High glucose (HG) increases angiotensin II (AngII) generation in mesangial cells (MC). Chymase, an alternative AngII-generating enzyme, is upregulated in the glomeruli of diabetic kidneys. In this study, we examined AngII synthesis by human MC via angiotensin-converting enzyme (ACE)-dependent and chymase-dependent pathways under normal glucose (NG, 5 mM) and HG (30 mM) conditions. NG cells expressed ACE and chymase mRNA. Under NG conditions the chymase inhibitor chymostatin reduced AngII levels in cell lysates and in the culture medium, and the ACE inhibitor captopril had no effect. HG induced a 3-fold increase in chymase mRNA and protein but not in ACE mRNA; however, HG induced a 10-fold increase in intracellular ACE activity. The increase in AngII generation induced by HG was found in the cell lysate but not in the culture medium. The rise in intracellular AngII was not prevented by captopril or by chymostatin. Moreover, captopril inhibited extracellular ACE activity but failed to block intracellular ACE activity; these results suggested that captopril was unable to reach intra-cellular ACE. Losartan did not change the intracellular AngII content in either NG or HG conditions, and this lack of change suggested that the increase in AngII was due to intracellular generation. Together these results suggest that chymase may be active in human MC and that both ACE and chymase are involved in increased AngII generation during the HG stimulus by different mechanisms, including an upregulation of chymase mRNA and a rise in intracellular ACE activity, favoring the generation and accumulation of intracellular AngII.

The effects of long-term diabetes in the presence of established nephropathy on tubular function remains poorly understood. We evaluated the levels of the main sodium and water transport proteins expressed in the kidney after long-term (8 weeks) of streptozotocin (STZ)-induced type 1 diabetes mellitus (DM) in untreated (D) and insulin (4 U/s.c./day)-treated (D+I) rats. D animals presented upregulation (∼4.5-fold) of Na/glucose cotransporter (SGLT1), whereas the α-subunit of the epithelial sodium channel (α-ENaC) and aquaporin 1 (AQP1) were downregulated (∼20 and 30% respectively) with no change in the Na/H exchanger (NHE3), Na/Cl cotransporter (TSC) and AQP2. Insulin replacement partially prevented these alterations and caused increases in the expression of α-ENaC and AQP2. These effects suggest an action of insulin in the tubular transport properties. The upregulation of SGLT1 may constitute a mechanism to prevent greater glucose losses in the urine but it may result in glucotoxicity to the proximal epithelial cells contributing to the diabetic nephropathy. The decrease of α-ENaC in D animals may compensate for the increased sodium reabsorption via SGLT1 resulting in discrete natriuresis. DM-induced polyuria was not due to changes in AQP2 expression.

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Treatment with PAG reduces the renal damage induced by CP. This effect seems to be related to the H2S formation and the restriction of the inflammation in the kidneys from PAG + CP-treated rats.

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.

BackgroundChronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection.MethodsThe present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined.ResultsHCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05).ConclusionsHCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.

Targeting individuals with altered expression of D antigen can be a good strategy for finding donors with RhCE variants. In our study 94·9% of the partial D samples revealed altered RHCE variant alleles and 5·7% of the samples with altered RHD allele predicted partial c, partial e and the lack of the high prevalence hr(B) and hr(S) antigens.

Chymase is an alternative pathway for angiotensin-converting enzyme in angiotensin II (Ang II) formation, and its expression is increased in human diabetic kidneys and in human mesangial cells (MCs) stimulated with high glucose. In addition, chymase activates transforming growth factor (TGF-β1) via an Ang II-independent pathway. The aim of this study was to evaluate the role of chymase on TGF-β1 activation in diabetic rats and in rat MCs (RMCs) stimulated with high glucose (HG). Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, intravenous). After 30 (D30) or 60 (D60) days, chymase activity and the expression of profibrotic markers were evaluated. RMCs were stimulated with HG in the presence or absence of 50 μmol/L chymostatin, a chymase inhibitor, or 100 nmol/L of losartan, an Ang II antagonist. Chymase activity and expression increased in D60 kidneys, with increased expression of fibronectin, type I and III collagen, TGF-β1 and Smad 3 and with no change in Smad 7 expression. RMCs exposed to HG presented increases in chymase activity and expression, together with upregulation in fibrosis markers and in the TGF-β1 signaling pathway. All these effects were reversed by chymostatin and by losartan, but type 1 angiotensin II receptor blockade did not interfere with the Smad 3 and 7 pathway. Similar to HG-stimulated RMCs, control RMCs treated with chymase responded with increased expression of TGF-β1, Smad 3 and fibrosis markers. These effects were reversed by chymostatin but not by losartan. The results indicate an important role for chymase in inducing fibrosis through TGF-β1 activation, parallel with Ang II effects.