A mathematical model of distal nephron acidification: diuretic effects

Through their action on the distal nephron (DN), diuretics may produce systemic acid-base disturbances: metabolic alkalosis with thiazides or loop diuretics and metabolic acidosis with amiloride. Enhanced acid excretion may be due to a local effect on the diuretic target cell (a shift of Na+ reabsorption from NaCl transport to Na+/H+ exchange), or an effect at a distance: namely, increases in luminal fluid flow or luminal Na+ concentration may enhance more distal proton secretion. Both local and distance effects are supported by micropuncture data. In the present work, mathematical models of the distal convoluted tubule (DCT)/connecting tubule (CNT) (Weinstein AM, Am J Physiol Renal Physiol 289: F721–F741, 2005), and cortical and medullary collecting ducts (CD) (Weinstein AM, Am J Physiol Renal Physiol 283: F1237–F1251, 2002) have been concatenated to yield a model of rat DN. Among the segments of this DN, the DCT-CNT is responsible for the major portion of distal acidification. Predictions from the model calculations include the following. 1) With increasing distal Na+ delivery, there is little change in net acid excretion, but a shift in acidification locus from shared DCT and CNT contributions, to DCT prominence. 2) Urinary acidification by thiazides is primarily local (in the DCT) via the shift in Na+ reabsorption from NaCl cotransport to entry via NHE2. Increased NaCl delivery to the CNT increases β-cell HCO3− secretion, and thus blunts urine acidification. 3) In contrast to conclusions drawn from the isolated CD model, inclusion of the CNT now reproduces the observed distal acidification defect found with ENaC block, so that this action of amiloride appears to be sufficient to produce “voltage-dependent” distal renal tubular acidosis. 4) The effect of furosemide to enhance distal urinary acidification is not reproduced by the model without major upregulation of CNT α-cell transport, perhaps as a result of increased luminal flow.

A mathematical model of the inner medullary collecting duct of the rat: pathways for Na and K transport

A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed representing Na+, K+, Cl−,[Formula: see text] CO2, H2CO3, phosphate, ammonia, and urea. Novel model features include: finite rates of hydration of CO2, a kinetic representation of the H-K-ATPase within the luminal cell membrane, cellular osmolytes that are regulated in defense of cell volume, and the repeated coalescing of IMCD tubule segments to yield the ducts of Bellini. Model transport is such that when entering Na+ is 4% of filtered Na+, approximately 75% of this load is reabsorbed. This requirement renders the area-specific transport rate for Na+ comparable to that for proximal tubule. With respect to the luminal membrane, there is experimental evidence for both NaCl cotransport and an Na+ channel in parallel. The experimental constraints that transepithelial potential difference is small and that the fractional apical resistance is greater than 85% mandate that more than 75% of luminal Na+ entry be electrically silent. When Na+delivery is limited, an NaCl cotransporter can be effective at reducing luminal Na+ concentration to the observed low urinary values. Given the rate of transcellular Na+ reabsorption, there is necessarily a high rate of peritubular K+recycling; also, given the lower bound on luminal membrane Cl− reabsorption, substantial peritubular Cl− flux must be present. Thus, if realistic limits on cell membrane electrical resistance are observed, then this model predicts a requirement for peritubular electroneutral KCl exit.

Peripheral blood mononuclear cells express mutated NCCT mRNA in Gitelman's syndrome: evidence for abnormal thiazide-sensitive NaCl cotransport.

Genetic analysis has demonstrated complete linkage between the human thiazide-sensitive sodium chloride cotransporter gene (NCCT or TSC) and Gitelman's syndrome (GS). Several genomic NCCT mutations have been reported. This study was performed to determine whether peripheral blood mononuclear cells (PBMC) express NCCT mRNA and whether defective PBMC NaCl cotransport could be demonstrated in GS. PBMC were isolated from two brothers with GS, their parents, and healthy control subjects. Northern analysis revealed that NCCT mRNA is expressed in PBMC. The sequence of full-length NCCT cDNA amplified from normal PBMC was identical to human renal NCCT cDNA. Two different mutations were detected in the patients' NCCT cDNA (compound heterozygote). In cDNA derived from the patient's maternal allele, exon 24 was deleted, resulting in a premature stop codon (after amino acid 920). cDNA derived from the patient's paternal allele had an additional 119-bp insertion between exons 3 and 4, generating a premature stop codon (after amino acid 187). The patient's genomic DNA had a previously described 5' splice site mutation in intron 24, GGT --> GTT (maternal allele), and a new 3' splice site mutation in intron 3, CAG --> CAA (paternal allele), which resulted in the activation of a nearby cryptic splice site in intron 3. The latter mutation was not present in 300 normal chromosomes. To determine the functional significance of these findings, chlorothiazide-inhibitable 22Na uptake was measured in PBMC from control subjects, the parents, and the patients with GS in the presence of bumetanide. In control PBMC, chlorothiazide inhibited 22Na uptake by approximately 9%. PBMC from the two patients with GS failed to respond to chlorothiazide. These results demonstrate that PBMC can be used for mutational analysis of NCCT mRNA in patients with GS. Furthermore, functional evidence is provided that the underlying cause of GS is defective NCCT NaCl cotransport.

Osmotic regulation of intestinal epithelial Na+-K+-Cl− cotransport: role of Cl− and F-actin

Previous data indicate that adenosine 3′,5′-cyclic monophosphate activates the epithelial basolateral Na+-K+-Cl−cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical Cl− loss (due to cell shrinkage or decreased intracellular Cl−). To further address the actin dependence of Na+-K+-Cl−cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed. Na+-K+-Cl−cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular Cl− concentration and also to activation of K+ and Cl− efflux pathways. Stabilization of actin with phalloidin blunted cotransporter activation by hypotonicity and abolished hypotonic activation of K+ and Cl− efflux. However, phalloidin did not prevent activation of cotransport by hypertonicity or isosmotic reduction of extracellular Cl−. Conversely, hypertonic but not hypotonic activation was attenuated by the microfilament disassembler cytochalasin D. The results emphasize the complex interrelationship among intracellular Cl− activity, cell volume, and the actin cytoskeleton in the regulation of epithelial Cl− transport.

Acute respiratory acidosis: large-dose furosemide and cerebrospinal fluid ions

NaCl cotransport carrier is known to be involved in transepithelial fluid absorption and secretion in various tissues. Recent studies indicate that Na-K-2Cl cotransport carrier also exists in the choroid plexus cells and that inhibition of the carrier decreases cerebrospinal fluid (CSF) production. In this study, we used large-dose intravenous furosemide, an inhibitor of Na-K-2Cl carrier, to determine the effects on cisternal CSF ionic composition in acute respiratory acidosis. In pentobarbital-anesthetized mechanically ventilated dogs, renal pedicles were ligated to prevent furosemide-induced diuresis. The experimental group (group II, n = 7) received 400 mg/kg of furosemide intravenously, and group I (control group, n = 7) received the vehicle. In group II, serial serum and CSF furosemide concentrations were approximately 10(-3) and 10(-5) mol/l, respectively. During 5 h of acute respiratory acidosis in both groups, the mean arterial PCO2 increased approximately 25 Torr, with comparable changes in CSF PCO2. In both groups, CSF [HCO3-] and [H+] rose approximately 3 meq/l and 20 neq/l, respectively. Changes in CSF [Na+], [K+], [Cl-], and [Na(+)-Cl-] were also similar and were not significantly different from each other when the two groups were compared. These data show that furosemide at the dose that inhibits NaCl cotransport carrier does not significantly alter ionic composition of cisternal CSF.