Tubuloglomerular feedback responses to acute contralateral nephrectomy R. C. BLANTZ,
0. W. PETERSON,
AND
S. C. THOMSON
Department of Medicine, University of California, San Diego, School of Medicine, La Jolla 92093; and San Diego Veterans Affairs Medical Center, La Jolla, California 92161
BLANTZ, R. C., 0. W. PETERSON,AND S. C. THOMSON. Tubuloglomerukzr feedback responses to acute contralateral nephrectomy. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F749-F756,1991.-After unilateral nephrectomy adaptive events must occur in the remaining kidney within the first 12-14 h in anticipation of an increasein glomerular filtration rate (GFR) and eventual renal hypertrophy. Utilizing micropuncture and microperfusion techniques in the rat, we have examined tubuloglomerular feedback (TGF) and single-nephron GFR (SNGFR) responseswhile the late proximal tubule was microperfused [late proximal tubule flow (V&l from 0 to 40 nl/min in 10 nl/min intervals at 2-4 and 12 h after contralateral nephrectomy. Urinary excretion increased,but SNGFR derived from distal collections was reduced, and early distal flow rate remained constant 2-4 h after nephrectomy. The operating point was shifted, suggestingactivation of TGF. The turning point half-maximal activity (VA) and slope were not statistically different when all nephron data were submitted to a curve-fitting procedure, but group mean data suggesteda quantitatively lower VN and steeper slope of the TGF profile. Twelve to fourteen hours after contralateral nephrectomy, valuesfor SNGFR at all microperfusion rates were increased, as were late proximal and early distal flow rates. The values for VIA and slope of TGF were not statistically different from control values. We conclude that TGF activity and sensitivity are not suppressedat 2 and 12 h after nephrectomy. Increased urinary excretion does not require TGF alterations. Changes in TGF may be adaptive to increasesin SNGFR and may not be causalto the increasein filtration rate after nephrectomy. glomerular filtration; tubular reabsorption AFTER REMOVAL of one kidney the remaining organ undergoes a prompt increase in whole kidney glomerular filtration rate (GFR) over a relatively short time period and a major increase in renal size (4, 6, 7, 9, 18). After contralateral nephrectomy the reabsorptive capacity of the kidney, as well as its excretory rate, increases considerably, and this increase in tubular reabsorption occurs at least in proportion to the increase in GFR (6, 12). These events occur prior to demonstrable hypertrophy, and the hypertrophy is primarily within tubular structures (7). The events that transpire after contralateral nephrectomy suggest that improved excretory function is not the only goal but rather improved reabsorptive capacity that represents an attempt to duplicate the filtration rate and reabsorptive capacity that existed when two kidneys were present. The temporal sequence of adjustments in GFR, tubular reabsorption, and increased urinary excretion has not been well defined.
Increased tubular reabsorptive capacity and increased GFR must be maintained in balance, and this coordinated process may play a significant role in contributing to renal hypertrophy. Under normal circumstances there exists a tight relationship between tubular reabsorption and GFR, as a conseque rice of both 1) gl .omerulotubular balance, whereby tubu lar reabsorption adapts to alterations in load into each nephron segment (18) and 2) tubuloglomerular feedback mechanisms (16, 21). The tubuloglomerular feedback system has been well defined over-the past several decades, utilizing micropuncture and tubular microperfusion techniques. Knowledge derived from several experimental studies suggests that increases in delivery of fluid to the distal nephron will elicit vasomotor responses, which decrease the filtration rate (1, 2,5, 16, 20,- 21, 23). For the GFR and single-nephron GFR (SNGFR) to rise after contralateral nephrectomy while flow to the distal nephron concurrently increases, adaptations in the tubuloglomerular feedback system must occur to permit this increase in filtration rate (1, 5, 10). Such adaptations of tubuloglomerular feedback mechanisms may be critical to the initial processes, which lead to increased GFR and tubular reabsorption and may be a requirement for the initiation of compensatory hypertrophy. These studies have been designed to examine the early phases of filtration and reabsorptive responses and the corresponding adaptations of the tubuloglomerular feedback mechanism 2 and 12 h after contralateral nephrectomy. METHODS
Experiments were performed in male Munich- Wistar rats of 200-275 g body wt, derived either from animals housed and raised at the Animal Research Facility at the San Diego Veterans Affairs Medical Center or purchased from a commercial supplier (Simonsen Labs, Gilroy, CA). Rats were fed a regular rat chow (24% protein) until 16 h before micropuncture evaluation and then were given free access to tap water up to the time of micropuncture. A right nephrectomy or sham procedure was performed at either 2 or 12 h before micropuncture evaluations. In 12-h nephrectomy studies animals were allowed access to food before the time of the brief surgical procedure. Animals were anesthetized with Brevital(lO0 mg/kg ip), a short-acting barbiturate anesthetic; under sterile conditions the right kidney was dissected free from its periF749
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renal fat, and the pedicle was exposed. The right renal artery and renal vein were doubly ligated with silk suture material as was the ureter, and the kidney was removed. The muscle and fur layers were closed with suture material and wound clips, respectively. The total period of anesthesia was ~15 min. Animals were awake and ambulating in their cages and drinking water within a period of 15 min after the surgical procedure. There were no infection complications, and fluid intake appeared adequate during the 12-h period. In the 2-h nephrectomy experiments, the right kidney was removed through a right subcostal incision under Inactin anesthesia (Byk Gulden) -2 h prior to the micropuncture measurements. The animals were studied during euvolemia after Inactin (100 mg/kg ip) anesthesia. Rats were subjected to preparatory surgery, as has been previously described from this laboratory (14, 15, 22). The rat was placed on a servo-controlled heating table and maintained at 37°C body temperature. The kidney was placed in a Lucite cup, packed with cotton, and sealed with agar. The kidney surface was superfused with sodium chloridesodium bicarbonate solution maintained at 37OC. During the surgery the rat was supplied with an infusion of 1.5 ml/h of isotonic sodium chloride-sodium bicarbonate solution. After the surgery was completed the animal was infused with littermate plasma at 1% body wt/h for the 1st h and thereafter at 0.15% body wt/h and [3H]inulin (100 &i/h) infused in sodium chloride-sodium bicarbonate throughout the experiment (14,15,22). Samples for SNGFR were obtained from the early distal tubule, and a late proximal collection was also obtained from the same nephron to ascertain the normal proximal-to-distal SNGFR difference, late proximal and early distal tubular flow rates, and proximal tubular and loop of Henle reabsorptive rates in sham control and postnephrectomy animals in which feedback microperfusion studies were performed (14, 22). Experimental protocol for renal microperfusion studies. Three types of renal micropuncture studies were performed: 1) the response of the tubuloglomerular feedback system to variations in late proximal flow rate utilizing a Hlimpel microperfusion pump and the corresponding proximal and distal tubular SNGFR and flow rates, 2) the variations in directly measured glomerular capillary hydrostatic pressure or stop-flow pressure in response to variations in late proximal flow rate, and 3) microperfusion studies that evaluated loop of Henle reabsorption between the late proximal tubule and the early distal tubule. Perfusion of single nephrons was performed utilizing artificial tubular fluid (ATF). The nephrons were selected at random on the kidney surface but required a minimum of three loops of proximal convoluted tubule and a distal tubular segment on the surface of the kidney (13, 15). Localization of tubular topography was performed after the injection of the nephron with FD & C green dye via a 3- to 5-pm tip pipette. For tubuloglomerular feedback studies a wax block was inserted into the late proximal tubule and the tubule was vented proximal to the block. A perfusion pipette (7-9 pm tip) attached to the microperfusion pump was inserted into the tubule distal to the wax block. Perfusion flow rates were applied in random order at 0, 10,20, 30, and 40 nl/min through
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the loop of Henle. A timed late proximal collection of -3 min duration was obtained after a period of at least 3-5 min of perfusion at the various late proximal flow rates to determine nephron filtration rates, utilizing the clearance rates of [3H]inulin, at all perfusion rates from the same nephron. Another set of experiments evaluated the alterations in glomerular capillary hydrostatic pressure (Po) with variations in flow rate from the late proximal tubule. These studies utilized either directly measured Po in surface glomeruli or estimates of Po utilizing stop-flow pressure (SFP) techniques (SFP + rI* = Po) where & is systemic oncotic pressure. Pressure was continuously monitored either as SFP or PG during perfusion from 0 to 40 nl/min in random order (13). These studies evaluating changes in Po were confined to the control and 2h postnephrectomy groups. It is a common custom to express tubuloglomerular feedback activity in terms of late proximal perfusion rate vs. the corresponding alteration in SNGFR measured in the same nephron. Although in most conditions this is a satisfactory method for evaluating tubuloglomerular feedback activity, changes in absolute or fractional loop of Henle reabsorption after contralateral nephrectomy might diminish the validity of evaluating tubuloglomerular feedback alterations with this approach. After contralateral nephrectomy at 2 h, it is possible that alterations in loop reabsorption might have occurred via alterations in neural traffic, prostaglandin generation, and/ or volume status (8, 10, 19). Studies were therefore performed to measure fractional and absolute loop of Henle reabsorption at flow rates between 10 and 30 nl/ min in and around the turning point of the late proximal response flow rate, thereby relating early distal flow rate to the corresponding changes in SNGFR in control twokidney animals and animals 2-h postcontralateral nephrectomy. A wax block was inserted into the late proximal tubule, and perfusion was established utilizing ATF lightly stained with FD & C dye as described previously (14). However, collections were made sequentially from the distal tubule of that same nephron. Fractional and absolute loop reabsorption and the early distal flow rate were evaluated at all perfusion rates. After perfusion had been established for a few minutes an early distal tubule collection (3 min) was obtained in a manner similar to the proximal free-flow studies. The volume of distal tubular fluid and the radioactive counts per minute (cpm) were evaluated and compared to the perfusate [3H]inulin cpm. The pump perfusion rate was verified from these data. In all studies whole kidney GFR and urinary volume flow rate were also assessed from left and right kidney when present. For purposes of quantifying tubuloglomerular feedback responses, an iterative computer program was utilized to generate best-fit curves (by least squares) of micropuncture data to an equation previously employed by others (5) to describe the apparent sigmoidal relationship between SNGFR and late proximal flow rate (V&. The behavior of this function is determined by four parameters that were then compared between groups. Curves were generated according to two protocols. 1) A curve was generated for each nephron for which at least four
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data points were available, and the parameters defining each curve were employed in statistical analysis for intergroup comparisons. Data from an entire individual nephron were excluded from the analysis if unable to be fit to the mode with ? > 0.75. Nephrons with five available data points were reanalyzed after exclusion of a single data point, if, and only if, inclusion of that data point resulted in a best-fit curve with ? c 0.75, the squared residual associated with that data point accounted for >50% of the residual sum of squares, and elimination of the point allowed an equation to be fit to the data with ? > 0.75. Analysis of the data by this method caused nephrons not manifesting a general decline in SNGFR with increasing perfusion rate to be excluded from the analysis but did provide data to which statistical analysis could be applied. 2) Group data were pooled before application of the mathematical model, and mean values were determined for SNGFR at each rate of loop perfusion. Data points falling further than 1.8 SD from the group mean at any individual rate of perfusion were excluded from the analysis. The mathematical model was subsequently applied to generate a single curve for each group representing the best fit to the recorded group means. By this method data from each nephron were able to be entered into the analysis, but no error of measurement could be assigned to the four defining parameters for use in intergroup comparison. Analysis and calculations. Nephron filtration rates (SNGFR) from proximal and distal tubular collections were computed as previously described (14, 22). Values of absolute proximal reabsorption, VLp, and early distal flow rate were computed based upon the distally derived SNGFR and the tubular fluid-to-plasma (TF/P) inulin concentrations in the respective proximal and distal collections. In loop of Henle perfusion studies, absolute loop reabsorption was calculated from the pump perfusion rate and verified by the collection rate of inulin, as determined from the measured volume was collected per minute and the TF/P inulin in early distal samples Proximal-to-distal SNGFR difference was computed as the proximal collection value minus the distal collection value for SNGFR in the same nephron unit. Statistical analysis. Variances were expressed as means + SE and statistical significance as P < 0.05. Paired evaluations within the same nephron were evaluated by paired t tests, and intergroup comparisons of all data were corrected for multiple groups by Tukey analysis (3) after analysis of variance. RESULTS
Urine volume and urinary sodium excretion increased significantly in both the 2- and 12-h postnephrectomy groups when compared with the appropriate sham control groups (Table 1). At the 2-h interval mean arterial pressures were equal in control and postnephrectomy animals; however, at 12 h the nephrectomized rats exhibited a significantly higher value for mean arterial pressure but exhibited values that were nearly identical to the 2-h animals. The GFR in euvolemic two-kidney sham control rats at micropuncture was 1.1 ml/min. The whole
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kidney GFR in the left kidney 2-4 h after contralateral nephrectomy was 1.03 ml/min (Table 1). By 12-15 h after nephrectomy there was a significant increase in whole kidney GFR to 1.48 ml/min, values higher than any other group. Therefore, the whole kidney GFR response is not immediate at 2-4 h, but increases are observed 12-14 h later. Table 2 depicts the nephron filtration rates and segmental flow rates and reabsorptive rates in sham control rats (14 nephrons in 5 rats) and in the 2-4 h (22 nephrons in 6 rats) and 12-h postnephrectomy animals (12 nephrons in 4 rats). Nephron filtration rates derived from proximal collections exhibited no significant differences between control sham and 2-h postnephrectomy animals. However, the distally derived value for SNGFR demonstrated a significantly lower value for SNGFR in the 2h postnephrectomy animals. Therefore, the proximal-todistal SNGFR difference was significantly greater than zero in the 2-h postnephrectomy animals but not in the control animals. Although early distal flow rates were nearly identical between control and 2-h postnephrectomy rats, the V Lp, which was calculated from the fractional reabsorption in late proximal collections and the distal SNGFR, exhibited a lower value in the 2-h postnephrectomy animals. This lower VLp was associated with a significantly lower absolute reabsorption within the loop of Henle, generating early distal flow rates that were similar. These data differ significantly from previous observations in the literature in several respects (6,11). These data suggest that increased urine flow rate and sodium excretion 2 h after nephrectomy are entirely the consequence of decreases in reabsorption in the most distal segments of the nephron, rather than the consequence of increased flow rates at the late proximal and early distal sites. Twelve hours after nephrectomy, the SNGFR derived from either proximal or distal collections was increased with respect to both control sham rats and 2-h postnephrectomy animals (Table 2). Glomerulotubular balance was exhibited within the proximal nephron in that absolute proximal reabsorption was increased at 12-14 h. VLp was significantly increased over both control and 2h postnephrectomy animals. The early distal flow rate was also significantly increased at this time. There was no proximal-to-distal SNGFR difference 12-14 h after nephrectomy. One could conclude from these data at 1214 h after nephrectomy that increased late proximal and early distal flow rate may have contributed to the increased salt and water excretion. However, as observed in 2-h postnephrectomy animals, this increase in urinary excretion can occur without the corresponding increases in early distal tubular flow rate. Tubuloglomerular feedback profiles. Tubuloglomerular feedback profiles of SNGFR responses to alterations in late proximal perfusion rate for control sham and 2-h postnephrectomy animals are depicted in Fig. 1. In control animals (13 nephrons in 5 rats) SNGFR decreased significantly in comparison to the 0 nl/min perfusion rate at perfusion rates of 30 nl/min and higher. In the 2h postnephrectomy animals (20 nephrons in 6 rats) this reduction occurred at the 20 nl/min perfusion rate. The overall visual impression from the curves depicted is a
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1. Micropuncture kidney excretory rates, GFR, and MAP in control and postcontralateral nephrectomized rats
TABLE
Urine flow rate, pl/min
UN,.,
peq/min
UKV, peq/min
GFR, ml/min
MAP, mmHg
Euvolemic control, two-kidney 2.4kO.l 0.14kO.02 0.61t0.10 1.10~0.05 112t7 Postnephrectomy, 2-4 h 3.9*0.2* 0.25t0.04* 1.70*0.17* 1.03kO.07 105t,3 Control sham, 12 h 3.4kO.3 0.24t0.05 1.27t0.14 1.13t0.08 103tl Postnephrectomy, 12 h 6.8rt0.2*? 0.69&0.13*7 2.43*0.19*t 1.48kO.l3*t 113t2* Values are means t SE. GFR, glomerular filtration rate; MAP, mean arterial pressure. * Significantly different from respective time control, P < 0.05; t significantly different from 2-4 h postnephrectomy, P < 0.05. UN,V and UkV, urinary excretion rates of Na and K, respectively.
2. The nephron filtrates rates, segmental nephron flow rates, and reabsorptive rates in sham control rats and rats 2-4 h and 12 h after contralateral nephrectomy
TABLE
n
Control sham 13 in 5 rats 22 in 6 rats 2 h Nephrex 12 in 4 rats 12 h Nephrex Values are means t SE; n, no. of absolute proximal reabsorption; ALR, 45
SNGFR, proximal
SNGFR, distal
APR
-
V8 0 rwn&l
.
23
l
-
0
10
VFdist
P-DA
ALR
37.4k2.2 33.821.4 13.4H.l 20.5k1.2 8.2t0.6 +3.6&2.0 11.3kO.7 35.2k1.3 27.7*1.0*? 11.8kO.6 15.9&0.8? 7.OAO.6 +7.5t0.6 8.9+0.4t 47.5+3.4-j-$ 46.2*3.3f$ 22.2k3.2.Q 25.3+1.5?$ 13.0&1.4Q +1.3*2.5$ 12.2+1.2$ nephrons. P-DA, change from proximal to distal; Vrprox, proximal flow rate; Vrdist, distal flow rate; APR, absolute loop reabsorption. * vs. proximal SNGFR; t vs. Control; $ vs. 2-h Nephrex.
* FO.05
25
V Fprox, nl/min
20 VLP
30
rb
(nmnin)
1. Tubuloglomerular feedback profiles relating late proximal flow rate (VLr; x-axis) to resulting nephron filtration rate (SNGFR) in sham control two-kidney rats (o) and animals 2-4 h after contralateral nephrectomy (0). Operating point (0) was shifted downward on curve 24 h after nephrectomy, suggesting activation of tubuloglomerular feedback. FIG.
slight leftward shift of the 2-h postnephrectomy profile, and the operating point is lower on the curve and exhibits an activated position when compared with the control operating point. These operating points fall close to the curve that is drawn through the mean values of SNGFR at all perfusion rates. To ensure that the VLp-to-SNGFR relationship was a valid indicator of alterations in tubuloglomerular feedback activity in these two groups, we also evaluated the
early distal flow rate in loop of Henle reabsorption responses to a range of perfusions between 10 and 30 nl/ min in both two-kidney controls and 2-4 h after acute nephrectomy. Absolute loop of Henle reabsorption evaluated at 10, 20, and 30 nl/min late proximal perfusion rate was not different between the 2-kidney control and 2- to 4-h postcontralateral nephrectomy rats. The resulting distal flow rate (Vn) and corresponding SNGFR are depicted in Fig. 2 labeled with the corresponding late proximal microperfusion rates. The major differences observed are not between the curves relating the perfusion early Vh to SNGFR but rather that the operating points (SNGFR) differ significantly. At identical freeflow values for early Vh the SNGFR is significantly diminished in the 2-h postnephrectomy animals. There was a tendency for a leftward shift in the relationship in the tubuloglomerular feedback profiles in the 2-h postnephrectomy group, and the operating point was sitting at a more activated portion of the tubuloglomerular feedback curve, significantly different from the control values for both VLp and SNGFR. We also assessed differences in tubuloglomerular feedback responses by examining the alterations in glomerular capillary hydrostatic pressure (PG) at perfusion rates of O-40 nl/min in control two-kidney and 2-h postnephrectomy animals. PG was essentially constant across this range of perfusion rates in these euvolemic animals. PG values at 10,20,30, and 40 nl/min perfusion rates were 50 t 4, 50 t 4, 50 t 4,50&5,and48+4 mmHg, respectively, in control rats and 55 t 1,56 t 1,55 t 1,54 t 1, and 55 t 1 mmHg 24 h after contralateral nephrectomy [not significant (NS)] vs. 0 nl/min. The same conclusions were derived from directly measured PG values and from stop-flow estimates. Only in hydropenic animals have we observed major reductions in PC with increasing perfusion rates (13). We have previously observed a dissociation between SNGFR feedback responses and feedback-induced alterations in glomerular capillary pressure (13). Tubuloglomerular feedback profiles for animals 12-14 h after nephrectomy (13 nephrons in 4 rats) are depicted
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0 Control-2K-2hrs V Control-2K12hrs 0
0 Control - 2 K 0 2 Hrs. Postnephrectomy I Operating points
38
n
l
* p
0. W. PETERSON,
AND
S. C. THOMSON
Department of Medicine, University of California, San Diego, School of Medicine, La Jolla 92093; and San Diego Veterans Affairs Medical Center, La Jolla, California 92161
BLANTZ, R. C., 0. W. PETERSON,AND S. C. THOMSON. Tubuloglomerukzr feedback responses to acute contralateral nephrectomy. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F749-F756,1991.-After unilateral nephrectomy adaptive events must occur in the remaining kidney within the first 12-14 h in anticipation of an increasein glomerular filtration rate (GFR) and eventual renal hypertrophy. Utilizing micropuncture and microperfusion techniques in the rat, we have examined tubuloglomerular feedback (TGF) and single-nephron GFR (SNGFR) responseswhile the late proximal tubule was microperfused [late proximal tubule flow (V&l from 0 to 40 nl/min in 10 nl/min intervals at 2-4 and 12 h after contralateral nephrectomy. Urinary excretion increased,but SNGFR derived from distal collections was reduced, and early distal flow rate remained constant 2-4 h after nephrectomy. The operating point was shifted, suggestingactivation of TGF. The turning point half-maximal activity (VA) and slope were not statistically different when all nephron data were submitted to a curve-fitting procedure, but group mean data suggesteda quantitatively lower VN and steeper slope of the TGF profile. Twelve to fourteen hours after contralateral nephrectomy, valuesfor SNGFR at all microperfusion rates were increased, as were late proximal and early distal flow rates. The values for VIA and slope of TGF were not statistically different from control values. We conclude that TGF activity and sensitivity are not suppressedat 2 and 12 h after nephrectomy. Increased urinary excretion does not require TGF alterations. Changes in TGF may be adaptive to increasesin SNGFR and may not be causalto the increasein filtration rate after nephrectomy. glomerular filtration; tubular reabsorption AFTER REMOVAL of one kidney the remaining organ undergoes a prompt increase in whole kidney glomerular filtration rate (GFR) over a relatively short time period and a major increase in renal size (4, 6, 7, 9, 18). After contralateral nephrectomy the reabsorptive capacity of the kidney, as well as its excretory rate, increases considerably, and this increase in tubular reabsorption occurs at least in proportion to the increase in GFR (6, 12). These events occur prior to demonstrable hypertrophy, and the hypertrophy is primarily within tubular structures (7). The events that transpire after contralateral nephrectomy suggest that improved excretory function is not the only goal but rather improved reabsorptive capacity that represents an attempt to duplicate the filtration rate and reabsorptive capacity that existed when two kidneys were present. The temporal sequence of adjustments in GFR, tubular reabsorption, and increased urinary excretion has not been well defined.
Increased tubular reabsorptive capacity and increased GFR must be maintained in balance, and this coordinated process may play a significant role in contributing to renal hypertrophy. Under normal circumstances there exists a tight relationship between tubular reabsorption and GFR, as a conseque rice of both 1) gl .omerulotubular balance, whereby tubu lar reabsorption adapts to alterations in load into each nephron segment (18) and 2) tubuloglomerular feedback mechanisms (16, 21). The tubuloglomerular feedback system has been well defined over-the past several decades, utilizing micropuncture and tubular microperfusion techniques. Knowledge derived from several experimental studies suggests that increases in delivery of fluid to the distal nephron will elicit vasomotor responses, which decrease the filtration rate (1, 2,5, 16, 20,- 21, 23). For the GFR and single-nephron GFR (SNGFR) to rise after contralateral nephrectomy while flow to the distal nephron concurrently increases, adaptations in the tubuloglomerular feedback system must occur to permit this increase in filtration rate (1, 5, 10). Such adaptations of tubuloglomerular feedback mechanisms may be critical to the initial processes, which lead to increased GFR and tubular reabsorption and may be a requirement for the initiation of compensatory hypertrophy. These studies have been designed to examine the early phases of filtration and reabsorptive responses and the corresponding adaptations of the tubuloglomerular feedback mechanism 2 and 12 h after contralateral nephrectomy. METHODS
Experiments were performed in male Munich- Wistar rats of 200-275 g body wt, derived either from animals housed and raised at the Animal Research Facility at the San Diego Veterans Affairs Medical Center or purchased from a commercial supplier (Simonsen Labs, Gilroy, CA). Rats were fed a regular rat chow (24% protein) until 16 h before micropuncture evaluation and then were given free access to tap water up to the time of micropuncture. A right nephrectomy or sham procedure was performed at either 2 or 12 h before micropuncture evaluations. In 12-h nephrectomy studies animals were allowed access to food before the time of the brief surgical procedure. Animals were anesthetized with Brevital(lO0 mg/kg ip), a short-acting barbiturate anesthetic; under sterile conditions the right kidney was dissected free from its periF749
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renal fat, and the pedicle was exposed. The right renal artery and renal vein were doubly ligated with silk suture material as was the ureter, and the kidney was removed. The muscle and fur layers were closed with suture material and wound clips, respectively. The total period of anesthesia was ~15 min. Animals were awake and ambulating in their cages and drinking water within a period of 15 min after the surgical procedure. There were no infection complications, and fluid intake appeared adequate during the 12-h period. In the 2-h nephrectomy experiments, the right kidney was removed through a right subcostal incision under Inactin anesthesia (Byk Gulden) -2 h prior to the micropuncture measurements. The animals were studied during euvolemia after Inactin (100 mg/kg ip) anesthesia. Rats were subjected to preparatory surgery, as has been previously described from this laboratory (14, 15, 22). The rat was placed on a servo-controlled heating table and maintained at 37°C body temperature. The kidney was placed in a Lucite cup, packed with cotton, and sealed with agar. The kidney surface was superfused with sodium chloridesodium bicarbonate solution maintained at 37OC. During the surgery the rat was supplied with an infusion of 1.5 ml/h of isotonic sodium chloride-sodium bicarbonate solution. After the surgery was completed the animal was infused with littermate plasma at 1% body wt/h for the 1st h and thereafter at 0.15% body wt/h and [3H]inulin (100 &i/h) infused in sodium chloride-sodium bicarbonate throughout the experiment (14,15,22). Samples for SNGFR were obtained from the early distal tubule, and a late proximal collection was also obtained from the same nephron to ascertain the normal proximal-to-distal SNGFR difference, late proximal and early distal tubular flow rates, and proximal tubular and loop of Henle reabsorptive rates in sham control and postnephrectomy animals in which feedback microperfusion studies were performed (14, 22). Experimental protocol for renal microperfusion studies. Three types of renal micropuncture studies were performed: 1) the response of the tubuloglomerular feedback system to variations in late proximal flow rate utilizing a Hlimpel microperfusion pump and the corresponding proximal and distal tubular SNGFR and flow rates, 2) the variations in directly measured glomerular capillary hydrostatic pressure or stop-flow pressure in response to variations in late proximal flow rate, and 3) microperfusion studies that evaluated loop of Henle reabsorption between the late proximal tubule and the early distal tubule. Perfusion of single nephrons was performed utilizing artificial tubular fluid (ATF). The nephrons were selected at random on the kidney surface but required a minimum of three loops of proximal convoluted tubule and a distal tubular segment on the surface of the kidney (13, 15). Localization of tubular topography was performed after the injection of the nephron with FD & C green dye via a 3- to 5-pm tip pipette. For tubuloglomerular feedback studies a wax block was inserted into the late proximal tubule and the tubule was vented proximal to the block. A perfusion pipette (7-9 pm tip) attached to the microperfusion pump was inserted into the tubule distal to the wax block. Perfusion flow rates were applied in random order at 0, 10,20, 30, and 40 nl/min through
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the loop of Henle. A timed late proximal collection of -3 min duration was obtained after a period of at least 3-5 min of perfusion at the various late proximal flow rates to determine nephron filtration rates, utilizing the clearance rates of [3H]inulin, at all perfusion rates from the same nephron. Another set of experiments evaluated the alterations in glomerular capillary hydrostatic pressure (Po) with variations in flow rate from the late proximal tubule. These studies utilized either directly measured Po in surface glomeruli or estimates of Po utilizing stop-flow pressure (SFP) techniques (SFP + rI* = Po) where & is systemic oncotic pressure. Pressure was continuously monitored either as SFP or PG during perfusion from 0 to 40 nl/min in random order (13). These studies evaluating changes in Po were confined to the control and 2h postnephrectomy groups. It is a common custom to express tubuloglomerular feedback activity in terms of late proximal perfusion rate vs. the corresponding alteration in SNGFR measured in the same nephron. Although in most conditions this is a satisfactory method for evaluating tubuloglomerular feedback activity, changes in absolute or fractional loop of Henle reabsorption after contralateral nephrectomy might diminish the validity of evaluating tubuloglomerular feedback alterations with this approach. After contralateral nephrectomy at 2 h, it is possible that alterations in loop reabsorption might have occurred via alterations in neural traffic, prostaglandin generation, and/ or volume status (8, 10, 19). Studies were therefore performed to measure fractional and absolute loop of Henle reabsorption at flow rates between 10 and 30 nl/ min in and around the turning point of the late proximal response flow rate, thereby relating early distal flow rate to the corresponding changes in SNGFR in control twokidney animals and animals 2-h postcontralateral nephrectomy. A wax block was inserted into the late proximal tubule, and perfusion was established utilizing ATF lightly stained with FD & C dye as described previously (14). However, collections were made sequentially from the distal tubule of that same nephron. Fractional and absolute loop reabsorption and the early distal flow rate were evaluated at all perfusion rates. After perfusion had been established for a few minutes an early distal tubule collection (3 min) was obtained in a manner similar to the proximal free-flow studies. The volume of distal tubular fluid and the radioactive counts per minute (cpm) were evaluated and compared to the perfusate [3H]inulin cpm. The pump perfusion rate was verified from these data. In all studies whole kidney GFR and urinary volume flow rate were also assessed from left and right kidney when present. For purposes of quantifying tubuloglomerular feedback responses, an iterative computer program was utilized to generate best-fit curves (by least squares) of micropuncture data to an equation previously employed by others (5) to describe the apparent sigmoidal relationship between SNGFR and late proximal flow rate (V&. The behavior of this function is determined by four parameters that were then compared between groups. Curves were generated according to two protocols. 1) A curve was generated for each nephron for which at least four
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data points were available, and the parameters defining each curve were employed in statistical analysis for intergroup comparisons. Data from an entire individual nephron were excluded from the analysis if unable to be fit to the mode with ? > 0.75. Nephrons with five available data points were reanalyzed after exclusion of a single data point, if, and only if, inclusion of that data point resulted in a best-fit curve with ? c 0.75, the squared residual associated with that data point accounted for >50% of the residual sum of squares, and elimination of the point allowed an equation to be fit to the data with ? > 0.75. Analysis of the data by this method caused nephrons not manifesting a general decline in SNGFR with increasing perfusion rate to be excluded from the analysis but did provide data to which statistical analysis could be applied. 2) Group data were pooled before application of the mathematical model, and mean values were determined for SNGFR at each rate of loop perfusion. Data points falling further than 1.8 SD from the group mean at any individual rate of perfusion were excluded from the analysis. The mathematical model was subsequently applied to generate a single curve for each group representing the best fit to the recorded group means. By this method data from each nephron were able to be entered into the analysis, but no error of measurement could be assigned to the four defining parameters for use in intergroup comparison. Analysis and calculations. Nephron filtration rates (SNGFR) from proximal and distal tubular collections were computed as previously described (14, 22). Values of absolute proximal reabsorption, VLp, and early distal flow rate were computed based upon the distally derived SNGFR and the tubular fluid-to-plasma (TF/P) inulin concentrations in the respective proximal and distal collections. In loop of Henle perfusion studies, absolute loop reabsorption was calculated from the pump perfusion rate and verified by the collection rate of inulin, as determined from the measured volume was collected per minute and the TF/P inulin in early distal samples Proximal-to-distal SNGFR difference was computed as the proximal collection value minus the distal collection value for SNGFR in the same nephron unit. Statistical analysis. Variances were expressed as means + SE and statistical significance as P < 0.05. Paired evaluations within the same nephron were evaluated by paired t tests, and intergroup comparisons of all data were corrected for multiple groups by Tukey analysis (3) after analysis of variance. RESULTS
Urine volume and urinary sodium excretion increased significantly in both the 2- and 12-h postnephrectomy groups when compared with the appropriate sham control groups (Table 1). At the 2-h interval mean arterial pressures were equal in control and postnephrectomy animals; however, at 12 h the nephrectomized rats exhibited a significantly higher value for mean arterial pressure but exhibited values that were nearly identical to the 2-h animals. The GFR in euvolemic two-kidney sham control rats at micropuncture was 1.1 ml/min. The whole
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kidney GFR in the left kidney 2-4 h after contralateral nephrectomy was 1.03 ml/min (Table 1). By 12-15 h after nephrectomy there was a significant increase in whole kidney GFR to 1.48 ml/min, values higher than any other group. Therefore, the whole kidney GFR response is not immediate at 2-4 h, but increases are observed 12-14 h later. Table 2 depicts the nephron filtration rates and segmental flow rates and reabsorptive rates in sham control rats (14 nephrons in 5 rats) and in the 2-4 h (22 nephrons in 6 rats) and 12-h postnephrectomy animals (12 nephrons in 4 rats). Nephron filtration rates derived from proximal collections exhibited no significant differences between control sham and 2-h postnephrectomy animals. However, the distally derived value for SNGFR demonstrated a significantly lower value for SNGFR in the 2h postnephrectomy animals. Therefore, the proximal-todistal SNGFR difference was significantly greater than zero in the 2-h postnephrectomy animals but not in the control animals. Although early distal flow rates were nearly identical between control and 2-h postnephrectomy rats, the V Lp, which was calculated from the fractional reabsorption in late proximal collections and the distal SNGFR, exhibited a lower value in the 2-h postnephrectomy animals. This lower VLp was associated with a significantly lower absolute reabsorption within the loop of Henle, generating early distal flow rates that were similar. These data differ significantly from previous observations in the literature in several respects (6,11). These data suggest that increased urine flow rate and sodium excretion 2 h after nephrectomy are entirely the consequence of decreases in reabsorption in the most distal segments of the nephron, rather than the consequence of increased flow rates at the late proximal and early distal sites. Twelve hours after nephrectomy, the SNGFR derived from either proximal or distal collections was increased with respect to both control sham rats and 2-h postnephrectomy animals (Table 2). Glomerulotubular balance was exhibited within the proximal nephron in that absolute proximal reabsorption was increased at 12-14 h. VLp was significantly increased over both control and 2h postnephrectomy animals. The early distal flow rate was also significantly increased at this time. There was no proximal-to-distal SNGFR difference 12-14 h after nephrectomy. One could conclude from these data at 1214 h after nephrectomy that increased late proximal and early distal flow rate may have contributed to the increased salt and water excretion. However, as observed in 2-h postnephrectomy animals, this increase in urinary excretion can occur without the corresponding increases in early distal tubular flow rate. Tubuloglomerular feedback profiles. Tubuloglomerular feedback profiles of SNGFR responses to alterations in late proximal perfusion rate for control sham and 2-h postnephrectomy animals are depicted in Fig. 1. In control animals (13 nephrons in 5 rats) SNGFR decreased significantly in comparison to the 0 nl/min perfusion rate at perfusion rates of 30 nl/min and higher. In the 2h postnephrectomy animals (20 nephrons in 6 rats) this reduction occurred at the 20 nl/min perfusion rate. The overall visual impression from the curves depicted is a
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1. Micropuncture kidney excretory rates, GFR, and MAP in control and postcontralateral nephrectomized rats
TABLE
Urine flow rate, pl/min
UN,.,
peq/min
UKV, peq/min
GFR, ml/min
MAP, mmHg
Euvolemic control, two-kidney 2.4kO.l 0.14kO.02 0.61t0.10 1.10~0.05 112t7 Postnephrectomy, 2-4 h 3.9*0.2* 0.25t0.04* 1.70*0.17* 1.03kO.07 105t,3 Control sham, 12 h 3.4kO.3 0.24t0.05 1.27t0.14 1.13t0.08 103tl Postnephrectomy, 12 h 6.8rt0.2*? 0.69&0.13*7 2.43*0.19*t 1.48kO.l3*t 113t2* Values are means t SE. GFR, glomerular filtration rate; MAP, mean arterial pressure. * Significantly different from respective time control, P < 0.05; t significantly different from 2-4 h postnephrectomy, P < 0.05. UN,V and UkV, urinary excretion rates of Na and K, respectively.
2. The nephron filtrates rates, segmental nephron flow rates, and reabsorptive rates in sham control rats and rats 2-4 h and 12 h after contralateral nephrectomy
TABLE
n
Control sham 13 in 5 rats 22 in 6 rats 2 h Nephrex 12 in 4 rats 12 h Nephrex Values are means t SE; n, no. of absolute proximal reabsorption; ALR, 45
SNGFR, proximal
SNGFR, distal
APR
-
V8 0 rwn&l
.
23
l
-
0
10
VFdist
P-DA
ALR
37.4k2.2 33.821.4 13.4H.l 20.5k1.2 8.2t0.6 +3.6&2.0 11.3kO.7 35.2k1.3 27.7*1.0*? 11.8kO.6 15.9&0.8? 7.OAO.6 +7.5t0.6 8.9+0.4t 47.5+3.4-j-$ 46.2*3.3f$ 22.2k3.2.Q 25.3+1.5?$ 13.0&1.4Q +1.3*2.5$ 12.2+1.2$ nephrons. P-DA, change from proximal to distal; Vrprox, proximal flow rate; Vrdist, distal flow rate; APR, absolute loop reabsorption. * vs. proximal SNGFR; t vs. Control; $ vs. 2-h Nephrex.
* FO.05
25
V Fprox, nl/min
20 VLP
30
rb
(nmnin)
1. Tubuloglomerular feedback profiles relating late proximal flow rate (VLr; x-axis) to resulting nephron filtration rate (SNGFR) in sham control two-kidney rats (o) and animals 2-4 h after contralateral nephrectomy (0). Operating point (0) was shifted downward on curve 24 h after nephrectomy, suggesting activation of tubuloglomerular feedback. FIG.
slight leftward shift of the 2-h postnephrectomy profile, and the operating point is lower on the curve and exhibits an activated position when compared with the control operating point. These operating points fall close to the curve that is drawn through the mean values of SNGFR at all perfusion rates. To ensure that the VLp-to-SNGFR relationship was a valid indicator of alterations in tubuloglomerular feedback activity in these two groups, we also evaluated the
early distal flow rate in loop of Henle reabsorption responses to a range of perfusions between 10 and 30 nl/ min in both two-kidney controls and 2-4 h after acute nephrectomy. Absolute loop of Henle reabsorption evaluated at 10, 20, and 30 nl/min late proximal perfusion rate was not different between the 2-kidney control and 2- to 4-h postcontralateral nephrectomy rats. The resulting distal flow rate (Vn) and corresponding SNGFR are depicted in Fig. 2 labeled with the corresponding late proximal microperfusion rates. The major differences observed are not between the curves relating the perfusion early Vh to SNGFR but rather that the operating points (SNGFR) differ significantly. At identical freeflow values for early Vh the SNGFR is significantly diminished in the 2-h postnephrectomy animals. There was a tendency for a leftward shift in the relationship in the tubuloglomerular feedback profiles in the 2-h postnephrectomy group, and the operating point was sitting at a more activated portion of the tubuloglomerular feedback curve, significantly different from the control values for both VLp and SNGFR. We also assessed differences in tubuloglomerular feedback responses by examining the alterations in glomerular capillary hydrostatic pressure (PG) at perfusion rates of O-40 nl/min in control two-kidney and 2-h postnephrectomy animals. PG was essentially constant across this range of perfusion rates in these euvolemic animals. PG values at 10,20,30, and 40 nl/min perfusion rates were 50 t 4, 50 t 4, 50 t 4,50&5,and48+4 mmHg, respectively, in control rats and 55 t 1,56 t 1,55 t 1,54 t 1, and 55 t 1 mmHg 24 h after contralateral nephrectomy [not significant (NS)] vs. 0 nl/min. The same conclusions were derived from directly measured PG values and from stop-flow estimates. Only in hydropenic animals have we observed major reductions in PC with increasing perfusion rates (13). We have previously observed a dissociation between SNGFR feedback responses and feedback-induced alterations in glomerular capillary pressure (13). Tubuloglomerular feedback profiles for animals 12-14 h after nephrectomy (13 nephrons in 4 rats) are depicted
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0 Control-2K-2hrs V Control-2K12hrs 0
0 Control - 2 K 0 2 Hrs. Postnephrectomy I Operating points
38
n
l
* p
