Donor-directed MHC class I antibody is preferentially cleared from sensitized recipients of combined liver/kidney transplants.

For patients with chronic renal and liver diseases, simultaneous liver and kidney transplantation (SLKT) is the best therapeutic option. The role of a pretransplant donor-specific antibody (DSA) in SLKT is unclear. We report the results of a retrospective review from 7/08 to 10/09 of SLKT at our institution. Monitoring of DSA was performed using single antigen bead assay. Between 7/08 and 10/09, there were six SLKT who had preformed DSA and positive XM (four class I and II DSA, one class I DSA only, one class II only). One-year patient and renal graft survival was 83%. Death-censored liver allograft survival was 100%. Acute humoral rejection (AHR) of the kidney occurred in 66% (three with both class I and II DSA and one with only class II DSA) of patients. In those with AHR, class I antibodies were rapidly cleared (p < 0.01) while class II antibodies persisted (p = 0.25). All patients who had humoral rejection of their kidney had preformed anticlass II antibodies. Liver allografts may not be fully protective of the renal allograft, especially with pre-existing MHC class II DSA. Long-term and careful follow-up will be critical to determine the impact of DSA on both allografts.

An unusual presentation of a Page kidney 24 days after transplantation: case report.

The Page kidney phenomenon is a well recognized entity where an extrinsically compressed kidney results in hypertension and loss of function. This compression is usually caused by a subcapsular hematoma secondary to blunt abdominal trauma or an invasive procedure such as a renal biopsy. We describe an unusual case involving the spontaneous development of a Page kidney 24 days after renal transplantation without any history of preceding trauma. The subcapsular hematoma was detected by a computerized tomographic scan performed as part of the work-up for acute allograft dysfunction. Prompt recognition and early intervention are essential if renal function is to be restored before irreversible damage occurs.

Prostaglandin E2 increases 7-pS Cl- channel density in the apical membrane of A6 distal nephron cells.

In A6 distal nephron cells, short-circuit current (Isc) was increased by basolateral exposure to prostaglandin E2 (PGE2; peak response at 1 microM). The effect was only partially abolished by either apical amiloride, an Na+ channel blocker, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl- channel blocker. In apical cell-attached patches, we observed a 7-pS Cl- channel with a linear current-voltage relationship, a reversal potential near resting membrane potential, and open probability > 0.5. The channel was blocked by diphenylamine-2-carboxylate, glibenclamide, and NPPB but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The frequency of observed Cl- channel activity increased 7-fold with 10-min exposure to PGE2 and 3.7-fold with longer (10-50 min) exposure to PGE2. The PGE2-induced increase in Cl- channel activity was due primarily to an increase in the number of functional channels. The following conclusions were made: 1) activation of apical, 7-pS Cl- channels in A6 cells accounts for the PGE2-induced increase in the amiloride-insensitive Isc, and 2) 7-pS Cl- channel activation was mediated via an increase in channel density without substantial effects on channel kinetics.

Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6).

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.

Prostaglandin E2 activates clusters of apical Cl- channels in principal cells via a cyclic adenosine monophosphate-dependent pathway.

UNLABELLED: We examined cell-attached patches on principal cells of primary cultured, rabbit cortical collecting tubules. Under basal conditions, apical 9-pS Cl(-)-selective channels were observed in 9% of patches (11/126), and number of channels times open probability (NP0) was 0.56 +/- 0.21. The channel had a linear current-voltage relationship, reversal potential (Erev) near resting membrane potential, a P0 (0.30-0.70) that was independent of voltage, and complicated kinetics (i.e., bursting) at hyperpolarized potentials. NP0 and channel frequency were increased after 30 min of basolateral exposure to 0.5 microM PGE2 (18/56), 10 microM forskolin (23/36), or 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP) (25/41). Increases in NP0 appeared to be mediated primarily through an increase in the number of observed channels per patch (N), not changes in P0. After these cAMP-increasing maneuvers, N was inconsistent with a uniform distribution of channels in the apical membrane (P < 0.001), but rather the channels appeared to be clustered in pairs. Apical 0.5 microM PGE2 (12/91), apical or basolateral 0.5 microM PGF2 alpha (8/110), or 0.25 microM thapsigargin (releaser of intracellular Ca2+ stores) (7/73) did not increase NP0 or channel frequency. CONCLUSIONS: (a) 9-pS Cl- channels provide a conductive pathway for apical membrane Cl- transport across principal cells. (b) Channel activation by basolateral PGE2 is mediated via a cAMP-, but not a Ca(2+)-dependent mechanism. (c) Apical channels are clustered in pairs. (d) With its low baseline frequency and Erev near resting membrane potential, this channel would not contribute significantly to transcellular Cl- flux under basal conditions. (e) However, cAMP-producing agonists (i.e., PGE2, arginine vasopressin) would increase apical Cl- transport with the direction determined by the apical membrane potential.

Inhibition of apical Na+ channels in rabbit cortical collecting tubules by basolateral prostaglandin E2 is modulated by protein kinase C.

UNLABELLED: We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. CONCLUSIONS: (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.

Regulation of renal epithelial sodium channels.

The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTP gamma S inhibits channel activity, whereas GDP beta S or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to 'feed-back inhibition' of apical Na+ conductance in tight epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the amiloride-blockable sodium channel from epithelial tissue.

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.