Recovery of renal function in undifferentiated connective tissue disease after treatment with angiotensin-converting enzyme inhibitors.

Angiotensin-converting enzyme (ACE) inhibitor therapy has been reported to improve patient survival and promote recovery of renal function in the renal crisis of systemic sclerosis. In addition, an ACE inhibitor and a calcium channel blocker have been reported to control hypertension and reverse dialysis-dependent renal failure in a patient with undifferentiated connective tissue disease. We treated a patient with undifferentiated connective tissue disease who developed hypertension, pulmonary compromise, and renal failure requiring prolonged dialysis therapy. Due to allergy, the patient's hypertension could not be treated with ACE inhibitors initially, yet pulmonary function improved and renal function partially recovered with tenormin and minipress. When blood pressure became refractory to tenormin and minipress after 14 months of peritoneal dialysis, the patient was treated with lisinopril alone. Pulmonary function has remained stable and the patient has been off renal replacement therapy for 26 months, with a further substantial increase in creatinine clearance following treatment with lisinopril. The delayed and sustained recovery of renal and pulmonary function in the present case suggests undifferentiated connective tissue disease, like systemic sclerosis, may benefit from therapy with ACE inhibitors.

Cl-/HCO-3 antiporter in red cell ghosts: a kinetic assessment with fluorescent probes.

The pH sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide were used to evaluate the Cl-/HCO-3 antiporter and proton and potassium conductances, respectively, in human red blood cell ghosts. Acidic, chloride-loaded ghosts alkalinized rapidly in pH 8.5 chloride-free media. Alkalinization could not be ascribed to conductive proton efflux with either depolarizing potassium influx or chloride efflux. Alkalinization was consequent to flux on the Cl-/HCO-3 antiporter: this process displayed saturation kinetics, competitive inhibition by external chloride, and inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The mean Kms for internal chloride, external bicarbonate, and external chloride were 2.19, 0.24, and 0.44 mM, respectively. These studies confirm both the asymmetry of this carrier and the high affinity for external HCO-3; however, the affinities for internal and external chloride are significantly greater than prior estimates. The Km for internal chloride (2.19 mM) was considerably lower than levels previously reported (20-65 mM) unless external (trans) chloride was raised above 2 mM. The present studies thus demonstrate and emphasize the critical importance of trans substrate concentration in assessing the kinetics of a carrier whose mobility is faster in the loaded than in the unloaded state.

Altered membrane ionic permeability in a rat model of chronic renal failure.

Acute elevations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentrations are known to increase ionic chloride permeability in diverse tissues. To determine if chronic endogenous increases in cAMP are associated with sustained alterations in membrane ionic permeabilities, renal cortical brush border membrane vesicles (BBMV) were prepared and red blood cells were harvested in a model of chronic renal failure, the 75% nephrectomized rat. Relative ionic permeabilities were determined using the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. These studies demonstrate that renal cortical homogenate and RBC cAMP concentrations are increased in chronic renal failure animals. In the same animals relative ionic chloride permeability (PCl/PK) was significantly increased in renal cortical BBMV and RBC ghosts: PNa/PK was not affected. This selective change in permeability results in a significant increase in PCl/PNa and hyperpolarization of BBMV of sufficient magnitude to stimulate Na(+)-dependent glutamine transport. The change in glutamine uptake was not consequent to an alteration in the kinetics of glutamine transport or delayed dissipation of the inward Na+ gradient. Renal hypertrophy per se did not effect renal homogenate cAMP concentration or relative ionic permeability of renal cortical BBMV prepared from kidneys of uninephrectomized animals fed a 40% protein diet. These studies demonstrate that relative ionic chloride permeability and tissue [cAMP] are chronically increased in diverse cells (renal proximal tubule and RBCs) in a rat model of renal failure. These findings suggest that membrane ionic permeability may be altered and electrogenic transport secondarily perturbed in renal failure in association with hormonally-induced chronic elevations of intracellular cAMP concentrations.

Hormonal modulation of ionic permeability in human red blood cells.

It has previously been reported that both exogenous adenosine cAMP analogs and forskolin-induced elevations in intracellular cAMP concentrations selective increase relative ionic chloride permeability in normal human red blood cells (RBC). A similar selectively increase in relative ionic chloride permeability was observed in untreated uremic subjects in whom endogenous RBC cAMP concentrations are chronically elevated. To detect which hormones might modulate RBC cAMP and ionic permeabilities, RBC were exposed to norepinephrine, epinephrine, and parathyroid hormone. Thereafter, RBC cAMP concentrations were measured by RIA and relative ionic permeabilities were determined in human RBC ghosts with the potential sensitive fluorescent probe diS-C3-(5). In ghosts prepared from normal RBC, norepinephrine and epinephrine significantly increased intracellular cAMP concentrations; in these ghosts, relative ionic chloride permeability (permeability of chloride/permeability of potassium (PCI/PK)), but not PNa/PK (permeability of sodium/permeability of potassium), was significantly increased. In contrast, exposure to parathyroid hormone did not affect either cAMP concentrations or relative ionic permeabilities. These results are consistent with the presence of adrenergic receptors and the absence of parathyroid hormone receptors in RBC. These studies demonstrate that hormonally induced changes in cAMP can modulate RBC relative ionic chloride permeability and suggest that, in uremic RBC, increased relative ionic chloride permeability could be consequent to elevated plasma levels of epinephrine or norepinephrine.

Modulation of ionic permeability in a nonpolarized cell: effect of cAMP.

To evaluate whether adenosine 3',5'-cyclic monophosphate (cAMP) modulates ionic permeabilities of nonpolarized cells, as reported in diverse polarized epithelia, relative ionic permeabilities were determined in human red cell ghosts by means of the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide. Relative ionic chloride permeability (PCl/PK), but not PNa/PK, was significantly increased in ghosts prepared from normal red blood cells (RBCs) exposed to cAMP analogues or forskolin, with the latter at a concentration that significantly increased intracellular cAMP concentration. As basal RBC cAMP concentrations of untreated uremic subjects were also increased, relative permeabilities of ghosts and unstimulated RBC cAMP concentrations were compared in normal, uremic, and dialyzed subjects, PCl/PK was significantly increased in uremic compared with normal subjects; PNa/PK was not altered. PCl/PK and RBC cAMP concentrations were indistinguishable in normal and dialyzed subjects. Neither the kinetics nor number of Cl(-)-HCO3- antiporters, assessed with the pH-sensitive probe acridine orange and the disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, respectively, were altered in uremic cells. These studies suggest that cAMP modulates ionic chloride permeability via increased chloride conductance of each Cl(-)-HCO3- antiporter or by activation/opening of new or existing channels in RBC membranes.

Hormonal regulation of rat renal proximal tubule brush-border membrane ionic permeability.

The effects on ionic permeability of toxins and hormones that activate or deactivate the guanine nucleotide regulatory (G) proteins that govern adenylate cyclase activity were examined in rat renal proximal tubule cell brush-border membranes. These studies demonstrate that activation of stimulatory G (Gs) proteins by cholera toxin or parathyroid hormone and deactivation of inhibitory (G (Gi) proteins by pertussis toxin result in a selective increase in Cl- permeability relative to that of K+ as determined with the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. In contrast, activation of Gi by angiotensin II significantly decreases relative Cl- permeability. The selective increase in relative Cl- permeability induced by parathyroid hormone results in an inside-negative potential in membrane vesicles exposed to an inward NaCl gradient that is of sufficient magnitude to stimulate electrogenic, Na(+)-dependent glucose transport. These data suggest that the relative ionic permeabilities of brush-border membranes are tonically regulated by the opposing effects of hormones that act via Gs or Gi proteins. Changes in membrane potential resulting from this regulation may play an important role in modifying transport in the proximal tubule.

Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel.

Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein. Ion channel activity was evaluated in planar lipid bilayers before and after fusion of uricase-containing proteoliposomes. In the presence of symmetrical solutions of urate and KCl, but absence of uricase, no current was generated when the voltage was ramped between +/- 100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified with a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl-. The channel was more selective to oxonate, an inhibitor of both enzymatic uricase activity and urate transport, than urate and it was equally selective to urate and pyrazinoate, an inhibitor of urate transport. With time, pyrazinoate blocked both its own movement and the movement of urate through the channel. Channel activity was also blocked by the IgG fraction of a polyclonal antibody to affinity purified pig liver uricase. These studies demonstrate that a highly selective, voltage dependent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.