Bicarbonate promotes BK-α/ß4-mediated K excretion in the renal distal nephron.

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-ß4 in acid/base-transporting intercalated cells (IC), we determined whether BK-ß4 knockout mice (ß4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-ß4 expression increased (Western blot). When WT and ß4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and ß4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with ß4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and ß4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in ß-IC, comparing WT and ß4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in ß4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of ß4KO. When WT on LNaHK were given NH(4)Cl in the drinking water, K excretion was reduced to the magnitude of ß4KO on LNaHK. These results show that WT, but not ß4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/ß4-mediated K secretion is promoted by bicarbonaturia.

Flow-sensitive K+-coupled ATP secretion modulates activity of the epithelial Na+ channel in the distal nephron.

The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K(+) efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-ß4 regulatory subunit, which is required for normal BK(Ca) channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-ß4 null mice. ENaC in BK-ß4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-ß4 null mice. Consequently, sodium excretion is impaired in BK-ß4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK(Ca) channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-ß4 null mice likely contributes to their elevated blood pressure.

Coupled ATP and potassium efflux from intercalated cells.

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (τ; 10 dynes/cm(2)) -induced, BK-dependent, K efflux. We found that 10 µM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P(2) receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited τ-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-ß4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and τ-induced K efflux. Similarly, carbenoxolone (25 µM), which blocks connexins, putative ATP pathways, blocked τ-induced K efflux and ATP secretion. Compared with BK-ß4(-/-) mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for τ-induced ATP release in IC.

Donor immune reconstitution after liver-small bowel transplantation for multiple intestinal atresia with immunodeficiency.

The syndrome of multiple intestinal atresia with immunodeficiency is a rare, invariably fatal congenital disorder. At 16 months of age, a child with this syndrome underwent liver-small bowel transplantation from a 1-of-6 HLA-matched donor. He acquired full enteral tolerance and normal liver function and has never shown evidence of allograft rejection. After mild graft-versus-host disease developed, studies revealed that more than 99% of his CD3(+) lymphocytes and 50% of his CD19(+) lymphocytes were of donor origin, whereas granulocytes and monocytes remained of recipient origin. He synthesizes polyclonal immunoglobulin G (IgG), IgA, and IgM and has developed antibodies to cytomegalovirus (CMV) and parainfluenza 3. His T lymphocytes are predominately CD3(+)CD4(-)CD8(-) with T-cell receptor gammadelta heterodimers and CD3(+)CD4(-)CD8(+) with CD8alphaalpha homodimers, populations consistent with an intraepithelial lymphocyte phenotypic profile. We postulate that he has engrafted a donor intestine-derived immune system and is incapable of rejecting his engrafted organs.

Quantitation of motexafin lutetium in human plasma by liquid chromatography-tandem mass spectrometry and inductively coupled plasma-atomic emission spectroscopy.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) methods were developed and validated for the evaluation of motexafin lutetium (MLu, lutetium texaphyrin, PCI-0123) pharmacokinetics in human plasma. The LC-MS/MS method was specific for MLu, whereas the ICP-AES method measured total elemental lutetium. Both methods were fast, simple, precise, and accurate. For the LC-MS/MS method, a closely related analogue (PCI-0353) was used as the internal standard (IS). MLu and the IS were extracted from plasma by protein precipitation and injected into an LC-MS/MS system configured with a C18 column and an electrospray interface. The lower limit of quantitation was 0.05 microg MLu mL(-1), with a signal-to-noise ratio of 15:1. The response was linear from 0.05 to 5.0 microg MLu mL(-1). For the ICP-AES method, indium was used as the IS. The sample was digested with nitric acid, diluted, filtered, and then injected into the ICP-AES system. Two standard curve ranges were validated to meet the expected range of sample concentrations: 0.5 to 50, and 0.1 to 10 microg Lu mL(-1). The LC-MS/MS and ICP-AES methods were validated to establish accuracy, precision, analyte stability, and assay robustness. Interday precision and accuracy of quality control samples were < or =6.3% coefficient of variation (CV) and within 2.2% relative error (RE) for the LC-MS/MS method, and < or =8.7% CV and within 4.9% RE for the ICP-AES method. Plasma samples from a subset of patients in a clinical study were analyzed using both methods. For a representative patient, over 90% of the elemental lutetium in plasma could be ascribed to intact MLu at early time points. This percentage decreased to 59% at 48 hours after dosing, suggesting that some degradation and/or metabolism of the drug may have occurred.