In this study we outlined the development of an enzymatic technique to activate plasma inactive renin by trypsin in rat plasma. Using this method, we reported the releasing mechanism of the trypsin-activable inactive renin which has not yet been clarified. Adult male Wistar rats (260-300g) were kept on regular diet (Na : 260mg/100g) unless explained and underwent operation under pentobarbital anesthesia (50mg/kg). Blood samples were obtained from conscious rats through the cannulae, which had been inserted into the left femoral arteries 24h before the experiments. After addition of excessive renin substrate which had been obtained from the 24 h-nephrectomized rat plasma, renin was measured by the commercial RIA-kit (Dainabot). Trypsin (Worthington) treatment (20mg/ml plasma for 10 min at 4°C) was followed by addition of SBTI (Sigma) (20mg/ml plasma). This condition maximally increased the rate of angiotensin I generation and did not alter the Km or optimum pH of the renin reaction. In this condition, trypsin reaction was completely inhibited by adding these concentrations of SBTI. The molecular weight of inactive renin (51,000) in the normal rat plasma estimated by Sephadex G-100 column (Pharmacia) was the same as that in the nephrectomized rat plasma. In conclusion, trypsin treatment of plasma (20mg/ml plasma for 10 min at 4°C) followed by SBTI (20mg/ml plasma) was justified for trypsin activation of rat plasma.
Using this method, we investigated the changes in active and inactive renin after bilateral nephrectomy in the salt-depleted rat. Active renin decreased rapidly after bilateral nephrectomy with a half life of 23.6±4.0 min. Inactive renin, on the other hand, increased gradually and reached to a plateau 24h after bilateral nephrectomy, and was kept unchanged during the following 24h. The infusion of mouse submandibular gland active renin or angiotensin II could not prevent the increase of plasma inactive renin in the nephrectomized rat. These suggest that there may be no feedback mechanisms between plasma inactive and active renin or angiotensin II.
Furthermore, we investigated the organ-related sources of plasma inactive renin which markedly increased after total nephrectomy. Simultaneous removals of submandibular glands but not of adrenal glands completely prevented the postnephrectomy increases of plasma inactive renin. But, removals of submandibular glands or adrenal glands alone were followed by no changes in the basal levels of plasma inactive renin. It is concluded that the increases of plasma inactive renin may be dependent on the presence of the submandibular glands, but not of the adrenal glands. However, the real sources of plasma inactive renin are still unknown.
