Analysis of improvements, full responses, remission and toxicity in rheumatoid patients treated with step-up combination therapy (methotrexate, cyclosporin A, sulphasalazine) or monotherapy for three years.

OBJECTIVE: To evaluate two monotherapies followed by step-up combination therapy with two or three complementary drugs in active rheumatoid arthritis (RA) in comparison with sulphasalazine (SSZ) alone. METHODS: One hundred and twenty-six consecutive patients with early active RA were enrolled in this open controlled clinical trial. The primary end-point was 50% improvement according to the ACR criteria (ACR50) at 6, 12 or 18 months. The secondary end-points were a full response (Magnusson criteria) and/or remission (ACR criteria) at 3 yr. Methotrexate (MTX) (group 1), cyclosporin A (CsA) (group 2) or SSZ (group 3) was used first. After 6 months, a combination of two drugs (CsA and MTX) was employed in groups 1 and 2. SSZ was added after 12 months if improvement was less than ACR50 with the combination. Group 3 continued with SSZ alone. RESULTS: After 6 months, 57% of patients in group 1, 31% of group 2 (MTX vs CsA, P=0.002) and 33% of group 3 (MTX vs SSZ, P=0.01) had reached ACR50 improvement according to intention-to-treat analysis. At month 12 after starting a drug combination, 67% of group 1 and 76% of group 2 had reached ACR50 compared with 24% of group 3. At the 18-month follow-up, 90% of group 1 and 88% of group 2 but only 24% of group 3 had reached ACR50. After 18 months, 62% of group 1, 60% of group 2 and 48% of group 3 showed side-effects and three, five and eight patients in the three groups respectively had dropped out of the study. At the 3-yr follow-up, 9% of the patients in groups 1 and 2 and 7% of group 3 were in remission according to the ACR criteria; according to the Magnusson criteria, 40% showed a full response in groups 1 and 2 but only 21% did so in group 3. CONCLUSION: MTX appears to be the fastest-acting agent. A step-up approach with MTX plus CsA plus SSZ led to a 50% improvement according to the ACR criteria in most patients. After 3 yr, 40% of patients receiving combination therapy and 21% of patients receiving monotherapy showed a full response, while 9 and 7% respectively attained remission.

Effect of endothelin 1 on proximal reabsorption and tubuloglomerular feedback.

Endothelin 1 (ET-1) is a powerful constrictor of the afferent glomerular artery, implicated in the occurrence of both functional and acute renal failure. Besides being produced by the endothelium, ET-1 is also secreted by proximal tubular cells, suggesting that it may act as an endoluminal messenger. The present study is intended to verify whether ET-1 may play a role in the tubuloglomerular feedback system. The experiments were performed in rat superficial glomeruli. In 25 nephrons we measured by the total collection technique the single-nephron glomerular filtration rate (SNGFR; nl/min) and reabsorption rates before (control) and during microinjection (MIJ) of ET-1 10(-7) M into the first proximal convolution or Bowman's space. The SNGFR rose from 27+/-3 to 61+/-11 nl/min (p<0.01), the percent proximal reabsorption rose from 43 to 74%, and the absolute reabsorption rose from 13+/-2 to 46+/-11 nl/min (p<0.01). In additional 23 nephrons the collections were performed at the earliest distal convolution accessible on the renal surface, while MIJ was performed in the last proximal convolution of the same nephrons. The SNGFR rose during MIJ from 22+/-3 to 40+/-6 nl/min (p<0.01), the percent reabsorption rose from 61 to 66% (p>0. 77), and the absolute reabsorption rose from 12+/-2 to 26+/-4 nl/min, (p<0.003). Exposure of the macula densa to intraluminally injected ET-1 causes an abrupt increase in SNGFR of the experimental nephron, in the absence of changes in systemic and renal hemodynamics. During proximal MIJ, ET-1 may have reached the macula densa during the time preceding the beginning of collection and interruption of the delivery of fluid to the distal nephron. ET-1 directly stimulates fractional and absolute volume reabsorption along the proximal tubule. Proximal secretion and/or filtration of ET-1 could represent a physiological mechanism to activate the tubuloglomerular feedback, eliciting a response opposite to that triggered by systemic and intrarenal infusion.

Systemic and intratubular effects of cyclosporin-A and tacrolimus on the rat kidney.

Cyclosporin-A and tacrolimus can cause hypertension and renal failure through endothelin receptors. The importance of tubular function was never investigated. The aim of this study was to compare the effects of intratubular injection of cyclosporin-A and tacrolimus with effects observed during systemic infusion. In 20 rats, either cyclosporin-A or tacrolimus was infused, 30 and 1 mg/kg i.v., respectively, in 30 min. Before and after administration, glomerular filtration rate, single nephron filtration rate, proximal and distal absolute reabsorption and percent reabsorption were measured by clearance and micropuncture techniques. In 22 other rats, single nephron filtration rate, absolute reabsorption, percent reabsorption, were measured at the last proximal and early distal tubules before and during intraluminal microinjection of either cyclosporin-A or tacrolimus. During cyclosporin-A and tacrolimus i.v. infusion, glomerular filtration rate fell from 536+/-43 to 448+/-37 microl/min (P<0.026) and from 408+/-33 to 284+/-81 microl/min (P<0. 02), single nephron filtration rate from 26.4+/-2.0 to 20.6+/-1.9 (P<0.002) and from 21.6+/-2.2 to 17.4+/-2.0 nl/min, respectively (P<0.02). The last proximal absolute reabsorption remained unchanged with cyclosporin-A (16.8+/-2.2 vs. 15.1+/-1.7 nl/min, P>0.444), but was slightly reduced by tacrolimus (14.4+/-1.7 vs. 11.3+/-1.7 nl/min, P<0.05). During microinjection, single nephron filtration rate was increased by cyclosporin-A (20+/-1 vs. 63+/-8 nl/min, P<0.0001), and tacrolimus (from 17+/-2 to 49+/-9 nl/min, P<0.0001), and so was reabsorption, independent of the sampling site. Cyclosporin-A and tacrolimus, indeed, raise single nephron filtration rate directly when injected intraluminally. Since this effect occurs in the direction opposite to that recorded during systemic infusion, it must be mediated through different pathways. The i.v. infusion of cyclosporin-A, but not tacrolimus, impairs glomerulo-tubular balance.

Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration.

Secretin is a vasoactive peptide capable of acting on transmembrane volume fluxes. We measured nephron filtration (SNGFR) and resorption during secretin microinjection (MIJ) into the tubular lumen or microperfusion (MP) into peritubular capillaries. In 24 rat nephrons, SNGFR, measured by collections from the distal tubule, rose from 25+/- 4 to 61+/-8 nl/min during MIJ of saline containing secretin 10(-9) M into the last convolution of the proximal tubule (LP). Percent and absolute resorptions rose from 70 to 90% and from 20+/-4 to 56+/-8 nl/min, respectively. During MIJ of secretin, 3 x 10(-)(9) M into the first convolution of the proximal tubule, SNGFR, measured at LP, rose from 32+/-4 to 61+/-8 nl/min, percent and absolute reabsorptions from 52+/-4 to 78+/-3% and from 16+/-2 to 50+/-7 nl/min, respectively (n = 30). During MP of secretin, 1.5x10(-9) M, SNGFR fell from 39+/-6 to 15+/-4, resorption from 19+/-4 to 9+/-2 nl/min, while percent resorption rose from 43+/-6 to 59+/-5% (n = 15). While all MIJ and MP changes were significant (P<0.001), paired pre- versus post-MIJ and MP values were not. Secretin is a powerful vasoconstrictor when perfused into peritubular capillary blood, unlike systemic and intra-arterial injections. When injected into the tubular lumen, it up-regulates SNGFR and increases reabsorption directly.

The effect of colchicine on proximal tubular reabsorption.

Aquaporins, expressed in the brush border membrane (BB) could play a pivotal role in glomerulo-tubular balance (GTB) by effecting adaptive changes of water permeability to the variations in the load of filtered solutes. Since aquaporin expression is modulated by microtubule-dependent trafficking between endoplasmic reticulum and cell membranes, we used the microtubule poison colchicine to assess the importance of aquaporins in mediating GTB. The effects of colchicine 1.6x10(-4)m on proximal tubule volume reabsorption was tested on 48 nephrons of ten rats by micropuncture techniques. Thirty proximal tubules were sampled from the last proximal convolution before, and recollected during and again after the microinjection (MIJ), into the early proximal convolution or Bowman's space, of colchicine added to a Ringer solution. We studied 18 proximal tubules in the same way before, during and after the microperfusion (MP) of colchicine added to an ultrafiltrate of plasma into the peritubular capillaries. During MIJ, SNGFR did not change significantly from baseline (17.7+/-1.3 vs 20.9+/-1.8 nl min(-1), P>0.12). Post-control values were superimposable upon their paired pre-MIJ controls, when available (15.8+/-1.3 vs 13.5+/-1.5 nl min(-1), P>0.25). The measurements of percentage reabsorption (49+/-5 during baseline, 45+/-7 during MIJ, and 55+/-5 in post-control, P>0.6) and absolute reabsorption (8.1+/-0.7, 11.1+/-2. 2, and 7.9+/-1.3 nl min(-1), respectively, P>0.18) were also unchanged. The three average measurements obtained in control conditions, during MP and again in post-MP control were not significantly different for SNGFR (19.8+/-3.0, 20.0+/-4.7, and 20. 2+/-3.5 nl min(-1), P>0.48), percentage (55+/-3, 59+/-5, and 47+/-3%, P>0.35) and absolute reabsorptions (12.5+/-2.2, 12.4+/-4.6, and 9. 4+/-1.0 nl min(-1), respectively, P>0.42). MIJ and MP of vehicles were devoid of any measurable effect. Colchicine does not acutely affect volume reabsorption in the proximal tubule. Aquaporin trafficking, if any, is not involved in mediating glomerulotubular balance in the proximal tubule, although aquaporin expression and function could still be important, although regulated by mechanisms different from microtubule-dependent shuttling between endoplasmic reticulum and BB.

The influence of furosemide on free water clearance.

BACKGROUND: Laboratory measurements of osmolality and electrolyte concentrations are useful as a source of clinical and pathophysiologic information on kidney function. We obtained different conditions of baseline diuresis in 54 rats, which were then treated with furosemide 10 mg/kg. From measurements of plasma (P) and urine (U) osmolality and Na, we calculated the free water clearance (CH2O) and the contributions of Na+ and non-Na(+)-solutes to its changes during diuretic administration. RESULTS: The results show that furosemide abolishes both urine diluting and concentrating ability, by reducing the contribution of non-Na(+)-solutes to Uosm and the formation of CH2O, while the contribution of Na+ to Posm remains unchanged. During furosemide the urines approach isosmoticity irrespective of the baseline Uosm with an asymptomatic function similar to that imposed by osmotic diuresis. It is suggested that the overflow to the concentrating sites of the nephron overwhelms their normal transport capacity independently of their baseline water permeability. The disruption of the transepithelial osmotic gradient caused by the drug impairs the transepithelial osmotic flow, leading to the excretion of isosmotic urines. CONCLUSIONS: The effect of non-Na(+)-solutes in the laboratory measurement of urine constituents can be reduced to that of plasma determination by diluting the urines according to empiric formulas derived from the present data.

Tubular reabsorption and sodium excretion during urine reinfusion.

BACKGROUND: The mechanisms responsible for the natriuresis that follows urine reinfusion was investigated in rats by clearance and micropuncture techniques. METHODS: In each animal two urine reinfusion periods (R1 and R2) were performed and compared to a non-urine-reinfusion, saline infusion period (S) sandwiched between them. RESULTS: Switching from urine reinfusion to an equivalent rate of saline loading was followed by a fall in Na excretion from 1.9 +/- 0.5 to 0.5 +/- 0.2% of filtered load, p < 0.002. Urine osmolality rose, and urine to plasma inulin concentration ratio rose significantly from 73 +/- 14 to 147 +/- 21 (p < 0.002). The changes in GFR, SNGFR, absolute and percent proximal reabsorption could not account for these findings. A reduced Na excretion coupled to increased urine osmolality indicates enhanced transport along a segment responsible for the urinary concentrating mechanism. Thus the data can be interpreted then as due to enhanced reabsorption along the ascending limb of Henle's loop. These changes were reversed by reinstituting urine reinfusion after the S period. The consensual changes in Na+ and K+ excretion excluded an effect of urine reinfusion on the distal exchange site. There was a continuous fall in proximal reabsorption from R1 (76 +/- 3%) to S (69 +/- 3%) to R2 (62 +/- 5%) which was inversely correlated with the changes in hematocrit (R = 0.49, p < 0.026). This indicates that part of the late diuresis and natriuresis was due to volume expansion. An osmotic effect of reinfused urine solutes was suggested by a late rise in plasma osmolality, from 312 +/- 13 to 323 +/- 8 mOsm/kg. Osmotic diuresis could have exerted additive effects upon those of volume expansion, accounting for the late fall in proximal reabsorption. CONCLUSIONS: We conclude that the acute effects of urine reinfusion are due to changes in transport of solutes and permeability to water along distal tubular segments. The changes in plasma osmolality during the last period of the present acute experiments, suggest the possibility that solute retention may be linked to the chronic effects of urine reinfusion.

Measurement of nephron filtration from distal and proximal tubules.

Nephron filtration rate (SNGFR), measured proximally by micropuncture using the technique of total collection of tubular fluid, may be spuriously high as the fall in intratubular pressure during collection may increase the net filtration pressure. Alternatively, the interrupted distal delivery of tubular fluid during proximal collection may up-regulate SNGFR by activating the tubulo-glomerular feedback. The accuracy of nephron filtration measurements was tested in 65 rats, by sampling from the distal and then from the proximal tubule of the same nephrons. Collections were also made in the presence of high concentrations of furosemide at the dose of 10 mg/kg i.v. In 290 nephrons, distal SNGFR was not significantly different from the paired measurements obtained from the proximal tubule (39+/-1 vs 39+/-1 nl/min, p > 0.83). During furosemide, the paired data were 42+/-2 vs 41+/-2 nl/min, p > 0.82, n=92. Different hydration conditions did not influence the results. The correlation between distally and proximally measured filtration rates held for the whole range of measurements (R=0.56; p < 0.0001). Under the present experimental conditions, SNGFR is not spuriously high when measured by total blockade of the proximal tubule. The accuracy of measurements and their reproducibility is not influenced by the site of sampling.

The site of action of furosemide.

To establish whether furosemide (F) acts on the proximal tubule beside the thick ascending limb of Henle's loop, we reviewed the data from 55 rats studied before and during the i.v. infusion of 10 mg kg-1 of furosemide. These animals were the object of previously published studies. SNGFR was 42.6+/-1.1 nl min-1 during baseline conditions (B), 42.7 nl min-1 during F (P>0.9). In 151 paired last proximal tubular samples, percentage reabsorption was 71.72 before and 71.72% after F (P=1.0). In 64 paired early distal tubular samples, percent reabsorption fell from 86% in B to 78% during F (P<0.0001). During F, the urine flow rate expressed as a percentage of GFR (24+/-2%) was significantly correlated (R=0.52, P<0.0001) with the percent delivery of filtrate out of the proximal tubule (26+/-3%). These data demonstrate that, even at this very high dosage, F does not act on the proximal tubule. It inhibits transport on the ascending limb of Henle's loop. By allowing the delivery of isotonic tubular fluid to the distal tubule, it causes a fall in volume absorption along the early distal convolution in proportion to its baseline water permeability. It increases urine flow by abolishing the interstitial hypertonicity and, consequently, the osmotically driven solvent flow across the distal epithelium and collecting ducts. Therefore the urine flow rate during F closely approximates volume delivery out of the proximal tubule.

The effect of reducing proximal tubular fluid delivery on the rate of filtration of single nephrons.

The rate of delivery of tubular fluid from the proximal tubule (PT) is thought to reset nephron filtration rate (SNGFR). In micropuncture experiments in rats we tested this hypothesis by reducing the efflux from the PT by simultaneously "double collecting" (DC) tubular fluid from the early distal tubule (DT) and from the last convolution of the PT of the same nephrons. SNGFR measured by total collection of tubular fluid was 34 +/- 3 nl/min at the DT and 34 +/- 3 nl/min at the PT (p > 0.97, n = 42). The simultaneous collection from proximal and distal sampling site was performed between these two paired measurements. It yielded an average SNGFR of 40 +/- 3 nl/min (p < 0.02). This may be due to the collection, at the distal site, of the extra amount of inulin stored between distal and proximal pipette, prior to starting the aspiration of tubular fluid. Since this error would decrease in longer collections, the difference in SNGFR between single and double collections was plotted against the duration of collections. In fact it was negatively correlated with the sampling time (p < 0.01), indicating no difference in SNGFRs for collections > 4 minutes. Reduction and complete interruption of the delivery of native proximal tubular fluid to the Macula Densa does not seem to influence the measurement of SNGFR. Filtration rate is not significantly different when measured within few minutes at the DT and PT of the same nephrons.

Proximal reabsorption with changing tubular fluid inflow in rat nephrons.

The relative contribution of intraluminal versus peritubular factors in mediating glomerulo-tubular balance (GTB) is still controversial. We modulated the load of tubular fluid to the proximal tubule of single nephrons of rats by injecting oil into the efferent arteries (EAO). In fifty nephrons the changes in reabsorption induced by obstruction occurred in the same direction as, and were significantly correlated with, the simultaneous changes in single nephron glomerular filtration rate (SNGFR) (y = -0.54 + 0.92x, R = 0.91, P < 0.0001). In an additional set of thirty-nine nephrons the load of tubular fluid was changed, during EAO, by partial collection from Bowman's space or from the early proximal convolution. Thus, the rate of tubular fluid delivery along the proximal tubule was changed in an experimental situation that prevented any modification in the oncotic pressure of peritubular capillaries. The changes in proximal deliveries during this experimental condition were significantly correlated with those during reabsorption (y = -2.87 + 0.71x, R = 0.82, P < 0.0001). These data demonstrate that GTB is fully expressed even when the native peritubular environment is kept constant while the rate of perfusion of proximal tubular segments with native tubular fluid is changed.

The validity of the recollection technique in micropuncture experiments on the rat kidney.

We tested the accuracy of the micropuncture technique by performing total collections of tubular fluid followed by immediate recollections from the same site. We studied thirty-one Wistar rats under different conditions of hydration and during maintenance infusions. To assure constancy of body fluid volumes the experiments were performed during continuous reinfusion of urine. Overall we performed 190 paired collections and immediate recollections from 147 proximal and 43 distal tubules. The mean values measured during the first collection were not different from the paired means obtained by recollection. Mean values (+/- S.E.M.) for first collection and recollection were: single nephron glomerular filtration rate (SNGFR), 34.7 +/- 1.3 vs. 34.2 +/- 1.3 nl min-1, P > 0.42; percentage reabsorption, 74 +/- 1 vs. 73 +/- 1, P > 0.53; absolute reabsorption, 26.4 +/- 1.2 vs. 25.9 +/- 1.2 nl min-1, P > 0.47; collection rate, 8.4 +/- 0.6 vs. 8.3 +/- 0.4 nl min-1, P > 0.92. When subdivided according to different physiological conditions and sampling sites, the results for the paired collection-recollection means were still not significantly different. The regression between collected and recollected SNGFRs had a slope of 0.96, r = 0.85, P < 0.0001. In order to exclude the possibility that urine reinfusion per se could obscure putative differences in collection-recollection pairs, the effect of urine reinfusion was separately evaluated. We measured the differences obtained in collection-recollection pairs before and during urine reinfusion in twenty-eight tubules from eleven rats, a sample size that allows the detection of 10% difference as significant. While SNGFR did not change, percentage reabsorption fell significantly from 76 +/- 3% before infusion to 60 +/- 3% during reinfusion, P < 0.003. We conclude that the recollection technique yields a reproducible estimate of SNGFR and tubular reabsorption, independent of the sampling site and of the technique used for fluid maintenance. Thus, it can be used to study the effect of different manoeuvres on the rate of proximal tubular transport. Urine infusion per se depresses proximal transport compared with that measured during maintenance with equivalent amounts of isotonic saline.

Consistency of nephron filtration measurements by collection of total tubular fluid from different proximal sites.

The stability of single nephron glomerular filtration rate (SNGFR) is assured by specific mechanisms such as the tubulo-glomerular feedback system and autoregulation. Studies on renal physiology rely heavily on the measurements of SNGFR, which are feasible only in animals. The measurement of SNGFR by collection of total tubular fluid may be influenced by the fall in intratubular hydrostatic pressure that may reflect the negative pressure applied to the sampling pipette. This effect may become more important with shortening of the distance between the sampling site and the Bowman space. We analysed this putative effect by performing collections of total tubular fluid from the late proximal (LP), and then from the early proximal (EP) segment of the same nephrons. In 128 paired collections LP-SNGFR averaged 35 (SEM 2) nl x min(-1), and was no different from the paired mean EP-SNGFR of 37 (SEM 2) nl x min(-1), P > 0.179. Then EP- and LP- SNGFR were significantly correlated (r = 0.77, P < 0.001). As expected, the respective paired means of absolute and percentage reabsorptions, and those of collection rates were significantly different. The average SNGFR computed from each LP and EP paired measurement was significantly correlated with the simultaneously measured kidney glomerular filtration rate, GFR (r = 0.60, P < 0.0001). The ratio of GFR to SNGFR indicated the expected number of glomeruli. These data would indicate that the sampling site does not influence the measurement of SNGFR in the proximal tubule when the total fluid collection technique is correctly performed. They also exclude a time-dependent activation of the macula densa capable of upregulating SNGFR within the interval elapsing between the beginning of LP and the completion of EP collections, which in our study averaged 4.4 (SEM 0.1) min.

Microinjection studies on the effect of furosemide on the rat nephron.

The tubular site of furosemide (F) action was studied by the technique of diuretic microinjection (MIJ) into proximal tubules of the rat nephron. F was injected into the last proximal superficial loop of 51 proximal tubules, at the concentration of 3 x 10(-4) mol/l in an infusate that contained 14C-inulin. Collections were performed at the early distal tubule before and after MIJ. Single nephron filtration rate (SNGFR) remained unchanged, while the percent of filtered volume reabsorbed up to the site of collection was 85+/-2 before, 79+/-2% after MIJ, p < 0.005. The calculated concentration of F in the collected distal tubular fluid during the post-MIJ measurements averaged 3 x 10(-5) mol/l. This experiment was repeated by injecting F into the early proximal convolutions of 43 nephrons, while the collections were performed at the last proximal segments. In these studies of proximal volume absorption, SNGFR was 34+/-3 before, 35+/-4 nl/min after F (p > 0.6). The respective percent reabsorptions were 70+/-3 and 73+/-3% (p +/- 0.3). In order to determine whether the technique per se was suitable to detect changes in reabsorption, the proximal MIJ study was repeated by using the carbonic anhydrase inhibitor dichlorphenamide 3 x 10(-5) mol/l in the microinjectate: while SNGFR remained unchanged, percent reabsorption fell from 63+/-5 to 45+/-7% during injection of the diuretic, p < 0.03. We conclude that the technique is adequate to examine the effects of drugs, and that F does not reduce proximal volume absorption at concentrations of 3 x 10(-5) mol/l. The loop diuretic decreases distal volume absorption by abolishing the osmotic gradient between blood and tubular fluid along the early distal convoluted tubule.

Segmental reabsorption measured by micropuncture and clearance methods during hypertonic sodium infusion in the rat.

BACKGROUND: We wanted to validate by direct measurements in rat tubules a technique used to calculate segmental volume absorption by each segment of the human nephron. METHODS: Experiments were performed on 17 rats during hypertonic Na infusion prior to and after frusemide administration. Tubular samples were taken from early distal and last proximal sites. The rate of filtration of single nephrons (SNGFR) was calculated by the technique of total collection of tubular fluid using labelled inulin as a marker. Reabsorption was computed by the tubular fluid to plasma (TF/P) inulin concentration ratio. RESULTS: SNGFR was 50 +/- 4 nl/min at the distal (n = 82), 51 +/- 3 nl/min at the proximal sampling site (n = 112, P > 0.65) during baseline conditions. Percent reabsorptions were 85 +/- 1 and 69 +/- 2% respectively (P < 0.0001). During frusemide these values were 52 +/- 6 nl/min and 76 +/- 2% at the distal, 49 +/- 5 nl/min and 66 +/- 2% at the proximal site. In 83 paired proximal collections, fractional (68 +/- 1 versus 67 +/- 1% P > 0.32), absolute reabsorption (34 +/- 2 versus 33 +/- 2, P > 0.50) and SNGFR (50 +/- 2 nl/min versus 50 +/- 3 nl/min, P > 0.99) were not different between baseline and frusemide. In 25 re-collections from the distal tubule these same values were 83 +/- 2% versus 76 +/- 2%, and 48 +/- 4 nl/min versus 55 +/- 6 nl/min respectively. Very similar results were obtained in 55 paired distal-proximal collections during baseline and 42 such pairs during frusemide. In the presence of the diuretic the fractional urine excretion was significantly correlated (R = 0.83, P < 0.0001) with fractional proximal delivery, Na+ resorption by Henle's loop was 22 +/- 2% calculated from clearance data and 23 +/- 1% of GFR from micropuncture data respectively. They were not significantly different (P > 0.70) and were significantly correlated (R = 0.57, P < 0.02). CONCLUSIONS: These data demonstrate that frusemide does not act proximally and that delivery beyond the proximal tubule approximates urine flow rate during the action of the drug. The values of segmental reabsorption along the nephron computed on clearance measurements are superimposable upon those obtained directly by micropuncture.

Micropuncture and clearance measurements of segmental reabsorption by the rat nephron.

BACKGROUND: We wanted to verify whether the calculations of segmental tubular reabsorption obtained during water diuresis were supported by direct micropuncture measurements. METHODS: Experiments were performed on 18 rats during baseline water diuresis (B) and after the administration of frusemide (F), 10 mg/kg, by whole-kidney clearance measurements and micropuncture collections from early distal (ED) and last proximal (LP) tubular segments. RESULTS: GFR was 957+/-79 in B, 1053+/-77 microl/min in F, P>0.013. SNGFR was 38+/-1 in 166 and 38+/-1 nl/min in 165 tubules respectively, P>0.77. In LP collections the percentage reabsorption was 71+/-2 in B and 76+/-2% during F (P>0.07) in 99 and 95 samples respectively. The absolute proximal reabsorption was not changed by F (27.6+/-1.5 versus 27.7+/-1.3 nl/min, P>0.96) The data were superimposable when the analysis was restricted to paired data. The difference between ED and LP resorption was 17+/-3 during B and fell significantly (P<0.008) to 5+/-3% during F, measured by clearance techniques, and the percentage of GFR excreted during F, measured by clearance techniques, and the percentage delivery of filtrate beyond the proximal tubule, measured independently by micropuncture, were not different (27+/-2 versus 24+/-2%, P>0.10), while they were significantly correlated (P<0.04). The calculations of segmental Na reabsorption along the different nephron segments by clearance techniques were not significantly different from and were significantly correlated with the reabsorptions measured directly by micropuncture. CONCLUSIONS: The present experiments validate the calculations of reabsorption by techniques applicable to human studies of clinical physiology.

The effect of intraluminal flow rate on glomerulotubular balance in the proximal tubule of the rat kidney.

To differentiate intraluminal from peritubular factors in mediating the glomerulotubular balance, single nephron filtration rate (SNGFR) and reabsorption were measured by total collection from the last proximal segment before, and during, externally applied mechanical compression of an early proximal convolution of ninety-three rat nephrons. SNGFR fell from 55.6 +/- 2.3 nl min-1 during control conditions to 34.5 +/- 2.0 nl min-1 during compression (P < 0.0001) in sixty tubules ('responders'). Absolute reabsorption fell from 41.8 +/- 2.0 to 28.4 +/- 1.8 nl min-1 (P < 0.0001), while percentage reabsorption rose from 75 +/- 2 to 82 +/- 2% (P < 0.0001). These effects were reversible and independent of whether the compression was applied before or after the control collection. In thirty-three proximal tubules the compression procedure was not successful ('non-responders'), SNGFR remaining unchanged (36.1 +/- 2.9 vs. 36.9 +/- 2.9 nl min-1, P > 0.3). Absolute and percentage reabsorptions rose slightly, albeit significantly, from 26.1 +/- 2.1 to 30.7 +/- 2.4 nl min-1 (P < 0.0001) and from 75 +/- 3 to 85 +/- 2% (P < 0.0001). Thus, the response of reabsorption to changes in intraluminal flow is composed of two different adaptive mechanisms. The slowing of flow is present in both sets of tubules and causes a slight increase in resorption; the change in filtration per se is present only in the first set of responders, and causes an SNGFR-dependent consensual change in resorption.

Micropuncture study of the effect of furosemide on proximal and distal tubules of the rat nephron.

The tubular effects of furosemide were studied by micropuncture and clearance techniques on 20 rats. Collections of tubular fluid (TF) from early distal (ED) and late proximal (LP) segments of the same nephrons and of different nephrons were performed during baseline conditions. Re-collections were taken from the same sites and new collections from different nephrons after 10 mg/kg furosemide. The glomerular filtration rate (GFR) was 1,309 +/- 212 microliters/min during baseline, and 1,348 +/- 199 microliters/min after furosemide (p > 0.89); while the urine flow rate rose from 36 +/- 8 to 167 +/- 30 microliters/min (p < 0.001). The nephron filtration rate (NFR) was not different in 46 paired distal (33.3 +/- 2.6 nl/min) versus proximal samples (34.2 +/- 2.9 nl/min, p > 0.72), neither was it different during baseline (37.2 +/- 1.4, n = 120) as compared to furosemide (37.2 +/- 2.7, n = 91, p > 0.99). The percent reabsorption (PR) at the ED sampling site was 87 +/- 4% during baseline, and 89 +/- 3% in 13 paired samples during furosemide (p > 0.47). PR at the LP sampling sites was 83 +/- 2% during baseline, and 80 +/- 2% in 26 paired samples during furosemide (p > 0.63). In 31 paired ED-LP collections, PR was 82 +/- 4 (ED) versus 72 +/- 4% (LP) during baseline, and 87 +/- 3 versus 74 +/- 6%, respectively, during furosemide. The respective collection rates were 4.6 +/- 1.0 versus 9.5 +/- 1.3 nl/min during baseline (p < 0.0001), 5.8 +/- 2.3 versus 8.7 +/- 3.0 nl/min during furosemide. The LP-ED differences obtained during baseline were not different from those measured during furosemide for the collection rate, PR and NFRs. The absolute LP resorption rate was not significantly different during baseline as compared to furosemide. Thus, furosemide did not affect the difference between ED and LP collection sites in collection rate, absolute and fractional reabsorption, in the absence of changes in GFR and NFR. These data indicate that furosemide acts solely along Henle's loop, where it blocks Na+ transport. The urine flow rate rises during furosemide because water abstraction along the distal tubule is reduced by the isotonicity of ED TF, and along the collecting ducts by the isotonicity of the medullary and papillary interstitium caused by the diuretic. We conclude that under the conditions of the present study, furosemide has no proximal effect.