Fanconi's syndrome and tubulointerstitial nephritis in association with L-lysine ingestion.

We report a case of a 44-year-old woman who developed Fanconi's syndrome in association with the oral ingestion of L-lysine. L-lysine is widely available in health food stores and has been recommended in the lay press for the treatment and prevention of recurrent herpes simplex. The development of a severe tubulointerstitial nephritis, and eventual progression to chronic renal failure, underscores the importance of this entity, heretofore unrecognized in humans.

Trimethoprim-sulfamethoxazole and hypouricemia.

BACKGROUND: Hypouricemia has been reported in a substantial fraction of patients with AIDS and attributed to an HIV-related renal urate transport defect. We tested the alternative hypothesis that hypouricemia was associated with the administration of high-dose trimethoprim-sulfamethoxazole (TMP-SMX). METHODS: Sociodemographic, clinical, and repeated laboratory data on 45 hospitalized patients with Pneumocystis carinii pneumonia (PCP) with and without HIV infection, were abstracted by a blinded reviewer. The primary outcome of interest was the percent change in serum uric acid concentration from baseline to hospital day 5 +/- 1. RESULTS: Subjects who received TMP-SMX were older (mean age 44.8 vs. 37.0, p = 0.02), less likely to be HIV-seropositive (61% vs. 94%, p = 0.01), and more likely to have received glucocorticoid therapy (75% vs. 35%, p = 0.01) than those who received pentamidine, dapsone-trimethoprim, clindamycin-primaquine, sulfadiazine-pyramethamine, or a combination of these agents. The administration of TMP-SMX was associated with a 37% +/- 12% reduction in serum uric acid concentration, adjusting for the effects of age, sex, race, HIV antibody status, renal function, serum sodium, and the use of diuretics and glucocorticoids (p = 0.005). CONCLUSION: Among a diverse cohort of hospitalized patients with PCP, treatment with high-dose TMP-SMX was strongly associated with a reduction in serum uric acid concentration over time.

Blood pressure control, proteinuria, and the progression of renal disease. The Modification of Diet in Renal Disease Study.

OBJECTIVE: To examine the relations among proteinuria, prescribed and achieved blood pressure, and decline in glomerular filtration rate in the Modification of Diet in Renal Disease Study. DESIGN: 2 randomized trials in patients with chronic renal diseases of diverse cause. SETTING: 15 outpatient nephrology practices at university hospitals. PATIENTS: 840 patients, of whom 585 were in study A (glomerular filtration rate, 25 to 55 mliters/min.1.73 m2) and 255 were in study B (glomerular filtration rate, 13 to 24 mliters/min.1.73 m2). Diabetic patients who required insulin were excluded. INTERVENTIONS: Patients were randomly assigned to a usual blood pressure goal (target mean arterial pressure, < or = 107 mm Hg for patients < or = 60 years of age and < or = 113 mm Hg for patients > or = 61 years of age) or a low blood pressure goal (target mean arterial pressure, < or = 92 mm Hg for patients < or = 60 years of age and < or = 98 mm Hg for patients > or = 61 years of age). MAIN OUTCOME MEASURES: Rate of decline in glomerular filtration rate and change in proteinuria during follow-up. RESULTS: The low blood pressure goal had a greater beneficial effect in persons with higher baseline proteinuria in both study A (P = 0.02) and study B (P = 0.01). Glomerular filtration rate declined faster in patients with higher achieved blood pressure during follow-up in both study A (r = -0.20; P < 0.001) and study B (r = -0.34; P < 0.001), and these correlations were stronger in persons with higher baseline proteinuria (P < 0.001 in study A; P < 0.01 in study B). In study A, the association between decline in glomerular filtration rate and achieved follow-up blood pressure was nonlinear (P = 0.011) and was stronger at higher mean arterial pressure. In both studies, the low blood pressure goal significantly reduced proteinuria during the first 4 months after randomization. This, in turn, correlated with a slower subsequent decline in glomerular filtration rate. CONCLUSIONS: Our study supports the concept that proteinuria is an independent risk factor for the progression of renal disease. For patients with proteinuria of more than 1 g/d, we suggest a target blood pressure of less than 92 mm Hg (125/75 mm Hg). For patients with proteinuria of 0.25 to 1.0 g/d, a target mean arterial pressure of less than 98 mm Hg (about 130/80 mm Hg) may be advisable. The extent to which lowering blood pressure reduces proteinuria may be a measure of the effectiveness of this therapy in slowing the progression of renal disease.

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity.

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.

Expression of Na(+)-H+ exchange and ATP-dependent proton extrusion in growing rat IMCD cells.

As the last step of urinary acidification, the inner medullary collecting duct (IMCD) is thought to secrete protons into the tubular lumens by means of a H(+)-translocating adenosinetriphosphatase (H(+)-ATPase). However, recent studies have also shown the existence of Na(+)-H+ exchange activity in IMCD cells. Although the physiological function of the antiporter in IMCD cells is unknown, activation of Na(+)-H+ exchange in other cell-culture systems has been suggested to be closely associated with the process of cell growth. Thus presence of Na(+)-H+ exchange may relate to the growth phase of these cells. To examine intracellular pH (pHi) regulation in growing IMCD cells, we studied proton transport by Na(+)-dependent and Na(+)-independent mechanisms by microfluorimetry using the pHi-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF/AM). Actively growing cells, defined by [3H]thymidine incorporations, demonstrated an amiloride-sensitive Na(+)-dependent pHi recovery after an acid load. No pHi recovery was evident in the absence of Na+, indicating the importance of Na(+)-H+ exchange for pHi recovery. However, when evaluated in quiescent cells, Na(+)-dependent pHi recovery appeared to be diminished. Instead, a Na(+)-independent pHi recovery which was inhibitable by ATP depletion and by 1 mM N-ethylmaleimide was present, suggesting function of a H(+)-ATPase. These findings indicate that Na(+)-dependent proton extrusion activity (Na(+)-H+ exchange) but not Na(+)-independent proton extrusion activity is expressed during the rapid growth phase of IMCD cells, whereas the more quiescent cells express Na(+)-independent ATP-dependent proton extrusion activity and a possibly less active Na(+)-H+ exchanger.

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.

Regulation of Na/H exchange in renal microvillus vesicles in chronic hypercapnia.

Several disturbances of acid-base balance, including chronic metabolic and respiratory acidoses and metabolic alkalosis, are associated with enhanced proximal tubule bicarbonate reabsorption. To determine whether augmented brush border Na/H exchange might mediate enhanced proximal tubule bicarbonate reabsorption in these disorders, we measured Na/H exchange activity in cortical brush border membrane vesicles (BBMV) prepared from rats and rabbits adapted to hypercapnia and other chronic acid-base disturbances. BBMV prepared from control animals and animals with chronic acid-base disturbances were similar as judged by marker enzymes, alkaline phosphatase, and ouabain-sensitive phosphatase. Despite profound respiratory acidosis, no increase in Na/H exchange activity could be detected in vesicles prepared from rats adapted to chronic (8 to 10 days) or subacute (24 hr) respiratory acidosis. In addition, vesicles prepared from rabbits exposed to chronic hypercapnia did not show increased Na/H exchange when compared with contemporaneous controls. By contrast, in agreement with previously published results, amiloride-sensitive sodium uptake was increased by 30% in vesicles derived from animals with ammonium chloride-induced acidosis compared with contemporaneous controls. Two models of chronic metabolic alkalosis were also studied; vesicles from alkalotic rats did not show any alteration in Na/H exchange. We conclude that metabolic acidosis, but not respiratory acidosis or metabolic alkalosis, leads to enhanced activity of the luminal Na/H exchanger.

Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells.

Epidermal growth factor (EGF) is a potent mitogen that rapidly activates plasma membrane Na+-H+ exchangers, thereby causing intracellular alkalinization. The rise in intracellular pH (pHi) may be an important signal for cell growth. However, recent studies have dissociated Na+-H+ exchange activity and/or alkalinization from cellular proliferation. We have studied the role of EGF in the growth of rat renal proximal tubule (PT) cells in primary culture and monitored the early effects of EGF on pHi in these cells using microfluorimetry and the pHi probe, 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). EGF increased DNA synthesis in growing PT cells and produced transient alkalinization (2-3 min) due to activation of Na+-H+ exchange. In contrast, in the absence of extracellular Na+, EGF administration caused pHi to decrease. This acidification was prevented by 2-deoxy-D-glucose and 6-aminonicotinamide, inhibitors of glucose utilization and the hexose monophosphate shunt (HMP), respectively. EGF was also found to stimulate HMP shunt activity in PT cells using an isotopic method for distinguishing between glucose utilization through the HMP shunt vs. glycolysis. Because EGF caused both cytoplasmic acidifying (HMP activation) and alkalinizing (Na+-H+ exchange activation) processes, we propose that the primary role for the activation of Na+-H+ exchange during growth may be to extrude acid from the cell in order to maintain pHi at levels permissive for cell growth.

cGMP mediates effects of atrial peptides on medullary collecting duct cells.

Atrial natriuretic peptides (ANP) stimulate renal Na+ excretion by poorly understood mechanisms, possibly involving direct inhibition of Na+ transport in the renal medulla. We have previously shown that human ANP 4-28 (hANP) inhibits Na+ entry-dependent O2 consumption (QO2) in rabbit inner medullary collecting duct (IMCD) cells. Because ANP actions in other tissues appear to be mediated by guanosine 3',5'-cyclic monophosphate (cGMP), the present studies examined the role of cyclic nucleotides in IMCD cell responses to ANP. 8-Bromo-cGMP (8-BrcGMP) diminished QO2 by 23.5 +/- 1.2% (SE) in IMCD cells but had no effect in cells derived from outer medullary collecting duct (OMCD); dibutyryl-adenosine 3',5'-cyclic monophosphate (cAMP) was without effect in IMCD cells. The inhibitory effect of BrcGMP was not additive with ANP, amiloride, or ouabain. Amphotericin, which enhances Na+ entry into cells, prevented the inhibitory effect of 8-BrcGMP. These results indicate that 8-BrcGMP, like ANP, inhibited Na+ entry in IMCD cells. hANP stimulated a 10-fold increase in cGMP in IMCD cells without altering IMCD cAMP levels or OMCD cGMP levels. Isobutyl methylxanthine, which inhibits phosphodiesterase activity, enhanced both cGMP accumulation and inhibition of QO2 by submaximal levels (10(-9) M) of ANP. Nitroprusside raised cGMP levels in both IMCD and OMCD cells but inhibited QO2 only in IMCD cells. We conclude that cGMP mediates the transport effects of ANP in IMCD cells. Our results indicate that cGMP may play an important role in the regulation of sodium transport in renal epithelia.

Use of monoclonal antibodies to culture rat proximal tubule cells.

Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation.

Coupling of Na-H exchange and Na-K pump activity in cultured rat proximal tubule cells.

Na-H exchange was studied using electron probe analysis and microfluorescent pH analysis of individual cells, in 3-day primary cultures of rat proximal tubule cells (RPTC) obtained from 40- to 50-day-old Sprague-Dawley rats. After Na-K pump inhibition, the initial rate of net Na influx was inhibited 87% by 1 mM amiloride. K influx, an estimate of Na-K pump activity, was increased approximately three times in cells containing high Na (0.114 mM K X mM P-1 X min-1) compared with control cells containing low Na (0.038 mM K X mM P-1 X min-1). Single cell measurements of RPTC loaded with the cytoplasmic pH indicator 5- (and -6) carboxy-4',5'-dimethylfluorescein indicated that there was reversible intracellular acidification in the absence of external Na or in the presence of amiloride. When intracellular acidification was induced by the addition and subsequent removal of NH4Cl, recovery of intracellular pH was inhibited in the absence of external Na or in the presence of amiloride. Using a similar protocol, it was found that after intracellular acidification, the rate of Na influx increased at least 5.9 times, and intracellular Na content was increased 3.15 +/- 0.64 times at 60 s. There was an initial 50% inhibition of Na-K pump activity within the first 60 s compared with control (nonacidified) RPTC, secondarily followed by an increase in Na-K pump activity. Amiloride (0.5 mM) inhibited the acidification-induced increase in Na influx, and persistent acidification led to a persistent inhibition of Na-K pump activity compared with control. In summary, these results demonstrate that Na-H exchange mediates the majority of net Na influx into RPTC under our basal conditions and is necessary for maintenance of intracellular pH homeostasis. In RPTC, Na-H exchange is further activated by intracellular acidification, leading to a net increase in intracellular Na content, which secondarily stimulates Na-K pump activity. The initial inhibition of Na-K pump activity may be due to a direct effect of intracellular acidification.

Atrial peptides inhibit oxygen consumption in kidney medullary collecting duct cells.

Atrial natriuretic peptides (ANP) stimulate renal Na+ excretion by poorly understood mechanisms, perhaps involving direct inhibition of Na+ transport in the kidney medulla. To examine the effects of ANP on renal cells directly, we prepared highly purified cell suspensions derived from inner and outer medullary collecting duct and thick ascending limb of rabbit kidney and monitored ouabain-sensitive oxygen consumption (QO2). Human ANP diminished QO2 by 27.4 +/- 1.6% (mean +/- SE) in inner medullary collecting duct cells but had no effect in cells derived from outer medullary collecting duct or thick ascending limb. The inhibitory effect of ANP was not additive with either amiloride or ouabain. ANP was without effect in the presence of amphotericin. These results indicate that ANP inhibited Na+ entry in inner medullary collecting duct cells. ANP-mediated inhibition of QO2 was dose dependent (Ki = 5.5 X 10(-10) M) and exhibited selectivity for peptide structure. These results suggest that atrial peptides enhance renal sodium excretion partly by direct inhibition of medullary collecting duct sodium transport.

Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.