Search

The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10+/+ wild-type (WT) and Kcnj10-/- knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression. [ABSTRACT FROM AUTHOR]

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-CI cotransporter (NCC) in the DCT. Immu-nostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj1(t+/+ mice (WT). This 40-pS K channel is absent in homozygous Kcnj10-/- (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism. [ABSTRACT FROM AUTHOR]

AbstractMany approaches targeting MUC1 for breast tumor immunotherapy have been attempted. However, preclinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in human. B7 molecules that bind CD28 provide an antigen-nonspecific signal, which, along with an antigen-specific signal, is crucial for T-cell activation. In the present study, we constructed a novel Bacillus Calmette-Guérin-based breast cancer vaccine that coexpressed four VNTRs (variable-number tandem repeats) of MUC1 and CD80 (rBCG-MVNTR4-CD80). The aim of our study was to enhance anti-MUC1 tumor immunity by vaccination of hu-PBL-SCID mice with the recombinant BCG vaccine. The inhibition effect on tumors from the mice immunized with rBCG-MVNTR4-CD80 significantly increased, compared with rBCG-MVNTR4, BCG-pDE22, and phosphate-buffered saline immunized mice (p< 0.05, p< 0.05, p< 0.05). ELISpot assays showed that there was a significant increase in interferon-gamma production in the splenocytes from the mice immunized with rBCG-MVNTR4-CD80. In addition, CD4 and CD8-positive lymphocytes in tumors from rBCG-MVNTR4-CD80-immunized animals were detected. These data showed that rBCG-MVNTR4-CD80 immunization elicited tumor-specific immune response, which closely related with the B7 molecule (CD80), indicating that the vaccine may be a good candidate for MUC1-positive breast cancer immunotherapy. [ABSTRACT FROM AUTHOR]