Fred S. Wright

Interference with feedback control of glomerular filtration rate by furosemide, triflocin, and cyanide.

Microperfusion experiments have shown that increases in flow rate of tubule fluid through the loop of Henle are followed by reductions in single nephron glomerular filtration rate (SNGFR) and stop-flow pressure (SFP) measured in the proximal tubule of the same nephron. Because changes in luminal sodium concentration are not consistently related to changes in SNGFR and SFP, we explored the possibility that a transport step at a flow-dependent distal-sensing site might be involved in feedback control of SNGFR. Because the macula densa cells of the distal tubule are adjacent to the glomerular vessels of the same nephrons, they could be the distal-sensing mechanism. We perfused superficial loops of Henle from late proximal to early distal segments in three groups of rats while measuring SFP in the proximal tubule of the same nephron, SNGFR in the proximal tubule of the same nephron, or flow rates of fluid, Na, K, and Cl emerging from the perfused loops. Perfusion solutions used were 0.15 NaCl, Ringer or Ringer with one of several inhibitors of electrolyte transport. Perfusion rates were 10 or 40 nl/min (also, zero during measurements of SFP and SNGFR). With Ringer alone the loop-flow rate increased from 10 to 40 nl/min, caused a decrease in SFP from 37.6 to 32.1 mm Hg, and a decrease in SNGFR from 29.9 to 18.7 nl/min. Concentrations of Na, K, and Cl in early distal fluid and absorption of Na and Cl along the loop segment were also increased when loop perfusion rate was increased. Decreasing the perfusion rate to zero had little effect on SFP or SNGFR. The SFP response to increased flow rate did not occur when the perfusion solution contained furosemide (10(-4) M). No reduction of the SFP response was seen with other diuretics tested (amiloride, acetazolamide, ethacrynic acid, mercaptomerin) or with 0.15 M NaCl alone. The SNGFR response to increased perfusion rate was reduced by furosemide, triflocin, and cyanide but not by amiloride. Na and Cl absorption by the perfused segment were inhibited by furosemide, triflocin, cyanide, and amiloride. Amiloride and acetazolamide, probably do not act in the ascending limb. Ethacrynic acid and mercaptomerin are known to be ineffective in rat nephrons. Thus, agents that could have inhibited NaCl absorption by macula densa cells interfered with the feedback mechanism.

Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.

Renal Mechanism of Trimethoprim-induced Hyperkalemia

Table. SI Units Hyperkalemia is increasingly being recognized in patients with human immunodeficiency virus (HIV) infection and the acquired immunodeficiency syndrome (AIDS) [1-7]. In this setting, it is likely that hyperkalemia is the result of inadequate renal potassium excretion. Three mechanisms could be responsible for renal potassium retention: adrenal insufficiency with inadequate production of aldosterone; acute renal failure with reduced glomerular filtration and damage to tubule cells; and inhibition of potassium secretion. Most attention has focused on the first two mechanisms [1-8]. We are aware, however, of three reports of reversible hyperkalemia [4, 9, 10] that suggest that a therapeutic agent may have a direct action on renal tubules to suppress potassium transport. A common factor in the three reports was the administration of trimethoprim for treatment of Pneumocystis carinii pneumonia. The purpose of our study was to test the hypothesis that trimethoprim causes hyperkalemia by a direct action on the distal nephron cells responsible for secreting potassium. Methods Study in Humans All patients receiving high-dose trimethoprim (20 mg/kg per day), with either sulfamethoxazole or dapsone, at the Yale-New Haven Hospital during a 4-month period, were included. As a part of the clinical management of these patients, serum measurements of sodium, potassium, and creatinine levels were recorded before, during, and after trimethoprim treatment. In some patients, increased serum potassium levels (>5.0 mmol/L) were identified while patients were receiving trimethoprim. In a group of these patients, we reviewed the clinical course and recommended further evaluation to search for causes of hyperkalemia. This evaluation included measurements of serum glucose, renin, aldosterone, and cortisol levels as well as osmolality; measurements of urinary sodium, potassium, chloride, glucose, and protein levels as well as osmolality; and examination of the urinary sediment. The tubule fluid/plasma concentration ratio for potassium in the cortical collecting duct (transtubular potassium gradient) [11, 12] was calculated from urine and serum values for potassium and osmolality as (K)urine/[K]serum)/([Osm]urine/[Osm]serum). The ability of cosyntropin to stimulate cortisol secretion was determined in patients whose cortisol level was < 552 nmol/L (<20 g/dL). Studies in Rats Male Sprague-Dawley rats, allowed free access to standard rat chow and tap water up to the time of the experiment, were anesthetized before surgical exposure of one kidney, as previously described [13]. The ureter was cannulated. Intravenous Infusion of Trimethoprim A salt solution (140 mmol/L sodium chloride, 4 mmol/L potassium chloride) was infused at 15 mL/h per kg body weight, and 45 minutes was allowed for equilibration after surgical preparation was completed. Subsequently, six 30-minute urine collections were obtained. The first 90 minutes was period I (collections 1 to 3), and the subsequent 90 minutes was period II (collections 4 to 6). The control and experimental groups of animals differed only in that at 90 minutes (after period I), 0.64 g/L of trimethoprim was added to the intravenous infusate (the trimethoprim infusion rate was 9.6 mg/h per kg body weight) in the experimental group and was maintained throughout period II. Sodium, potassium, and chloride concentrations were measured in urine [13]. The urine flow rate was measured, and the excretion rates for fluid, sodium, potassium, and chloride were calculated. Microperfusion of Distal Tubules Microperfusion experiments were done as described previously [13, 14]. Distal tubules were perfused with an artificial tubule fluid with or without trimethoprim (the composition of the perfusion solutions is given in Table 1. A perfusion pipette was positioned at the upstream end of the tubule, and a collection pipette was positioned at the downstream end. The perfusion pump was set to deliver 15 nL/min. A paired design was used as follows: After the first tubule fluid sample was collected, both the collection pipette and the perfusion pipette were removed: Then a second perfusion pipette containing a different solution [the order of solutions was alternated] and a second collection pipette were used to collect a second tubule fluid sample. The volume of collected samples was measured. Sodium, potassium, chloride, and inulin concentrations in perfused and collected fluids were measured, as described previously [14]. Osmolality and pH of bulk solutions were measured [14]. The perfusion rate was calculated from the collection rate and the inulin concentrations. Net fluid transport was calculated as the difference between perfusion and collection rates. Net transport rates for sodium, potassium, and chloride levels by each distal tubule were determined. Transepithelial voltage across the wall of the late distal tubule was measured, as previously described [15]. In experiments designed to test the effect of different concentrations of trimethoprim on transepithelial voltage, a higher perfusion rate (30 compared with 15 nL/min) was used to minimize changes in luminal ion composition along the length of the perfused tubule. Table 1. Distal Tubule Flow Rates, Collected Ion Concentrations, and Transport Rates with Control and Trimethoprim Solutions* Statistical Analysis Results were analyzed using the t-statistic. A P value of less than 0.05 (95% CI) was statistically different. Results Human Studies Records from 30 consecutive inpatients who were treated with high-dose trimethoprim at Yale-New Haven Hospital between 10 July and 18 November 1992 were reviewed. No patients were excluded from this study. All patients were HIV positive and were treated for presumed or confirmed P. carinii pneumonia. Twenty-three of the patients were treated with trimethoprim-sulfamethoxazole, and seven were treated with trimethoprim-dapsone. On average, the length of the trimethoprim treatment period was 5.3 2.79 days (mean SD; range, 1 to 13 days). Figure 1 shows that the serum potassium concentration increased by 0.6 mmol/L (CI, 0.29 to 0.95 mmol/L) during treatment with trimethoprim. When the drug was discontinued, the potassium concentration decreased to pretreatment values. In 15 of 30 patients (50%), the serum potassium concentration was more than 5.0 mmol/L on at least 1 day during trimethoprim treatment. Severe and potentially life-threatening hyperkalemia (potassium > 6.0 mmol/L) occurred in three patients (10%). The serum creatinine concentration was slightly higher during trimethoprim treatment than during recovery (mean difference, 15.9 mmol/L; CI, 5.3 to 26.5 mmol/L [0.18 mg/dL; CI, 0.06 to 0.30 mg/dL]). None of the patients were taking nonsteroidal anti-inflammatory drugs, converting-enzyme inhibitors, or potassium-sparing diuretics. Figure 1. Effect of trimethoprim on serum potassium concentration in patients with AIDS. Renal and adrenal function were evaluated in seven patients during hyperkalemia (potassium > 5 mmol/L). The mean serum potassium concentration was 5.9 0.9 mmol/L, and the urinary potassium concentration was 11.3 5.8 mmol/L (mean SD). Oliguria was not present in any of the patients, and the serum creatinine concentration was not increased above baseline (mean difference, 17.7 mmol/L; CI, 9.72 to 43.3 mmol/L [0.2 mg/dL; CI, 0.11 to 0.49 mg/dL]). The transtubular potassium gradient calculated for the cortical collecting duct [11, 12] was 1.9 1.1 (mean SD). This value was low (expected range was 6 to 11) for a plasma potassium concentration of >5 mmol/L. In three patients, the transtubular potassium gradient was calculated both during and after treatment with trimethoprim. After discontinuation of trimethoprim, the transtubular potassium gradient increased to normal values in all three patients (mean difference, 4.5; CI, 1.4 to 7.5). The urinary sodium concentration was 103 65.7 mmol/L, and there was mild hyponatremia (132 2.8 mmol/L). The plasma cortisol (497 152 nmol/L, supine [18.0 5.5 g/dL, supine]); renin (0.667 0.25 ng/[L x s], supine [2.4 0.9 ng/mL per hour, supine]); and aldosterone (535 264 pmol/L, supine [19.3 9.5 ng/dL, supine]) levels were all high normal or increased during hyperkalemia. In two patients with borderline serum cortisol levels (221 and 469 nmol/L [8 and 17 g/dL]), cosyntropin stimulation test results were normal. Glucose levels were all within the normal range. Rat Studies The effect of trimethoprim on potassium and sodium excretion rates in the whole kidney is shown in Figure 2. Compared with the control group, trimethoprim decreased potassium excretion by 572 nmol/min (CI, 299 to 845 nmol/min). The reduction in potassium excretion during trimethoprim infusion was 40% (CI, 21% to 60%) of the control rate measured in period II. Although sodium excretion in the control group increased with time, trimethoprim caused a twofold larger increase in sodium excretion. The difference between these changes, 1192 nmol/min (CI, 240 to 2142), was statistically significant. The increase in sodium excretion during trimethoprim infusion was 46% (CI, 9% to 83%) of the control rate measured in period II. There was no effect of trimethoprim on urine flow or chloride excretion rate (Appendix 1). Appendix Table 1. Figure 2. The scales depict the change () in ion excretion rate with time (period II minus period I) in control and experimental (Trimethoprim) animals. Panel A Panel B Table 1 gives flow rates, lumen ion concentrations, and ion transport rates from in vivo microperfusion of distal tubules of rats. Figure 3 shows that 1 mmol/L of trimethoprim inhibited net potassium secretion by 59% (CI, 26% to 92%). The rate of net sodium absorption did not decrease. Rates of net chloride and water absorption were also not affected. Figure 3. Net potassium transport during perfusion of 14 distal tubules with control and trimethoprim (TMP) solutions. Figure 4 shows the effect of trimethoprim concent

Modification of Feedback Influence on Glomerular Filtration Rate by Acute Isotonic Extracellular Volume Expansion

The behavior of the feedback mechanism, that causes glomerular capillary pressure and filtration rate to decrease when tubule fluid flow rate through the loop of Henle of the same nephron is increased, was examined in rats before and during isotonic extracellular fluid volume expansion. The loop of Henle was perfused from the late proximal tubule at either 10 or 40 nl/min while proximal fluid was collected to measure single nephron filtration rate (SNGFR), while proximal stop-flow pressure (PSF) was measured, or while fluid was collected from the early distal tubule to assess reabsorption of fluid and electrolytes by the loop of Henle. During control periods increasing loop perfusion caused SNGFR to decrease 37%, PSF to decrease 19%, and absorption of fluid, sodium and chloride by the loop of Henle to increase. After 1 h of infusion of isotonic NaCl solution the same change in loop flow causes a 19% decrease in SNGFR and an 8% decrease in PSF. Fluid absorption by the loop of Henle did not increase with increased loop perfusion. Increases in Na and Cl absorption were similar to the increases in control periods. The smaller decreases in SNGFR and PSF indicate that acute volume expansion decreases the sensitivity of the feedback response. The mechanism of this decrease in gain could involve interference with local generation or action of angiotensin, or a change in the composition or pressure of interstitial fluid tending to dilate the pre-glomerular resistance vessels.

Calcium Transport by the Proximal Tubule

Taken together the results of these in vivo microperfusion experiments indicate that calcium absorption by the proximal tubule depends on more than one transport mechanism. We have observed that net calcium flux is affected by changes in calcium ion activity (even with constant total calcium concentration) and in transepithelial voltage. This sensitivity of calcium flux to changes in electrochemical driving force points to a diffusional component of calcium transport. Ng et al. (1984) have recently concluded that simple diffusion accounts for the majority of calcium absorption by superficial proximal convoluted tubules of the rabbit. The pathway for this diffusional component may involve paracellular channels. The permeability of this pathway appears to be as high for calcium as it is for sodium, potassium and chloride. Calcium flux is also affected by changes in osmotic water flow. The effect of changes in volume flow on calcium transport occurs even in the absence of concentration changes in bulk solutions. Thus, it does not appear to be the result of changes in passive driving forces secondary to dilution or concentration of tubule fluid. At present we are not able to distinguish between two other possible mechanisms: solute polarization in a microscopic unstirred fluid layer adjacent to the cell membrane, or true entrainment of calcium in the stream of osmotically driven water flow (solvent drag). Either mechanism could provide an additional component of total calcium transport independent of changes in bulk phase ion concentrations and electrical driving forces. A third component of total calcium absorption appears to involve active transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of potassium ion-exchanger electrode for microanalysis.

Numerous constituents of renal tubule fluid have been measured in vitro after collecting small volumes in micropipets. Some of these (hydrogen, sodium, and potassium ions) have also been measured in vivo by direct puncture of surface tubules with micro-electrodes (1–6). Similarly the intracellular ionic composition of renal tubule cells has been estimated chemically in digests of renal tissue by several workers (7–9) and, recently, some results of direct measurements with potassium sensitive electrodes have been reported (6,9). Knowledge of cellular and luminal potassium concentrations in mammalian renal tubules, especially in the distal portion of the nephron, is crucial to an understanding of the mechanisms by which potassium is transported and electrical potentials are generated. In the course of attempting in vivo measurements of intracellular and intratubular potassium activity we have been confronted by the special problems attending measurements in the mammalian distal tubule.

Chapter 6 Overview: Renal Potassium Transport along the Nephron

Publisher Summary This chapter discusses the renal potassium (K) handling along the nephron. Renal mechanisms for excretion of K indicate that K excretion depends on secretion of K at a distal tubule site and that the secretory process behaves as though it involves an exchange of reabsorbed Na for secreted K. The idea that a distal secretory process provides the major route by which K gains access to the urine and is ultimately excreted has been confirmed in several respects. The rate of distal secretion is variable and is changed from near zero to rates exceeding the K filtration rate by factors that change urinary excretion in the same direction. K is not totally reabsorbed by the proximal tubule and by the ascending limb of the loop of Henle, but the quantity of K left unreabsorbed at the beginning of the distal secretory region does not vary over as wide a range as the quantity reaching the collecting duct or the final urine.