The Renal Medulla and Mechanisms of Hypertension in the Spontaneously Hypertensive Rat Kim Solez, MD, Robert J. D'Agostini, BA, Ronald A. Buono, BA, Nancy Vernon, BA, Ann L. Wang, BA, Paul M. Finer, BA, and Robert H. Heptinstall, MD
A significant number of offspring from brother-sister matings of N'IH-Okamoto-Aoki spontaneously hypertensive rats (SHRs) were found to be normotensive at 20 weeks of age. Over 20% of the animals that were hypertensive at this age had mild-to-moderate unilateral hydronephrosis at the time of sacrifice. In over 90% of the rats that did not develop hypertension spontaneously, ligation of one ureter raised blood pressure above 150 mm Hg within 2 weeks. In those rats made hypertensive by obstructing one ureter and in those that developed hypertension with accompanying naturally occurring hydronephrosis, subcutaneous implants of fragmented renal medulla from unrelated normal rats decreased blood pressure to normotensive levels. In contrast, medullary implants had no significant effect in rats developing hypertension spontaneously without hydronephrosis. Renal inner medullary plasma flow was low in the obstructed kidneys of hydronephrotic hypertensive SHRs but was elevated in the kidneys of nonhydronephrotic hypertensive SHRs. The hypertension in hydronephrotic SHRs appears to be related to an impairment of the antihypertensive function of the renal medulla. Such an impairment of medullary antihypertensive function does not appear to play a significant role in the hypertension in SHRs without hycdronephrosis. (Am J Pathol 85:555-568, 1976) THE SPON-TAN-EOUSLY HYPERTENSIVE RAT (SHR) developed by
Okamoto and Aoki 1 has been widelv studied as a model of human essential hypertension, and various ideas have been advanced to explain the mechanism for the elevation of blood pressure. The following are examples of some of the factors implicated by various authors: decreased intracellular cyclic AMP,23 altered calcium binding in vascular smooth muscle,' increased mvocardial contractility,5 increased cardiovascular reactivity to prostaglandin F2a,6 decreased renal production of prostaglandins £-2 and A2,7 increased adrenocortical activity,8 abnormalities of the kallikrein-kinin system,8 altered dopamine B-hvdroxvlase activity,10 or an intrinsic renal defect in ion transport 11,12 perhaps provoked by increased sympathetic nerve activity. 13 The present experiments were undertaken to study the effects of implantation of renal medullarv tissue from unrelated rats into SHRs to see if a deficiency of the antihvpertensive factors produced by the renal medulla From the Department of Pathology. The Johns Hopkins University School of medicine and Hospital. Baltimore. Mlarvland Supported by Grants HL-07835 and GM-(041 from the US Public Health Service Accepted for publication July 2.3. 1976. Address reprint requests to Dr. Robert H Heptinstall. Department of Pathology. The Johns Hopkins Hospital, Baltimore. MD 21205
555
556
SOLEZ ETAL
American Journal of Pathology
plays a part in producing the elevation of pressure in this strain of rat. During these experiments it was noted that 2 rats behaved differently from the other 14 in that they responded to implantation with a dramatic fall in pressure whereas there w.as little change in the others. Inspection of the kidneys of these 2 rats revealed unilateral hvdronephrosis, a feature not observed in the other 14 SHRs. It was further noted early in the study that not all rats had become hypertensive by 20 Xveeks of age. Considering the possibility that a certain number of rats in the breeding colony became hypertensive by the development of hvdronephrosis, it wvas decided to simulate spontaneously developing hydronephrosis by tying off one ureter in that population of rats that had not developed hypertension by 20 weeks. It was found that hypertension could be produced in a high proportion of these rats by tving off one ureter. In order to explore the mechanism of hypertension in this group, the effect of implantation of medullary tissue was studied and compared Xwith the effect of implantation in rats that developed hypertension naturally, i.e., without the development of spontaneous hvdronephrosis. To test the possibility that anx impairment of medullary antihvpertensive function observed wvas caused by medullary ischemia, inner medullary plasma flov- was determined in the various groups studied. Materials and Methods General Consideratons, Terminolgy for Rats, Ureteric Ligation Adult Okamoto-Aoki SHRs bred from brother-sister pairs from the :33rd filial generation of the National Heart and Lung Institute colony (supplied by Dr. Carl Hansen) were used in this stud!-. The breeding pairs and their offspring were housed in filter-top plastic cages and given Purina Rat Chow and w-ater ad libitum. The general term spontaneously hypertensive rat (SHR) is used for all of the rats in the colony. The SHRs which became hypertensive spontaneously are designated TSHRs (true SHRs). Some of these TSHRs had unilateral h%-dronephrosis. Comparison studies were carried out in Kyoto-Wistar rats (Taconic Farms). Holtzman rats, and Sprague-Daw ley rats (Charles River) housed under similar conditions. Both males and females were studied. Ligation of the left ureter 1 cm above the ureteroxesical junction was carried out under ether anesthesia in :38 SHRs that failed to develop hypertension spontaneously by 20 to 30
weeks of age. The animals of this group which became hypertensive within 2 Weeks after initial ligation are referred to as HO-SHRs (hypertensive-obstructed SHRs).
Medullary Implants and Blood Chemistry and Histologic Studies Subcutaneous allotransplantation of normal renal medulla from 160 to 220-g female Holtzman rats to 20- to 70-week-old TSHRs (N = 16) or HO-SHRs (N=10) was carried out as described b%- N\uirhead et al."4 \ledullarv tissue from 3 Holtzman donors (six kidnevs(
was used for each SHR recipient. As a control for the implants of medullary tissue, implants of cortical tissue were also carried out in TSHRs (N=6) and HO-SHRs (N=l0) using the technique of \lIuirhead et al." Rats w-ere killed by exsanguination under ether
Vol. 85, No. 3 December 1976
RENAL MEDULLA AND HYPERTENSION
557
anesthesia 3 weeks after implantation of cortical or medullary tissue. Blood was taken from the inferior vena cava for serum urea nitrogen and creatinine determinations w-hich were carried out as previously described.'5 Paraffin-embedded kidney tissue which had been fixed in 10%5 buffered formalin was cut at 4 u and stained with hematoxylin and eosin. In addition to these histologic studies on rats which had received implants. gross and microscopic examination of the kidneys was carried out in 141 SHRs which were killed by exsanguination under ether anesthesia at 12 to 70 weeks of age. Blod Pmsure Measurements Indirect systolic blood pressure measurements were made on rats lightly anesthetized with ether using the indirect tail cuff method, as previously described.,' using apparatus from Narco Biosvstems, Houston, Texas. When tail pulse tracings of suitable magnitude appeared, systolic blood pressure was determined five times and the average of these five determinations was taken as the blood pressure value for the day. Rats with sy stolic pressure of 150 mm Hg or greater were considered hypertensive. Blood pressure was determined at least twice a week in the SHRs from age 3 months until sacrifice. For :3 weeks following implantation of renal tissue or ligation of one ureter, blood pressure w-as determined dailv. Medullary Plasma Flow Determination
The method used for deterrnining medullary plasma flows has been discussed in detail in a previous publication from this laboratory.15 The method is based on the fact that the transit time for circulating albumin in the renal papilla for the normal rat is about 40 seconds. If the radioactive albumin is allowsed to circulate through the kidney for 30 seconds, the accumulation rate of papillary radioactivity during this period is proportional to plasma flow rate, and by comparing this to the average radioactivity in the arterial blood, an accurate estimation of medullary plasma flos may be obtained. Inner medullary plasma flow was determined in TSHRs without hydronephrosis. TSHRs with spontaneous unilateral hydronephrosis, HO-SHR rats one day after left ureteral ligation, HO-SHR rats 2 w-eeks after left ureteral ligation, normotensive SHRs. normal Holtzman rats, normal Sprague-Dawley rats, and normal Wistar-Kyoto rats (normotensive rats of the strain from which the SHRs were developed by selective inbreeding). Albumin Transit Tume The effective transit time for circulating albumin in the inner medulla of TSHRs and normal Holtzman rats was determined using methods previously described 1s by study ing the volume of distribution of 15I-albumin in the papilla 13, :30, 45, and 60 seconds after radioactive albumin had been allowed to circulate through the kidney. This determination
was necessary to establish the appropriateness of the :30-second infusion time for the determinations of inner medullary plasma flos-.15 Statistal Methods Student's t test was used to evaluate the significance of differences betw een means.
Results Hypertenson
The offspring of four F,,, mating pairs (obtained directly from the National Heart and Lung Institute) and six F.4 mating pairs were studied.
American Journal of Pathology
SOLEZ ET AL
558
The females in three of the F3 mating pairs had systolic blood pressures of 120 to 140 mm Hg at 20 weeks. The remaining females and all the males of both the Fu and F. breeding pairs had systolic pressure above 150 mm Hg at this age. Average systolic blood pressures in the total progenv of the F., and F. mating pairs at 20 weeks of age was 144.2 ± 2.1 mm Hg in the males (N=140) and 118.2 ± 2.9 mm Hg in the females (N=98). The total incidence of hypertension in the offspring (both sexes combined) of individual mating pairs at 20 weeks of age varied between 26.1 % (6 of 23 rats) and 72.0% (20 of 27 rats) in the offspring of the F. pairs and between 64.3% (9 of 14 rats) and 100% (24 of 24 rats) in the offspring of the FS pairs. Some of the rats remained normotensive for up to 18 months. In 17 SHRs that represented a random sampling of our colony, we observed that blood pressures determined in the restrained unanesthetized rat were 22 ± 7 mm Hg higher than those obtained on the previous or following day under ether anesthesia using the same equipment (see Discussion). This difference was significant using a paired t test (P < .01). Effects of Algenic Medulay and Cortal lmpnts and Left Ureteral Ligation
In TSHRs without spontaneous hydronephrosis (Text-figure 1), there was a significant difference (P
A significant number of offspring from brother-sister matings of N'IH-Okamoto-Aoki spontaneously hypertensive rats (SHRs) were found to be normotensive at 20 weeks of age. Over 20% of the animals that were hypertensive at this age had mild-to-moderate unilateral hydronephrosis at the time of sacrifice. In over 90% of the rats that did not develop hypertension spontaneously, ligation of one ureter raised blood pressure above 150 mm Hg within 2 weeks. In those rats made hypertensive by obstructing one ureter and in those that developed hypertension with accompanying naturally occurring hydronephrosis, subcutaneous implants of fragmented renal medulla from unrelated normal rats decreased blood pressure to normotensive levels. In contrast, medullary implants had no significant effect in rats developing hypertension spontaneously without hydronephrosis. Renal inner medullary plasma flow was low in the obstructed kidneys of hydronephrotic hypertensive SHRs but was elevated in the kidneys of nonhydronephrotic hypertensive SHRs. The hypertension in hydronephrotic SHRs appears to be related to an impairment of the antihypertensive function of the renal medulla. Such an impairment of medullary antihypertensive function does not appear to play a significant role in the hypertension in SHRs without hycdronephrosis. (Am J Pathol 85:555-568, 1976) THE SPON-TAN-EOUSLY HYPERTENSIVE RAT (SHR) developed by
Okamoto and Aoki 1 has been widelv studied as a model of human essential hypertension, and various ideas have been advanced to explain the mechanism for the elevation of blood pressure. The following are examples of some of the factors implicated by various authors: decreased intracellular cyclic AMP,23 altered calcium binding in vascular smooth muscle,' increased mvocardial contractility,5 increased cardiovascular reactivity to prostaglandin F2a,6 decreased renal production of prostaglandins £-2 and A2,7 increased adrenocortical activity,8 abnormalities of the kallikrein-kinin system,8 altered dopamine B-hvdroxvlase activity,10 or an intrinsic renal defect in ion transport 11,12 perhaps provoked by increased sympathetic nerve activity. 13 The present experiments were undertaken to study the effects of implantation of renal medullarv tissue from unrelated rats into SHRs to see if a deficiency of the antihvpertensive factors produced by the renal medulla From the Department of Pathology. The Johns Hopkins University School of medicine and Hospital. Baltimore. Mlarvland Supported by Grants HL-07835 and GM-(041 from the US Public Health Service Accepted for publication July 2.3. 1976. Address reprint requests to Dr. Robert H Heptinstall. Department of Pathology. The Johns Hopkins Hospital, Baltimore. MD 21205
555
556
SOLEZ ETAL
American Journal of Pathology
plays a part in producing the elevation of pressure in this strain of rat. During these experiments it was noted that 2 rats behaved differently from the other 14 in that they responded to implantation with a dramatic fall in pressure whereas there w.as little change in the others. Inspection of the kidneys of these 2 rats revealed unilateral hvdronephrosis, a feature not observed in the other 14 SHRs. It was further noted early in the study that not all rats had become hypertensive by 20 Xveeks of age. Considering the possibility that a certain number of rats in the breeding colony became hypertensive by the development of hvdronephrosis, it wvas decided to simulate spontaneously developing hydronephrosis by tying off one ureter in that population of rats that had not developed hypertension by 20 weeks. It was found that hypertension could be produced in a high proportion of these rats by tving off one ureter. In order to explore the mechanism of hypertension in this group, the effect of implantation of medullary tissue was studied and compared Xwith the effect of implantation in rats that developed hypertension naturally, i.e., without the development of spontaneous hvdronephrosis. To test the possibility that anx impairment of medullary antihvpertensive function observed wvas caused by medullary ischemia, inner medullary plasma flov- was determined in the various groups studied. Materials and Methods General Consideratons, Terminolgy for Rats, Ureteric Ligation Adult Okamoto-Aoki SHRs bred from brother-sister pairs from the :33rd filial generation of the National Heart and Lung Institute colony (supplied by Dr. Carl Hansen) were used in this stud!-. The breeding pairs and their offspring were housed in filter-top plastic cages and given Purina Rat Chow and w-ater ad libitum. The general term spontaneously hypertensive rat (SHR) is used for all of the rats in the colony. The SHRs which became hypertensive spontaneously are designated TSHRs (true SHRs). Some of these TSHRs had unilateral h%-dronephrosis. Comparison studies were carried out in Kyoto-Wistar rats (Taconic Farms). Holtzman rats, and Sprague-Daw ley rats (Charles River) housed under similar conditions. Both males and females were studied. Ligation of the left ureter 1 cm above the ureteroxesical junction was carried out under ether anesthesia in :38 SHRs that failed to develop hypertension spontaneously by 20 to 30
weeks of age. The animals of this group which became hypertensive within 2 Weeks after initial ligation are referred to as HO-SHRs (hypertensive-obstructed SHRs).
Medullary Implants and Blood Chemistry and Histologic Studies Subcutaneous allotransplantation of normal renal medulla from 160 to 220-g female Holtzman rats to 20- to 70-week-old TSHRs (N = 16) or HO-SHRs (N=10) was carried out as described b%- N\uirhead et al."4 \ledullarv tissue from 3 Holtzman donors (six kidnevs(
was used for each SHR recipient. As a control for the implants of medullary tissue, implants of cortical tissue were also carried out in TSHRs (N=6) and HO-SHRs (N=l0) using the technique of \lIuirhead et al." Rats w-ere killed by exsanguination under ether
Vol. 85, No. 3 December 1976
RENAL MEDULLA AND HYPERTENSION
557
anesthesia 3 weeks after implantation of cortical or medullary tissue. Blood was taken from the inferior vena cava for serum urea nitrogen and creatinine determinations w-hich were carried out as previously described.'5 Paraffin-embedded kidney tissue which had been fixed in 10%5 buffered formalin was cut at 4 u and stained with hematoxylin and eosin. In addition to these histologic studies on rats which had received implants. gross and microscopic examination of the kidneys was carried out in 141 SHRs which were killed by exsanguination under ether anesthesia at 12 to 70 weeks of age. Blod Pmsure Measurements Indirect systolic blood pressure measurements were made on rats lightly anesthetized with ether using the indirect tail cuff method, as previously described.,' using apparatus from Narco Biosvstems, Houston, Texas. When tail pulse tracings of suitable magnitude appeared, systolic blood pressure was determined five times and the average of these five determinations was taken as the blood pressure value for the day. Rats with sy stolic pressure of 150 mm Hg or greater were considered hypertensive. Blood pressure was determined at least twice a week in the SHRs from age 3 months until sacrifice. For :3 weeks following implantation of renal tissue or ligation of one ureter, blood pressure w-as determined dailv. Medullary Plasma Flow Determination
The method used for deterrnining medullary plasma flows has been discussed in detail in a previous publication from this laboratory.15 The method is based on the fact that the transit time for circulating albumin in the renal papilla for the normal rat is about 40 seconds. If the radioactive albumin is allowsed to circulate through the kidney for 30 seconds, the accumulation rate of papillary radioactivity during this period is proportional to plasma flow rate, and by comparing this to the average radioactivity in the arterial blood, an accurate estimation of medullary plasma flos may be obtained. Inner medullary plasma flow was determined in TSHRs without hydronephrosis. TSHRs with spontaneous unilateral hydronephrosis, HO-SHR rats one day after left ureteral ligation, HO-SHR rats 2 w-eeks after left ureteral ligation, normotensive SHRs. normal Holtzman rats, normal Sprague-Dawley rats, and normal Wistar-Kyoto rats (normotensive rats of the strain from which the SHRs were developed by selective inbreeding). Albumin Transit Tume The effective transit time for circulating albumin in the inner medulla of TSHRs and normal Holtzman rats was determined using methods previously described 1s by study ing the volume of distribution of 15I-albumin in the papilla 13, :30, 45, and 60 seconds after radioactive albumin had been allowed to circulate through the kidney. This determination
was necessary to establish the appropriateness of the :30-second infusion time for the determinations of inner medullary plasma flos-.15 Statistal Methods Student's t test was used to evaluate the significance of differences betw een means.
Results Hypertenson
The offspring of four F,,, mating pairs (obtained directly from the National Heart and Lung Institute) and six F.4 mating pairs were studied.
American Journal of Pathology
SOLEZ ET AL
558
The females in three of the F3 mating pairs had systolic blood pressures of 120 to 140 mm Hg at 20 weeks. The remaining females and all the males of both the Fu and F. breeding pairs had systolic pressure above 150 mm Hg at this age. Average systolic blood pressures in the total progenv of the F., and F. mating pairs at 20 weeks of age was 144.2 ± 2.1 mm Hg in the males (N=140) and 118.2 ± 2.9 mm Hg in the females (N=98). The total incidence of hypertension in the offspring (both sexes combined) of individual mating pairs at 20 weeks of age varied between 26.1 % (6 of 23 rats) and 72.0% (20 of 27 rats) in the offspring of the F. pairs and between 64.3% (9 of 14 rats) and 100% (24 of 24 rats) in the offspring of the FS pairs. Some of the rats remained normotensive for up to 18 months. In 17 SHRs that represented a random sampling of our colony, we observed that blood pressures determined in the restrained unanesthetized rat were 22 ± 7 mm Hg higher than those obtained on the previous or following day under ether anesthesia using the same equipment (see Discussion). This difference was significant using a paired t test (P < .01). Effects of Algenic Medulay and Cortal lmpnts and Left Ureteral Ligation
In TSHRs without spontaneous hydronephrosis (Text-figure 1), there was a significant difference (P
