Urinary Kallikrein in Dahl-Iwai Salt-Sensitive and -Resistant Rats

Abstract

This study was designed to evaluate the differences between the renal kallikrein in newly established Dahl-Iwai rats under salt loading and that of Sprague-Dawley rats (SD). Urinary kallikrein quantity and activity was markedly lower in Dahl-Iwai rats than in SD even during the control period. Moreover, kallikrein quantity and activity in Dahl-Iwai salt-sensitive rats (SS) were clearly diminished in comparison with salt-resistant rats (SR). The kallikrein activity/quantity ratio was also lower in SS and SR than in SD during the control period. After salt loading, systolic blood pressure increased only in SS. Kallikrein activity in SS and SR, and kallikrein quantity in SS were increased, whereas those in SD did not change. Although the kallikrein activity/quantity ratio in SR reached the same level in SD after salt loading, that in SS was lower throughout the experiment. These results suggest that Dahl-Iwai rats are less able hereditarily to produce renal kallikrein and that there may exist structurally abnormal kallikrein that may have a lower activity. Different kinetics of renal kallikrein between SS and SR by salt loading might be explained by kallikrein inhibitors or abnormal kallikrein or nonkallikrein kininogenase. These different kinetics of renal kallikrein may play some role on blood pressure elevation in SS. Am J Hypertens 1997;10:73S-77S © 1997 American Journal of Hypertension, Ltd.

Dahl rat, Sprague-Dawley rat, kallikrein, hypertension, salt

The renal kallikrein-kinin system has been postulated to play a role in the renal water sodium metabolism and blood pressure regulation, and defects in this system could contribute to the pathogenesis of clinical hypertension. In fact, extensive epidemiologic studies have documented that an inverse correlation exists between blood pressure and urinary kallikrein levels.^1,,,,,–,7 Reduced renal kallikrein levels have also been described in a number of genetically hypertensive rats.^8,9 The inbred Dahl/Rapp salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats represent one of the best characterized and most widely studied models of salt-sensitive hypertension.^10,11 Carretero et al¹² reported that renal kallikrein excretion and its activity was much lower in salt-sensitive rats than salt-resistant rats, although they were not fed a high salt diet. Furthermore, Rapp et al¹³ and Arbeit et al¹⁴ showed the possibility that there could be genetically different kallikrein or kallikrein-binding inhibitors in SS/Jr. However, some investigators failed to confirm a hypertensive phenotype in salt loaded SS/Jr, and it was found out that there was genetic contamination in SS/Jr.^15,16 It seems to be possible that this genetic contamination affected the results of renal kallikrein-kinin in Dahl/Rapp, which many investigators described before.

Recently, Dahl-Iwai rat strains were newly established as an inbred strain,¹⁷ which is considered more purebred. To clarify the contribution of renal kallikreinkinin system in blood pressure elevation of Dahl rats, we used Dahl-Iwai rats and studied the time course of urinary kallikrein quantity and its activity, and compared them with those in Sprague-Dawley rats.

Materials and Methods

Female Dahl-Iwai salt-sensitive rats (SS, n = 8), salt-resistant rats (SR, n = 8), and Sprague-Dawley rats (SD, n = 8) at 4 weeks of age were placed in metabolic cages (Nalge, New York, NY) individually and kept in a temperature-controlled (25°C) room that was illuminated between 7:00 and 19:00. All rats were fed normal rat chow (1% NaCl) for the first week (control period) and then switched to high salt chow (8% NaCl) for an additional 3 weeks. Systolic blood pressure (SBP) and body weight were measured and 24-h urine samples were collected in the 2 days of every weekend. SBP was evaluated by the tail-cuff method, urinary sodium concentration was measured by the ion electrode method (Shimadzu clinical ion meter, Shimadzu, Kyoto). The urinary kallikrein quantity (KALq) was measured by direct radioimmunoassay as described by Shimamoto et al.¹⁸ Urinary kallikrein activity (KALa) was assessed by the kininogenase method.¹⁹

Statistical Analysis

All values were expressed as means ± standard error. Data were subjected to one-way ANOVA with repeated measures or two-way repeated ANOVA. The Bonferroni test was applied to the means after the one-way ANOVA. A value of P < .05 was considered statistically significant.

Results

There was no difference in SBP between any of the groups during the control period (SD: 123 ± 5 mm Hg; SR: 117 ± 2 mm Hg; SS: 119 ± 4 mm Hg). Two weeks after starting the high salt diet, SBP rose significantly (P < .01) and became hypertensive (177 ± 5 mm Hg) only in the SS group at the third week (Fig. 1). On the other hand, in SR and SD, SBP rose slightly but not to hypertensive levels. SBP showed no difference in any week between the SR and SD groups.

Change of SBP in SS, SR, and SD groups.

Figure 1.

Urine volume (UV) and urinary sodium excretion (U_NaV) were increased in all groups after the high salt diet. UV and U_NaV in SD were significantly lower than those in other groups. Body weight was more in SD than SS and SR throughout the experiment.

As shown in Fig. 2, KALq in SS and SR were significantly lower than that in SD during the control period. Moreover KALq in the SS group was much lower than in SR (SD: 197.6 ± 19.9 μg/kg/day; SR: 110.9 ± 7.8 μg/kg/day; P < .05 v SD, SS: 47.5 ± 8.4 μg/kg/day, P < .05 v SD and SR). KALq of SD and SR did not change during salt loading, but KALq of SS increased gradually and became not different from SR 2 weeks after starting the high salt load.

Change of urinary kallikrein quantity (KALq) and urinary kallikrein activity (KALa) in SS, SR, and SD groups.

Figure 2.

KALa was significantly lower in the SS and SR groups than in the SD group during the control period, and in addition KALa in SS was significantly lower than in SR (Fig. 2; SD: 52.2 ± 4.1 μg/kg/min/day; SR: 17.9 ± 2.9 μg/kg/min/day, P < .01 v SD, SS: 8.5 ± 1.3 μg/kg/min/day, P < .01 v SD, P < .05 v SR). KALa in SD did not alter after salt load, and KALa in both SS and SR were increased significantly, whereas KALa in SS and SR was significantly lower than in SD.

Fig. 3 shows the comparison of KALa/KALq ratio in SS, SR, and SD groups. The ratio of kininogenase activity to kallikrein quantity was significantly lower in both SS and SR than in SD at the control period (P < .05), and there was no difference in SS and SR. Although there came to be no difference between SR and SD groups at the second week after salt load, the ratio was significantly lower only in SS throughout the experiment (P < .05).

Comparisons of KAL activity/KAL quantity ratio in SS, SR, and SD groups.

Figure 3.

Discussion

In this study, the SBP of SS was increased time-dependently by a high salt diet, whereas hypertension did not appear in SR or SD (Fig. 1). This conformed to many former reports about Dahl/Rapp strains. Urinary kallikrein quantity and activity were markedly lower in both SS and SR rats than in SD rats during the control period (Fig. 2); moreover the quantity and activity in SS were clearly diminished compared to those in SR. These data imply that renal kallikrein production in Dahl-Iwai strains is less than in SD rats, and that it is also less in SS than in SR. This diminished renal kallikrein production seems to be genetic, because these abnormalities were observed although the rats were normotensive. Furthermore, the kallikrein activity/quantity ratio was significantly lower in both SS and SR than in SD during the control period and there was no difference between SS and SR (Fig. 3). The data concerning the control period indicate that Dahl-Iwai strains have abnormal kallikrein that has a low activity, or have kallikrein inhibitors that may inactivate kallikrein. Chao et al²⁰ showed the kallikrein-binding protein that inhibits kallikrein activity in serum, urine, and various tissues in the rat (spontaneously hypertensive rat and Wistar-Kyoto rat) by Western blot analysis and radioimmunoassay. They succeeded in the purification of a human tissue kallikrein inhibitor named kallistatin.²¹ However, we have no data about kallistatin in Dahl-Iwai strains. As the next step, kallistatin should be evaluated in this model by Western blot or radioimmunoassay.

It is well known that urinary kallikrein is increased by a low sodium diet, mineralocorticoid excess,²² or furosemide. In our study, kallikrein quantity did not change in SD or SR rats after salt loading, whereas in SS it was significantly increased time-dependently and was at the same level of SR at 3 weeks (Fig. 2). There have been no reports of increases in kallikrein quantity in humans and rats as a result of chronic salt loading. In SS, kallikrein quantity actually increased after salt load, whereas the kallikrein activity/quantity ratio decreased in SR. Considering these different kinetics of kallikrein, it seems probable that there are structural differences of kallikrein between SS and SR, and that abnormal kallikrein in SS, whose activity is lower than normal kallikrein, is inducible by salt loading. The abnormal kallikrein in Dahl/Jr was described by Rapp et al.¹³ They showed different urinary kallikrein between SS/Jr and SR/Jr and concluded that there existed a structural difference of kallikrein between SS/Jr and SR/Jr and that this different kallikrein may have different activities.

We cannot eliminate some other possibilities, for example, kallikrein inhibitors. If not only urinary kallikrein itself but also kallikrein inhibitors are increased simultaneously in SS by salt loading, it might be compatibly explained that kallikrein activity and the kallikrein activity/quantity are still lower in SS than in SR or SD. In SR, kallikrein quantity did not change but kallikrein activity increased after salt loading. Furthermore, after the second week the kallikrein activity/quantity ratio reached the same level as SD. These data may indicate that the effects of kallikrein-binding inhibitor itself was diminished through unknown mechanisms by salt loading in SR. On the other hand, the existence of nonkallikrein kininogenase, as reported by Chao et al,²³ might also be considered. They observed that SD rat have nonkallikrein kininogenase (esterase A) in the urine, which is about a quarter the amount of kallikrein produced on a normal sodium diet. And Sustarsic et al²⁴ also found esterase A in SR/Jr. If nonkallikrein kininogenase are stimulated by salt loading, we can understand the increase in kininogenase activity even without an increase in kallikrein quantity in SR after salt loading. As a result of these possibilities, there may be a protective effect at onset or on development of hypertension in SR caused by increasing kinin-generating activity.

In summary, Dahl-Iwai rat are hereditarily less able to produce renal kallikrein. SS may have abnormal kallikrein or kallikrein inhibitor and SR may also have kallikrein inhibitors or nonkallikrein kininogenase, and all the above could be affected by salt loading. These different kinetics of renal kallikrein between SS and SR by salt loading could partly explain why hypertension occurs. To clarify these abnormalities, further studies are needed.