Renal Transport Of Anionic Diuretics

Three steps are necessary to effect tubular secretion of organic anions. First, the anionic compound must be delivered to the basolateral surface of the proximal tubule cell; second, the anionic compound must be transported into the cell across the basolateral membrane; and third, the anionic compound must be transported across the brush border membrane into the tubule lumen. Most investigations of these processes relating to anionic diuretic secretion have used furosemide and chlorothiazide as the probes and it is assumed that other loop diuretics and acetazolamide are secreted in a similar manner.

The first step in the process of tubule secretion is the delivery of the diuretic to the interstitial space surrounding the basolateral portion of the proximal tubule cell. Binding between the diuretic and albumin is central to this process. Inoue and associates have shown that the attenuated furosemide response observed in analbuminemic rats results almost entirely from impaired delivery of furosemide to the tubule lumen [7]. In these rats urinary furosemide recovery and diuretic response are significantly lower than in normal rats, although sodium excretion plotted as a function of urinary furosemide concentration is normal (Fig. 1). The essential role of albumin as a carrier for furosemide in this setting is demonstrated by the finding that a mixture of equal molar amounts of albumin with furosemide prior to intravenous diuretic administration significantly increased diuretic response in analbuminemic rats but had no additional effect in normal rats. The effects of this mixture was not due to the oncotic effects of the albumin since administration of the same amount of albumin prior to diuretic infusion resulted in no improvement in natriuretic response in either animal group. Thus diuretic albumin binding is a necessary step for diuretic secretion in the kidney. Albumin may also play an important role in the regulation of anionic diuretic secretion [2], Albumin stimulates renal organic anion transport in a dose-dependent manner up to a concentration of 1.0 g/dl. This effect is independent of peritubular oncotic pressure and independent extent of anion-albumin binding. High concentration of albumin may also inhibit anion transport. The mechanism for the regulatory effect of albumin on renal anion secretion and whether this is important the regulation of diuretic secretion is yet to be determined. Nonetheless, albumin is an important component for the initial steps of renal secretion of anionic diuretics.

The transport of anionic compounds from the interstitial space across the basolateral cell membrane has been extensively examined. Early studies used

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Urinary furosemide excretion, (ig/kg/hr

FIGURE 1. One-hour urinary sodium and furosemide excretion rates following intramuscular administration of various doses of furosemide to normal (open circles) and analbuminemic (closed circles) rats. Urinary furosemide recovery was low in analbuminemic rats compared with normal rats but sodium excretion as a function of urinary furosemide excretion was not different between groups. Adapted from Inoue et al (1987, Fig. 3, p. 199) with permission.

para-aminohippurate (PAH) as the probe for this process because PAH is as avidly secreted as hippurate, the principal endogenous anion secreted by the dog, PAH is not metabolized, and PAH is easily measured. These studies demonstrated that in the kidney anionic transport was localized to the proximal tubule with greater activity in juxtamedullary than cortical nephrons. Within individual proximal tubules the density of transporters seem to be greatest in the S2 segment although there may be some species heterogeneity. Kinetic evaluation of PAH transport in the rabbit juxtamedullary and cortical proximal tubules suggests that there is unequal distribution of transporters with a common substrate affinity along the proximal tubule subsegments. The S2 segment has a maximal transport rate five- to sevenfold greater than the S, or S3 segment and this difference is due to differences in basolateral membrane transport [5, 6, 10]. However, more recent studies suggest that there are multiple renal anion transport systems which can operate exchanging organic for organic substrates, organic for inorganic substrates, or inorganic for inorganic substrates depending on the intra- and extracellular availability of the anionic substrates themselves (Fig. 2). Fortunately for our understanding of renal diuretic secretion the anionic transport system which transports PAH is also the one primar-

Lumen

Proximal Tubule Cell

Peritubular Space

Dicarboxylati

Lumen

Dicarboxylati

Diuretic or

hco3"

Peritubular Space

Diuretic or

Dicarboxyk

Dicarboxyk

Dicarboxylates

Diuretic or or

hco3"

FIGURE 2. Tertiary active transport system for anionic diuretics. At the basolateral border of proximal tubule cells organic anions such as PAH or diuretic are transported across the cell membrane in exchange for dicarboxylate intermediates of the Kreb's cycle. Dicarboxylate intermediate enters by cotransport with sodium at either cell surface. Energy for both transport processes is provided by the NaK ATPase pump (solid circle). At the brush border membrane the diuretic exits the cell in exchange for tubular anion including chloride or bicarbonate or by passive diffusion.

ily responsible for the transport of acetazolamide, the loop diuretics, and the thiazide diuretics.

Transport across the proximal tubule basolateral membrane occurs in the form of bidirectional anion exchange between the pericellular and intracellular compartments. Specific anions can be moved in either direction based on their affinity for the transporter and concentration in each compartment. For the proximal tubule transporter it appears that in vivo the usual intracellular anionic substrates exchanged for the interstitial anions are dicarboxylate components of the Krebs cycle, usually alpha ketoglutarate or glutarate. These dicarboxylates can either be manufactured in the cell or more frequently added to the cytoplasm from tubule fluid or blood by cotransport with sodium. The tight association of these two distinct transport systems led to the initial consideration that PAH transport may be sodium dependent. However, in rat renal basolateral membrane vesicles inwardly directed gradients of lithium, potassium, and rubidium are as effective as sodium in stimulating PAH uptake in the presence of glutarate [9], while sodium in the absence of glutarate was ineffective in stimulating PAH transport. PAH uptake is also accelerated in vesicles preloaded with glutarate compared to glutarate-free vesicles. Therefore it appears that glutarate cotransported into the cell with sodium is then exchanged with extracellular PAH, leading to accumulation of PAH in the vesicle. Subsequent studies have shown that in spite of the sharing of dicarboxylate substrates between the two transport systems, the affinity for other anionic compounds is not identical. It seems that while all substrates for the sodium dicarboxylate cotransporter are accepted by the dicarboxylate PAH exchanger, the latter transporter handles a number of organic and inorganic anions not accepted by the sodium dicarboxylate cotransporter. Probenecid and thiazide diuretics are examples of such compounds which compete with PAH for uptake by the baso-lateral PAH dicarboxylate exchanger but do not interact with the sodium dicarboxylate cotransporter. The energy for these exchanges is provided by Na/K ATPase and possibly other ATPases located in the basolateral membrane. These produce an outside-in transmembrane electrochemical cation gradient which powers the sodium dicarboxylate cotransporter. The entire basolateral membrane transport process has been referred to as tertiary active transport.

The mechanism for the exit of anionic compounds from the cell into the tubular lumen have also been intensively studied. Studies have demonstrated that this last step across the luminal brush border membrane is in part due to an electroneutral anion exchanger which can operate in organic-organic, organic-inorganic, inorganic-inorganic substrate exchange modes using a variety of substrates including even HCO" and Cl~ from the tubule lumen in exchange for intracellular substrates [11]. The structural specificity for this transporter is diverse. Furosemide, bumetanide, probenecid, and penicillin show higher affinity for the brush border transporter than does PAH. Additionally, the capacity for transport is greater along the brush border than the basolateral membrane at least in the dog. Transport at this location is also less sensitive to inhibition by probenecid than is transport at the basolateral membrane. Transport across the brush border membrane also occurs by simple diffusion down an electrochemical gradient determined by both the pH and the flow rate of the tubule fluid.

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