of the patient was excellent, with no local recurrence three years after surgery. To the best of our knowledge, the current study outlines the first pediatric case of chondrolipoma arising in the finger.

Boron neutron capture therapy for clear cell sarcoma.

Clear cell sarcoma of tendons and aponeuroses (CCS) is a rare, malignant tumor arising in lower extremities with no effective treatment other than wide surgical resection. Here described is a case of primary CCS in the peroneal tendon of the right foot of a 54-year-old woman enrolled to undergo BNCT. The tumor mass post-BNCT disappeared totally without damage to other normal tissue, demonstrating, for the first time, the potential efficacy of BNCT in complete local control of CCS.

Prognostic influence of the treatment approach for pulmonary metastasis in patients with soft tissue sarcoma.

Soft tissue sarcomas (STSs) are rare heterogeneous malignancies of mesenchymal origin. Pulmonary metastases develop in approximately 50% of the patients with high-grade STS, being the major cause of mortality in patients with metastatic STS. Pulmonary metastasectomy has been reported to contribute to long-term survival; however, an appropriate treatment has not been established. We aimed to identify factors associated with post-metastasis survival in STS patients with pulmonary metastasis and determine the appropriate treatment for each patient. We retrospectively reviewed the records of metastatic STS patients treated between 2000 and 2017 and analyzed the clinico-pathologic variables to identify factors associated with the survival. The median survival after pulmonary metastasis was 20.6 months, and the 1-, 3-, and 5-year survival rates were 68.6%, 36.0%, and 25.1%, respectively. The survival was significantly greater in patients who underwent pulmonary metastasectomy than in those without surgery (38.9 months vs. 10.5 months; p < 0.0001). Among those who did not undergo surgery, the survival was significantly greater in patients who received chemotherapy than in those without chemotherapy (19.1 months vs. 6.3 months, p = 0.037). Multivariate analysis identified pulmonary metastasectomy as the most important prognostic factor for post-metastasis survival (Hazard ratio 5.623; 95% Confidence Interval 2.733-11.572; p < 0.0001). In conclusion, pulmonary metastasectomy was the most important prognostic factor for post-metastasis survival in patients with metastatic STS. In addition, chemotherapy could prolong survival in patients who were not eligible for pulmonary resection. Although we should carefully weigh the risks and benefits, appropriate treatment for pulmonary metastases could contribute to long-time survival.

Predicting the survival of patients with bone metastases treated with radiation therapy: a validation study of the Katagiri scoring system.

BACKGROUND: The selection of radiation therapy dose fractionation schedules for bone metastases is often based on the estimation of life expectancy. Therefore, accurate prognosis prediction is an important issue. It is reported that the Katagiri scoring system can be used to predict the survival of patients with bone metastases. We aimed to assess prognostic factors and validate the Katagiri scoring system in patients who were treated with radiation therapy for bone metastases. MATERIALS/METHODS: We retrospectively reviewed data of all patients who were treated with radiation therapy for bone metastases between 2004 and 2013. Age, sex, Karnofsky performance status (KPS), Eastern Cooperative Oncology Group performance status (ECOG PS), primary site (lesions and characteristics), visceral metastases, laboratory data, previous chemotherapy, and multiple bone metastases were analyzed for associations with overall survival (OS). Katagiri scores were calculated for each patient and were used to compare OS. RESULTS: Out of the 616 patients included in this analysis, 574 had died and 42 remained alive. The median follow-up time for survivors was 42 months. Univariate analysis revealed that age (P = 0.604) and multiple bone metastases (P = 0.691) were not significantly associated with OS. Multivariate analysis revealed that sex, ECOG PS, KPS, primary characteristics, visceral metastases, laboratory data, and previous chemotherapy were significantly associated with OS. The survival rates at 3, 6, 12, and 24 months, categorized by Katagiri score, were as follows: score 0-3, 94.4, 77.8, and 61.1%, respectively; score 4-6, 67.7, 48.7, and 31.2%, respectively; and score 7-10, 39.1, 22.1, and 9.0%, respectively (P < 0.001). CONCLUSION: Sex, ECOG PS, KPS, primary characteristics, visceral metastases, laboratory data, and previous chemotherapy were significant predictors of survival in patients with bone metastases. The Katagiri scoring system was significantly correlated with OS and can help us select the optimal dose-fractionation.

Huge chronic expanding hematoma of the iliac bone following multiple hip surgeries: a case report.

BACKGROUND: Chronic expanding hematoma is a rare entity resulting from trauma or surgery. This condition usually occurs in soft tissue, such as the trunk or extremities, while chronic expanding hematoma arising from bone has not been reported previously. We describe an unusual case of a huge intraosseous chronic expanding hematoma arising from the ilium, which had grown over a 40-year period following hip surgeries. CASE PRESENTATION: A 57-year-old Japanese woman presented with a 1.5-year history of right hip pain. She had a history of bilateral developmental dysplasia of the hip and had undergone bilateral arthroplasties in childhood. A physical examination revealed a large, firm, immobile mass at her right ilium. Based on radiographic findings, a type of slow-growing bone tumor was suspected, and an incisional biopsy was performed. A histopathologic examination revealed large amounts of old clotted blood within the lesion, and the capsule of the lesion was composed of dense, fibrous, connective tissue. There was no evidence of neoplasia, and chronic expanding hematoma was suspected. The lesion was resistant to conservative treatment, and so we performed an internal hemipelvectomy (including the capsule of the mass) and a reconstruction by hip transposition 2.5 years after the incisional biopsy. There was no recurrence of chronic expanding hematoma at the most recent follow-up of 1 year and 8 months postoperatively. CONCLUSIONS: A chronic expanding hematoma is characterized by its persistence and increasing size more than 1 month after the trauma or surgical event suspected of causing hemorrhage. To the best of our knowledge, this is the first report of chronic expanding hematoma arising from bone. We performed internal hemipelvectomy and hip transposition, and there has so far been no recurrence. This disease may be considered a differential diagnosis for bone tumor when the patient has a history of surgery or trauma, regardless of how many years have passed since the index event.

Transcutaneous carbon dioxide application suppresses bone destruction caused by breast cancer metastasis.

Hypoxia plays a significant role in cancer progression, including metastatic bone tumors. We previously reported that transcutaneous carbon dioxide (CO2) application could decrease tumor progression through the improvement of intratumor hypoxia. Therefore, we hypothesized that decreased hypoxia using transcutaneous CO2 could suppress progressive bone destruction in cancer metastasis. In the present study, we examined the effects of transcutaneous CO2 application on metastatic bone destruction using an animal model. The human breast cancer cell line MDA-MB-231 was cultured in vitro under three different oxygen conditions, and the effect of altered oxygen conditions on the expression of osteoclast-differentiation and osteolytic factors was assessed. An in vivo bone metastatic model of human breast cancer was created by intramedullary implantation of MDA-MB-231 cells into the tibia of nude mice, and treatment with 100% CO2 or a control was performed twice weekly for two weeks. Bone volume of the treated tibia was evaluated by micro-computed tomography (µCT), and following treatment, histological evaluation was performed by hematoxylin and eosin staining and immunohistochemical staining for hypoxia-inducible factor (HIF)-1α, osteoclast-differentiation and osteolytic factors, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclast activity. In vitro experiments revealed that the mRNA expression of RANKL, PTHrP and IL-8 was significantly increased under hypoxic conditions and was subsequently reduced by reoxygenation. In vivo results by µCT revealed that bone destruction was suppressed by transcutaneous CO2, and that the expression of osteoclast-differentiation and osteolytic factors, as well as HIF-1α, was decreased in CO2-treated tumor tissues. In addition, multinucleated TRAP-positive osteoclasts were significantly decreased in CO2-treated tumor tissues. Hypoxic conditions promoted bone destruction in breast cancer metastasis, and reversal of hypoxia by transcutaneous CO2 application significantly inhibited metastatic bone destruction along with decreased osteoclast activity. The findings in this study strongly indicated that transcutaneous CO2 application could be a novel therapeutic strategy for treating metastatic bone destruction.

Synergistic Effects of a Smac Mimetic with Doxorubicin Against Human Osteosarcoma.

BACKGROUND/AIM: Second mitochondria-derived activator of caspase (Smac) is a proapoptogenic mitochondrial protein that antagonizes inhibitors of apoptosis proteins (IAPs), resulting in induction of apoptosis. In the present study we investigated the effects of a Smac mimetic in combination with doxorubicin against osteosarcoma. MATERIALS AND METHODS: In vitro effects of the combination of a Smac mimetic AT-406 and doxorubicin on cell proliferation and apoptosis in osteosarcoma cell lines were examined using cell proliferation assays, flow cytometry, and immunoblot analyses. For in vivo experiments, human osteosarcoma xenografts were treated with combination of the two substances, and tumor volume and apoptotic activity in treated tumors were assessed. RESULTS: In vitro studies revealed that combination of the two substances significantly inhibited osteosarcoma proliferation with decreased cIAP1 expression and induced apoptosis in osteosarcoma cells. Combination of the two substances significantly suppressed osteosarcoma growth in vivo. Moreover, decreased cIAP1 expression and increased apoptotic activity were observed in tumors treated by their combination of the substances. CONCLUSION: The Smac mimetic AT-406 showed an apoptotic effect and a synergistic antitumor effect with doxorubicin on osteosarcoma. The combination of AT-406 and doxorubicin may serve as a novel therapeutic strategy for osteosarcoma treatment.

AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway.

The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator­activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

Antitumor effect of YM155, a novel small-molecule survivin suppressant, via mitochondrial apoptosis in human MFH/UPS.

Survivin is a member of the inhibitor of apoptosis family, which is known to inhibit mitochondrial apoptosis. Survivin is highly expressed in cancers and plays an important role in cancer cell survival, and increased survivin expression is an unfavorable prognostic marker in cancer patients. YM155, a novel small-molecule survivin suppressant, selectively suppresses survivin expression, resulting in the induction of apoptosis in various malignancies. However, the roles of survivin in human malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (MFH/UPS) have not been studied. In the present study, we examined survivin expression in human musculoskeletal tumor tissues, and the effect of survivin inhibition by siRNA or YM155 on apoptotic activity in human MFH/UPS cell lines. In tumor tissues, mRNA expression of survivin was significantly higher in MFH/UPS samples than in benign schwannomas. Moreover, in vitro studies revealed that both survivin siRNA and YM155 suppressed survivin expression and inhibited MFH/UPS cell proliferation in a dose- and a time-dependent manner. Further, the numbers of apoptotic cells significantly increased with YM155 treatment. in vivo, tumor volume in YM155-treated groups was significantly reduced without significant bodyweight loss. Increased apoptotic activity along with decreased survivin expression was also observed in YM155-treated tumors. The findings in this study strongly suggest that survivin suppressants, including YM155, contribute to the suppression of human MFH/UPS cell growth via promoting mitochondrial apoptosis, and that survivin may be a potent therapeutic target for the novel treatment of human MFH/UPS.

Transcutaneous application of CO2 enhances the antitumor effect of radiation therapy in human malignant fibrous histiocytoma.

Sarcomas are relatively resistant because of hypoxia. We previously demonstrated that the transcutaneous CO(2) therapy reduced hypoxic conditions in human malignant fibrous histiocytoma (MFH). Therefore, we hypothesized that transcutaneous CO(2) therapy could enhance the antitumor effect of radiation therapy in human MFH. Our purpose was to evaluate the effects of transcutaneous CO(2) therapy on the antitumor efficacy of X-ray irradiation using MFH. First, in an in vitro study, we assessed apoptotic activity and reactive oxygen species (ROS) production using flow cytometric and immunoblot analysis at 24 h after X-ray irradiation under three different oxygen conditions (normoxic, reoxygenated and hypoxic). In addition, in the in vivo study, 24 male athymic BALB/c nude mice with MFH tumors that were inoculated in the dorsal subcutaneous area were randomized into four groups: control, CO(2), X-ray irradiation and combination (CO(2) and X-ray irradiation). Treatments were performed twice weekly for 2 weeks, four times in total. Tumor volume was calculated. All tumors were excised and apoptotic activity, ROS production, related proteins and HIF-1α expression were assessed using flow cytometric and immunoblot analysis. The in vitro study revealed that X-ray irradiation induced increased apoptosis and ROS production in MFH cells under normoxic and reoxygenated conditions relative to hypoxic conditions (P<0.01). In the in vivo study, tumor volume in the combination group was reduced to 28, 42 and 47% of that in the control, CO(2), and X-ray groups, respectively (P<0.05). Apoptotic activity and ROS production in the combination group were strongly increased with decreasing HIF-1α expression relative to the control, CO(2) and X-ray groups. The transcutaneous CO(2) system enhanced the antitumor action of X-ray irradiation and could be a novel therapeutic tool for overcoming radio-resistance in human malignancies.

Intraoperative platelet-rich plasma does not improve outcomes of total knee arthroplasty.

This randomized controlled study was conducted to assess the effects of platelet-rich plasma (PRP) on outcomes of total knee arthroplasty (TKA). Forty patients who underwent unilateral TKA were evaluated prospectively; 20 received intraoperative PRP and 20 served as control subjects. The results showed no significant differences in reduction of bleeding, range of motion, swelling around the knee joint, muscle power recovery, pain, Knee Society Scores, and Knee Injury and Osteoarthritis Outcome Score between the 2 groups. Additionally, no distinct clinical characteristics were found in patients who received intraoperative PRP. Therefore, we conclude that intraoperative PRP does not improve outcomes of TKA.

Recombinant human erythropoietin ameliorated endothelial dysfunction and macrophage infiltration by increasing nitric oxide in hypertensive 5/6 nephrectomized rat aorta.

Recombinant human erythropoietin (rHuEPO), used clinically for renal anemia, reportedly exhibits pleiotropic properties in various tissues. To test whether it ameliorates vascular injury, rHuEPO (75U/kg) was administered subcutaneously every 3days for 10days to 5/6 nephrectomized hypertensive rats (5/6Nx) treated with 1% NaCl. rHuEPO had no effect on increased systolic blood pressure or decreased hematocrit values, but normalized levels of proteinuria and creatinine clearance. Vasodilation in response to acetylcholine in the aortic ring was impaired in the 5/6Nx, and improved by treatment with rHuEPO. Immunohistochemical analysis revealed that the infiltration of adventitial areas by macrophages and expression of osteopontin were enhanced in the 5/6Nx aorta and the overexpression was suppressed by rHuEPO. rHuEPO also attenuated medial hyperplasia. Akt signaling was activated by the increased expression of phosphorylated Akt and GSK-3ß in aorta from rHuEPO-treated 5/6Nx. rHuEPO restored plasma NOx (NO(2)(-)+NO(3)(-)) levels and endothelial nitric oxide synthase (eNOS) content in the 5/6Nx aorta. Treatment with an eNOS substrate, l-arginine, which caused a similar increase in plasma NOx levels as the rHuEPO treatment, resulted in a normalization of endothelial dysfunction and vascular inflammation. These results suggest that a low dose of rHuEPO exerted vasoprotective effects in rats with hypertensive renal failure.

Chronic treatment with recombinant human erythropoietin exerts renoprotective effects beyond hematopoiesis in streptozotocin-induced diabetic rat.

Recombinant human erythropoietin (rHuEPO), which has been used clinically for the management of renal anemia, is reported to exert pleiotropic beneficial properties against acute ischemic/reperfusion injury in various tissues. To investigate the hypothesis that chronic treatment with rHuEPO might ameliorate diabetic nephropathy beyond hematopoiesis, rHuEPO (150 U/kg, subcutaneously) was administered three times per week to the streptozotocin-induced diabetic rats for 4 weeks. Streptozotocin (65 mg/kg, intravenously) significantly increased urinary protein excretion and collagen deposition in glomerular and tubulointerstitial areas in the kidney, which were attenuated by rHuEPO. rHuEPO normalized the levels of creatinine clearance, serum creatinine, and blood urea nitrogen of diabetic rats. RT-PCR analysis revealed that the expressions of mRNA for transforming growth factor-beta, osteopontin and adhesion molecules were enhanced in the diabetic rat kidney and that the overexpression of these molecules was suppressed by rHuEPO. rHuEPO exerted antioxidant properties by inhibiting renal activation and overexpression of NADPH oxidase. We found the activation of the Akt signaling pathway by the increased expression of phosphorylated Akt and GSK-3beta and a reduction of TUNEL-positive apoptotic cell death in renal tissue from rHuEPO-treated diabetic group. We also demonstrated that rHuEPO restored the endothelial nitric oxide synthase (eNOS) content in the diabetic rat kidney. On the other hand, treatment with rHuEPO did not affect blood glucose level, blood pressure, or hematocrit in diabetic rats. These results suggest that chronic treatment with rHuEPO attenuated renal injury beyond hematopoiesis and regulated apoptosis and eNOS expression, which might be due to the activation of Akt pathway.