The p70S6K/PI3K/MAPK feedback loop releases the inhibition effect of high-dose rapamycin on rat mesangial cell proliferation.

Glomerular mesangial cell (MC) proliferation is one of the causative factors of glomerular diseases and one of their prominent pathological features. Rapamycin can inhibit MC proliferation and slow the progression to chronic renal fibrosis. The present study was designed to observe the role of rapamycin in MC proliferation and to explore the mechanism by which rapamycin acts on Akt and MAPK/ERK1/2 pathways in mesangial cells. MTT assay and flow cytometry were used to evaluate the proliferation and the cell cycle phase of glomerular mesangial cells respectively. The mRNA expression level of p70S6K was detected by RT-qPCR. Western blotting was performed to determine p70S6K, PI3K/Akt, and PI3K/MAPK protein expression. We found that rapamycin could reduce mesangial cell proliferation and arrest the cell cycle in the G1 phase, however the inhibition effect of 1000 nmol/L rapamycin was not higher than that in the 100 nmol/L group. The results of western blotting showed that 1000 nmol/L rapamycin more significantly inhibited the phosphorylation of p70S6K than 100 nmol/L, suggesting there should be another signaling pathway that activates the proliferation of MCs. Moreover, our results revealed that 1000 nmol/L rapamycin led to Raf1-MEK1/2-ERK pathway activation through a p70S6K-PI3K-mediated feedback loop in MCs. This study demonstrated that high-dose rapamycin leads to ERK1/2 activation through a p70S6K/PI3K/MAPK feedback loop in rat MCs, thus reducing the inhibitory effect of rapamycin on MC proliferation.

Identification of Two Novel Truncated Transcripts of Janus Kinase 2 Gene.

Jak2 is a nonreceptor tyrosine kinase that plays a critical role in signal transduction through an abundance of receptors, such as erythropoietin receptor. In this paper, we report two previously unknown transcripts of Jak2 gene. One transcript deletes the 77nt of 3' end exon 10 of the Jak2 gene, resulting in a frameshift that introduces a stop codon in the downstream exon and produces a truncated protein of 421 amino acids if translated. The other transcript skips the entire exon 10, leading to a premature stop codon in the adjacent exon 11, producing a truncated protein of 414 amino acids if translated. Therefore, the physiological significance of the expression of two novel transcripts in healthy volunteers and patients with myeloproliferative neoplasms, acute leukemia, and chronic myeloid leukemia needs to be investigated further.

Intermedin ameliorates IgA nephropathy by inhibition of oxidative stress and inflammation.

IgA nephropathy (IgAN) is the most frequent form of glomerulonephritis worldwide. The role of oxidative stress and inflammation in the pathogenesis of IgAN has been reported. Intermedin (IMD) is a newly discovered peptide that is closely related to adrenomedullin. We have recently reported that IMD can significantly reduce renal ischemia/reperfusion injury by diminishing oxidative stress and suppressing inflammation. The present study was designed to explore whether IMD ameliorates IgAN via oxidative stress- and inflammation-dependent mechanisms. Our results showed that IMD administration resulted in the prevention of albuminuria and ameliorated renal pathomorphological changes. These findings were associated with (1) decreased renal TGF-ß1 and collagen IV expression, (2) an increased SOD level and reduced MDA level, (3) the inhibition of the renal activation of NF-κB p65 and (4) the downregulation of the expression of inflammatory factors (TNF-α, MCP-1 and MMP-9) in the kidney. These results indicate that IMD in the kidney protects against IgAN by reducing oxidative stress and suppressing inflammation.

Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms.

Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F.

[Identification of a novel aberrant spliceosome of MPL gene (MPLL391-V392ins12)in patients with myeloproliferative neoplasms].

OBJECTIVE: To identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN). METHODS: MPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR). RESULTS: A novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people. CONCLUSION: MPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

The Akt/mTOR/p70S6K pathway is activated in IgA nephropathy and rapamycin may represent a viable treatment option.

IgA nephropathy (IgAN) is one of the most frequent forms of glomerulonephritis, and 20 to 40% of patients progress to end-stage renal disease (ESRD) within 20 years of disease onset. However, little is known about the molecular pathways involved in the altered physiology of mesangial cells during IgAN progression. This study was designed to explore the role of mTOR signaling and the potential of targeted rapamycin therapy in a rat model of IgAN. After establishing an IgA nephropathy model, the rats were randomly divided into four groups: control, control+rapamycin, IgAN and IgA+rapamycin. Western blotting and immunohistochemistry were performed to determine phospho-Akt, p70S6K and S6 protein levels. Coomassie Brilliant Blue was utilized to measure 24-h urinary protein levels. The biochemical parameters of the rats were analyzed with an autoanalyzer. To evaluate IgA deposition in the glomeruli, FITC-conjugated goat anti-rat IgA antibody was used for direct immunofluorescence. Cellular proliferation and the mesangial matrix in glomeruli were assayed via histological and morphometric procedures. Our results showed that p70S6K, S6 and Akt phosphorylation were significantly upregulated in IgAN rats, and rapamycin effectively inhibited p70S6K and S6 phosphorylation. A low dose of the mTOR inhibitor rapamycin reduced proteinuria, inhibited IgA deposition, and protected kidney function in an IgAN rat model. Low-dose rapamycin treatment corresponded to significantly lower cellular proliferation rates and a decreased mesangial matrix in the glomeruli. In conclusion, the Akt/mTOR/p70S6K pathway was activated in IgAN, and our findings suggested that rapamycin may represent a viable option for the treatment of IgAN.

MDM4 overexpressed in acute myeloid leukemia patients with complex karyotype and wild-type TP53.

Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10-15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.

[Effects of adiponectin on endoplasmic reticulum stress-mediated apoptosis and cell cytoskeleton and its mechanism of podocytes cultured in high glucose].

OBJECTIVE: To explore the effects of adiponectin (APN) on high glucose-induced endoplasmic reticulum stress (ERS)-mediated apoptosis and cytoskeleton. METHODS: The conditionally immortal human glomerular podocytes were divided into normal glucose, mannitol, high glucose and high glucose with adiponectin groups. Flow cytometry was employed to assess cell apoptosis.Real-time polymerase chain reaction (PCR) and Western blot were used to detect the expressions of such ERS molecules as GRP78, CHOP and caspase 12 and desmin and TRPC6. Immunofluorescent staining was used to detect the changes in the skeleton of podocyte. RESULTS: (1) The apoptosis rate in high glucose group is significantly higher than the other groups (26.15% ± 1.38% vs 2.39% ± 0.58%, 4.84% ± 0.87%, 16.71% ± 1.15%, all P < 0.05). Compared with control group, the mRNA and protein expressions of GRP78, CHOP and caspase 12 were all up-regulated significantly in high glucose group (P < 0.05). The high glucose with adiponectin group could reduce the podocyte apoptosis by 10% and down-regulated the mRNA and protein expressions of GRP78, CHOP and caspase 12 versus high glucose group (all P < 0.05). (2) High glucose-induced podocyte caused the up-regulated expressions of TRPC6 and desmin (all P < 0.05) and it was inhibited by adiponectin (all P < 0.05). Additionally, immunofluorescent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and absent tensile fiber. Adiponectin prevented the F-actin cytoskeleton disruption under high glucose. CONCLUSION: Adiponectin plays a protective role in high glucose-induced podocyte through reducing endoplasmic reticulum stress-induced apoptosis and blunting the injury of cytoskeleton.

The short isoform of the long-type PML-RARA fusion gene in acute promyelocytic leukaemia lacks sensitivity to all-trans-retinoic acid.

Alternative splicing is associated with human disease. In acute promyelocytic leukaemia (APL) patients with the long (L)-type promyelocytic leukaemia-retinoic acid receptor α fusion gene (PML-RARA), three alternative splicing isoforms can be detected: E5(+)E6(+), E5(-)E6(+), and E5(-)E6(-). This study is the first to demonstrate that alternative splicing of L-type PML-RARA is associated with time to achieve complete remission (CR) in APL. Higher expression of the E5(-)E6(-) isoform, the short isoform, was related to longer time to achieve CR. Each isoform was constructed into recombinant lentiviral vector and transfected into U937 cells. Compared with the E5(-)E6(+) and E5(+)E6(+) groups, the U937 cells with E5(-)E6(-) showed lower sensitivity to all-trans-retinoic acid treatment.

A tetraploid minimally differentiated acute myeloblastic leukemia with extensive erythrophagocytosis: a case report and literature review.

Tetraploidy is a rare chromosome number aberration in de novo acute myeloid leukemia (AML), and may be associated with erythrophagocytosis by leukemic blast cells. We report a 48-year-old female patient with minimally differentiated acute myeloblastic leukemia (AML-M0) exhibiting tetraploidy and erythrophagocytosis. The karyotype was 46,XX[2]/92,XXXX[18]. Bone marrow aspirate smears showed large and prominent nuclei, with erythrophagocytosis in leukemic cells. Fluorescence in situ hybridization using RUNX1 dual color break probes detected four fusion signals, accounting for 95 % (190/200), in one interphase nucleus. The mutations of TP53 and the fusion genes RUNX1/ETO, CBFß/MYH11, and PML/RARα were all negative. This patient showed a poor response to chemotherapy, and died 66 days after the onset. To our knowledge, this is the first reported case of AML-M0 with tetraploidy and erythrophagocytosis and without additional chromosome aberrations. This case of tetraploid AML with poor prognosis suggests that further biological study of more cases of tetraploid AML will be of great importance in improving the understanding and prognosis of this tetraploid AML.