Renal and nephron hemodynamics spontaneously hypertensive rats
in
WILLIAM J. ARENDSHORST AND WILLIAM H. BEIERWALTES Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 of intrarenal hydrostatic+pressures in models of chronic hypertension have been lacking. Azar et al. (5,6) reported micropuncture data for intrarenal pressures in two groups of hypertensive rats; unfortunately, the data conflict. In 9-mo-old rats with hypertension induced by chronic ingestion of a high sodium diet, pressures in superficial vessels and nephrons were elevated above normal, and segmental resistances seemed to be reduced. In contrast, 17. to l&wk-old rats with genetic hypertension had normal or reduced intrarenal hydrostatic pressures, and resistances were elevated. Whether these differences reflect the two models of hypertension studied, the disparities in age, or the severity of the disease process remains to be determined. Clearly, further investigation is needed to better understand renal hemodynamics during chronic hypertension. Because of the apparent similarities between the hypertensive disease in humans and in genetically hypertensive rats, we studied 12.wk-old spontaneously hypertensive rats (SHR) which had established hypertension. Clearance and micropunture techniques were used to characterize whole kidney and individual nephron hemodynamics in nondiuretic Wistar-Kyoto rats (WKY) and in two groups of SHR. One SHR group had a mean arterial pressure averaging 158 mmHg; in the other SHR group we constricted the aorta to acutely reduce renal renal blood flow; renal vascular resistance;glomerular filtration perfusion pressure to 114 mmHg, a level similar to that rate; arterial pressure;capillary pressure; autoregulation; geof the normotensive WKY. netic hypertension
ARENDSHORST,WILLIAMJ.,ANDWILLIAM H. BEIERWALTES. Renal and nephron hemodynamicsin spontaneouslyhypertensive rats. Am. J. Physiol. 236(3): F246F251, 1979 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 5(3): F246-IF251, 1979.-Renal and nephron hemodynamicswere compared between anesthetized, nondiuretic, spontaneously hypertensive ;ats (SHR) and Wistar-Kyoto rats (WKY). Although the mean arterial pressurewas higher in SHR than in WKY, 158vs. 114 mmHg, glomerular filtration rate (GFR) and renal blood flow (RBF) were similar in both groups. So were intrarenal hydrostatic pressures,single nephron GFR (SNGFR), and single nephron blood flow (SNBF). Accordingly, the increasedrenal vascular resistance (RVR) in SHR was due to predominant preglomerular vasoconstriction. Xn a second group of SHR, SHR-AC, the femoral arterial pressurewas reduced acutely to 114 mmHg by means of aortic constriction above the renal arteries. The meanvalues for GFR, RBF, SNGFR, SNBF, and intrarenal hydrostatic pressures resembled those in SHR, whereas RVR was lessin SHR-AC. These autoregulatory adjustments of RVR were again largely limited to the preglomerular vasculature. Efferent arteriolar resistancewas similar in all three groups.We conclude that the enhancedRVR in 1% wk-old SHR is primarily a consequenceof a physiological, autoregulatory responseof afferent arteriolar resistanceto the elevated arterial pressure.Further, RVR in SHR is not fixed and constant but respondsappropriately to reductions in renal perfusion pressure.
METHODS MECHANISMS RESPONSIBLE FOR changes in renal hemodynamics during the development and maintenance of chronic hypertension are poorly understood. Most of our knowledge of this subject during essential hypertension is based on indirect clearance studies. Although it is well established that renal vascular resistance (RVR) is increased in subjects with essential hypertension, the intrarenal site of this vasoconstriction is less certain. Some investigators have postulated that the enhanced resistance occurs predominantly in afferent arterioles (16,19), whereas others have proposed efferent arteriolar constriction (15). Lowenstein et al. (19) published suggestive data in 1970. They measured an elevated, wedged, renal venous pressure in hypertensive patients and believed it reflected a uniform increase in glomerular capillary, proximal intratubular, and peritubular capillary pressures. Until recently, however, direct measurements
THE
F246
A total of 28 male SHR (217 * 30 (SD) g body wt; 12 A 1 wk) and 13 male WKY (218 t 31 g body wt; 11 * 2 wk) of the Okamoto-Aoki strain (22, 23) were studied. The SHR were bred locally, brother-sister, from an original stock of SHR of F36-a generations, obtained’ from Dr. Carl Hansen at the National Institutes of Health. Most of the WKY were bred locally; some were purchased from Charles River Breeding Laboratories (Wilmington, MA). The renal hemodynamics of the WKY were similar overall, regardless of the source. The rats, deprived of food overnight but allowed free access to water prior to the experiment, were anesthetized by an intraperitoneal injection of sodium pentobarbital(50 mg/ kg body wt) and placed on a heating table that maintained body temperature at 37.38OC. Immediately after the induction of anesthesia, femoral arterial blood was sampled for an initial or presurgical determination of
0363-6127/79/0000-0000!$01.25
Copyright 0 1979 the American Physiological Smiety
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
RENAL HEMODYNAMlCS
F247
IN GENETIC HYPERTENSION
hematocrit and plasma protein concentration. The left kidney was exposed through an abdominal incision for micropuncture as previously described (3,4), and the left ureter was catheterized with PE-10 polyethylene tubing. Femoral arterial pressure was monitored with a Statham P23Db pressure transducer connected to a Hewlett-Packard recorder. Our experience with normotensive rats, the experience of others (1,18,20), and our preliminary studies of SHR all indicated that hematocrit increases and plasma protein concentration decreases in systemic blood during and following the surgical preparation required for mil cropuncture. We have recently documented a progressive loss of fluid and protein from the vasculaturi using radioactively labeled rat albumin and red blood cells (7). To compensate, we employed the following protocol to maintain hematocrit and plasma protein concentra@on near the baseline values obtained before surgery. Heparinized plasma from donor rats was infused intravenously to 1.25 ml/100 g body wt during surgery and maintained at 10 $/mm for the duration of each experiment. Whether the donor was $HR or WKY did not affect the results. Saline (0.85% NaCl) containing [3H-]inulin (ICN, Irvine, CA), 85 pCi/lOO g body wt per h, and para-aminohippurate (PAH) (Eastman Kodak Co., Rochester, NY), 3-4 mg/lOO g body wt per h, was infused into a jugular vein at a rate of 10 @nin during a nondiuretic period. Micropuncture and clearance measurements followed a l-h stabilization period. Urine was collected for 60 min, femoral arterial blood was sampled at the beginning and end of each clearance period. Hematocrit was measured in heparinized capillary tubes. Blood was sampled from the left renal vein at the end of the experiment to determine PAH extraction. In another, similarly prepared group of SHR, SHRAC, we evaluated renal and nephron function after the renal perfusion pressure had been reduced to within the normotensive range. A ligature encircling the abdominal aorta above the renal arteries was constricted to maintain femoral arterial pressure at approximately 115 mmHg, the average pressure in WKY, for the duration of an experiment. Tubular fluid from two to five separate late proximal and early distal convolutions was collected using sharpened, siliconized, glass pipettes with external tip diameters of 7-14 p. The collection and measurement procedures were performed as previously described (3). The last superficial coils of proximal convoluted tubules were localized with the aid of intravenous injections of 0.05 ml of 5% buffered solution of FD&C green dye (Keystone Aniline and Chemical Co., Chicago). “Late” proximal convolutions were also identified by intraluminal injections of an oil droplet or green dye (3); similar results were obtained with both methods of localization. “Early” distal convolutions were identified as those in which the dye injected intravenously first appeared on the kidney surface after its transit through the loop of Henle. The duration of tubular fluid collection from proximal and distal convolutions averaged 2.7 t 0.7 and 3.7 =t 1.1 min, respectively; these times were similar for each group of rats. Hydrostatic pressure was measured
in random, surface proximal and distal convolutions and in postglomerular vessels with sharpened glass pipettes (3-7 pm, OD), filled with 2 M NaCl, and a continuously recording electronic servo-nulling apparatus (3,4). Since glomeruli were not accessible for direct micropuncture, glomerular capillary pressure was estimated from the sum of stop-flow hydrostatic pressure, measured in the earliest accessible coil of a proximal tubule as previously described (3,4), and colloid osmotic pressure of systemic plasma. Radioactivity in the tubular fluid, urine, and plasma samples was measured in a liquid scintillation spectrometer. The concentration of PAH in the urine and plasma samples was determined by an adaptation of Bratton and Marshall’s method (9). A Zeiss PMQ II flame-emission spectrophotometer W8s used to measure theLrinary sodium concentration . We determined plasma protein concentration by an adaptation of the Lowry technique (lo), using rat plasma , total protein standards, and calculated the colloid osmotic pressure according to the LandisPappenheimer equation (17). Glomerular filtration rate (GFR) was measured by the clearance of inulin, and renal plasma flow (RPF) was determined from the clearance and extraction of PAH. The ratio of GFR/RPF was equated with the filtration fraction (FF). Renal blood flow (RBF) was determined from RPF/ (1 - hematocrit), and renal vascular resistance was calculated as arterial pressure/RBF. Clearance data are reported for the left kidneys, which were studied with micropuncture techniques. Single nephron glomerular filtration rate (SNGFR) was calculated as the fluidto-plasma (F/P) inulin concentration ratio times the tubular fluid flow rate (measured in nanoliters per minute). merent and efferent arteriolar resistances were estimated from the reduction in hydrostatic pressure across and the single nephron blood flow (SNBF) through these vascular segments. The latter was based on SNGFR and kidney FF, assuming FF to be similar for superficial nephrons and the kidney, as others have shown to be the case for SHR and WKY (5,13). Student’s t tests for paired and unpaired variates were done for analysis of significance. Results considered statistically significant had P values of less than 0.05. The values given were calculated from the means during each observation period in an individual animal. RESULTS
The SHR had a greater mean arterial pressure than did the normotensive WKY (Table 1). Femoral arterial pressure in SHR-AC averaged 114 mmHg, which was similar to that of WKY but appreciably less than that of hypertensive SHR. Despite these differences in renal perfusion pressure, the average .GFR and RBF were similar in each group. Mean RPF, the same in SHR and SHR-AC, was greater in WKY; therefore, FF was elevated in both SHR groups. Total RVR was related to arterial pressure, being significantly greater in SHR but comparable in WKY and SHR-AC. To facilitate comparisons among the groups, these results are summarized in Fig. 1. The similarity of GFR. and RBF in both SHR groups indicates that RVR responded in an appropriate
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
F248
W. J. ARENDSHORST
1. Renal function iiz spontaneously hypertensive rats and Wistar- Kyo to rats
TABLE
WKY
Arterial pressure, -Hg Glomerular filtration rate, ml/min Renal blood flow, ml/min Renal vascular resistance,
114 k6
P
in
WILLIAM J. ARENDSHORST AND WILLIAM H. BEIERWALTES Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 of intrarenal hydrostatic+pressures in models of chronic hypertension have been lacking. Azar et al. (5,6) reported micropuncture data for intrarenal pressures in two groups of hypertensive rats; unfortunately, the data conflict. In 9-mo-old rats with hypertension induced by chronic ingestion of a high sodium diet, pressures in superficial vessels and nephrons were elevated above normal, and segmental resistances seemed to be reduced. In contrast, 17. to l&wk-old rats with genetic hypertension had normal or reduced intrarenal hydrostatic pressures, and resistances were elevated. Whether these differences reflect the two models of hypertension studied, the disparities in age, or the severity of the disease process remains to be determined. Clearly, further investigation is needed to better understand renal hemodynamics during chronic hypertension. Because of the apparent similarities between the hypertensive disease in humans and in genetically hypertensive rats, we studied 12.wk-old spontaneously hypertensive rats (SHR) which had established hypertension. Clearance and micropunture techniques were used to characterize whole kidney and individual nephron hemodynamics in nondiuretic Wistar-Kyoto rats (WKY) and in two groups of SHR. One SHR group had a mean arterial pressure averaging 158 mmHg; in the other SHR group we constricted the aorta to acutely reduce renal renal blood flow; renal vascular resistance;glomerular filtration perfusion pressure to 114 mmHg, a level similar to that rate; arterial pressure;capillary pressure; autoregulation; geof the normotensive WKY. netic hypertension
ARENDSHORST,WILLIAMJ.,ANDWILLIAM H. BEIERWALTES. Renal and nephron hemodynamicsin spontaneouslyhypertensive rats. Am. J. Physiol. 236(3): F246F251, 1979 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 5(3): F246-IF251, 1979.-Renal and nephron hemodynamicswere compared between anesthetized, nondiuretic, spontaneously hypertensive ;ats (SHR) and Wistar-Kyoto rats (WKY). Although the mean arterial pressurewas higher in SHR than in WKY, 158vs. 114 mmHg, glomerular filtration rate (GFR) and renal blood flow (RBF) were similar in both groups. So were intrarenal hydrostatic pressures,single nephron GFR (SNGFR), and single nephron blood flow (SNBF). Accordingly, the increasedrenal vascular resistance (RVR) in SHR was due to predominant preglomerular vasoconstriction. Xn a second group of SHR, SHR-AC, the femoral arterial pressurewas reduced acutely to 114 mmHg by means of aortic constriction above the renal arteries. The meanvalues for GFR, RBF, SNGFR, SNBF, and intrarenal hydrostatic pressures resembled those in SHR, whereas RVR was lessin SHR-AC. These autoregulatory adjustments of RVR were again largely limited to the preglomerular vasculature. Efferent arteriolar resistancewas similar in all three groups.We conclude that the enhancedRVR in 1% wk-old SHR is primarily a consequenceof a physiological, autoregulatory responseof afferent arteriolar resistanceto the elevated arterial pressure.Further, RVR in SHR is not fixed and constant but respondsappropriately to reductions in renal perfusion pressure.
METHODS MECHANISMS RESPONSIBLE FOR changes in renal hemodynamics during the development and maintenance of chronic hypertension are poorly understood. Most of our knowledge of this subject during essential hypertension is based on indirect clearance studies. Although it is well established that renal vascular resistance (RVR) is increased in subjects with essential hypertension, the intrarenal site of this vasoconstriction is less certain. Some investigators have postulated that the enhanced resistance occurs predominantly in afferent arterioles (16,19), whereas others have proposed efferent arteriolar constriction (15). Lowenstein et al. (19) published suggestive data in 1970. They measured an elevated, wedged, renal venous pressure in hypertensive patients and believed it reflected a uniform increase in glomerular capillary, proximal intratubular, and peritubular capillary pressures. Until recently, however, direct measurements
THE
F246
A total of 28 male SHR (217 * 30 (SD) g body wt; 12 A 1 wk) and 13 male WKY (218 t 31 g body wt; 11 * 2 wk) of the Okamoto-Aoki strain (22, 23) were studied. The SHR were bred locally, brother-sister, from an original stock of SHR of F36-a generations, obtained’ from Dr. Carl Hansen at the National Institutes of Health. Most of the WKY were bred locally; some were purchased from Charles River Breeding Laboratories (Wilmington, MA). The renal hemodynamics of the WKY were similar overall, regardless of the source. The rats, deprived of food overnight but allowed free access to water prior to the experiment, were anesthetized by an intraperitoneal injection of sodium pentobarbital(50 mg/ kg body wt) and placed on a heating table that maintained body temperature at 37.38OC. Immediately after the induction of anesthesia, femoral arterial blood was sampled for an initial or presurgical determination of
0363-6127/79/0000-0000!$01.25
Copyright 0 1979 the American Physiological Smiety
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
RENAL HEMODYNAMlCS
F247
IN GENETIC HYPERTENSION
hematocrit and plasma protein concentration. The left kidney was exposed through an abdominal incision for micropuncture as previously described (3,4), and the left ureter was catheterized with PE-10 polyethylene tubing. Femoral arterial pressure was monitored with a Statham P23Db pressure transducer connected to a Hewlett-Packard recorder. Our experience with normotensive rats, the experience of others (1,18,20), and our preliminary studies of SHR all indicated that hematocrit increases and plasma protein concentration decreases in systemic blood during and following the surgical preparation required for mil cropuncture. We have recently documented a progressive loss of fluid and protein from the vasculaturi using radioactively labeled rat albumin and red blood cells (7). To compensate, we employed the following protocol to maintain hematocrit and plasma protein concentra@on near the baseline values obtained before surgery. Heparinized plasma from donor rats was infused intravenously to 1.25 ml/100 g body wt during surgery and maintained at 10 $/mm for the duration of each experiment. Whether the donor was $HR or WKY did not affect the results. Saline (0.85% NaCl) containing [3H-]inulin (ICN, Irvine, CA), 85 pCi/lOO g body wt per h, and para-aminohippurate (PAH) (Eastman Kodak Co., Rochester, NY), 3-4 mg/lOO g body wt per h, was infused into a jugular vein at a rate of 10 @nin during a nondiuretic period. Micropuncture and clearance measurements followed a l-h stabilization period. Urine was collected for 60 min, femoral arterial blood was sampled at the beginning and end of each clearance period. Hematocrit was measured in heparinized capillary tubes. Blood was sampled from the left renal vein at the end of the experiment to determine PAH extraction. In another, similarly prepared group of SHR, SHRAC, we evaluated renal and nephron function after the renal perfusion pressure had been reduced to within the normotensive range. A ligature encircling the abdominal aorta above the renal arteries was constricted to maintain femoral arterial pressure at approximately 115 mmHg, the average pressure in WKY, for the duration of an experiment. Tubular fluid from two to five separate late proximal and early distal convolutions was collected using sharpened, siliconized, glass pipettes with external tip diameters of 7-14 p. The collection and measurement procedures were performed as previously described (3). The last superficial coils of proximal convoluted tubules were localized with the aid of intravenous injections of 0.05 ml of 5% buffered solution of FD&C green dye (Keystone Aniline and Chemical Co., Chicago). “Late” proximal convolutions were also identified by intraluminal injections of an oil droplet or green dye (3); similar results were obtained with both methods of localization. “Early” distal convolutions were identified as those in which the dye injected intravenously first appeared on the kidney surface after its transit through the loop of Henle. The duration of tubular fluid collection from proximal and distal convolutions averaged 2.7 t 0.7 and 3.7 =t 1.1 min, respectively; these times were similar for each group of rats. Hydrostatic pressure was measured
in random, surface proximal and distal convolutions and in postglomerular vessels with sharpened glass pipettes (3-7 pm, OD), filled with 2 M NaCl, and a continuously recording electronic servo-nulling apparatus (3,4). Since glomeruli were not accessible for direct micropuncture, glomerular capillary pressure was estimated from the sum of stop-flow hydrostatic pressure, measured in the earliest accessible coil of a proximal tubule as previously described (3,4), and colloid osmotic pressure of systemic plasma. Radioactivity in the tubular fluid, urine, and plasma samples was measured in a liquid scintillation spectrometer. The concentration of PAH in the urine and plasma samples was determined by an adaptation of Bratton and Marshall’s method (9). A Zeiss PMQ II flame-emission spectrophotometer W8s used to measure theLrinary sodium concentration . We determined plasma protein concentration by an adaptation of the Lowry technique (lo), using rat plasma , total protein standards, and calculated the colloid osmotic pressure according to the LandisPappenheimer equation (17). Glomerular filtration rate (GFR) was measured by the clearance of inulin, and renal plasma flow (RPF) was determined from the clearance and extraction of PAH. The ratio of GFR/RPF was equated with the filtration fraction (FF). Renal blood flow (RBF) was determined from RPF/ (1 - hematocrit), and renal vascular resistance was calculated as arterial pressure/RBF. Clearance data are reported for the left kidneys, which were studied with micropuncture techniques. Single nephron glomerular filtration rate (SNGFR) was calculated as the fluidto-plasma (F/P) inulin concentration ratio times the tubular fluid flow rate (measured in nanoliters per minute). merent and efferent arteriolar resistances were estimated from the reduction in hydrostatic pressure across and the single nephron blood flow (SNBF) through these vascular segments. The latter was based on SNGFR and kidney FF, assuming FF to be similar for superficial nephrons and the kidney, as others have shown to be the case for SHR and WKY (5,13). Student’s t tests for paired and unpaired variates were done for analysis of significance. Results considered statistically significant had P values of less than 0.05. The values given were calculated from the means during each observation period in an individual animal. RESULTS
The SHR had a greater mean arterial pressure than did the normotensive WKY (Table 1). Femoral arterial pressure in SHR-AC averaged 114 mmHg, which was similar to that of WKY but appreciably less than that of hypertensive SHR. Despite these differences in renal perfusion pressure, the average .GFR and RBF were similar in each group. Mean RPF, the same in SHR and SHR-AC, was greater in WKY; therefore, FF was elevated in both SHR groups. Total RVR was related to arterial pressure, being significantly greater in SHR but comparable in WKY and SHR-AC. To facilitate comparisons among the groups, these results are summarized in Fig. 1. The similarity of GFR. and RBF in both SHR groups indicates that RVR responded in an appropriate
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
F248
W. J. ARENDSHORST
1. Renal function iiz spontaneously hypertensive rats and Wistar- Kyo to rats
TABLE
WKY
Arterial pressure, -Hg Glomerular filtration rate, ml/min Renal blood flow, ml/min Renal vascular resistance,
114 k6
P
