Renin extraction

A correlation between the activity of G-6-PDH and extractable renin in rat kidneys has been reported, suggesting that the macula densa cells and also the juxtaglomerular apparatus are parts of a system related to the formation of renin (H6). 

The site of formation of renin is not known, although the indirect and circumstantial evidence favors slightly the juxtaglomerular apparatus rather than the tubules as a source (18). Crude renin, however, is extracted readily from renal cortex by saline extraction, acidification, and precipitation with ammonium sulfate and sodium chloride. Other active protein substances are likewise extracted, and their separation from renin is often a matter of considerable difficulty. The renin substrate, an arglobulin, is found in blood serum and is probably formed by the liver. It can be easily salted out of beef serum as a crude preparation. 

Under the proper stimulus renin is released into the circulating blood where it can be identified, especially in that from the renal vein. It acts rapidly upon its specific substrate, splitting the protein into peptides, one or more of which have been called hypertensin or angiotonin. Hypertensin has been concentrated but not obtained in pure form. It is the effector substance of renin, constricting arterioles and raising blood pressure. The action of hypertensin is abolished by hypertensinase, an enzyme found in blood and renal extracts. Fortunately, the latter is destroyed by heat and alkalinity. 

An unidentified substance, named nephrin, was described by Enger (19). Nephrin is a pressor material having a prolonged action, obtained from extracts of renal cortex, but not present in any other tissues. It was also found in urine and in blood and was said to have been present in increased amounts both in hypertensive dogs and in patients with hypertension, eclampsia, nephritis, and other hypertensive conditions. Its action was not intensified by cocaine nor affected by ergotoxin and it did not act upon the guinea pig uterine muscle. As it was dialyzable, it probably was not renin. If this work is substantiated, nephrin represents yet another renal pressor substance probably of nonprotein nature. Its relation to hypertension has not been substantiated. 

The renal pressor mechanism—renin and hypertensin—acts in acute hypertension and in acute renal ischemic states, but apparently not in chronic hypertension. The other mechanisms shown to be active in chronic hypertension are vasoexcitor-vasodepres-sor material relationship pherentasin, a pressor substance found only in human hypertension amines resulting from the insufficient oxidation of amino acids, which are increased in human hypertension and norepinephrine (Sympathin E), which largely reproduces the hemodynamic picture of chronic hypertension. Most of the known pressor substances, with the notable exception of norepinephrine, come from disturbances of, or are extracted from, the kidneys. The large number of pressor substances which have been obtained suggests that many may represent different stages of metabolism of certain parent substances, and that their effectors may be fewer in number and simpler in structure. The chemical identification and purification of most of these substances leave much to be desired, and their phafmacology has in most cases been inadequately studied. The whole problem, however, may soon become simplified. 

In 1898, the Finnish physiologist Robert Tigerstedt and his Swedish student Per Bergman coined the term renin to signify the soluble factor that was responsible for the dramatic rise in blood pressure observed upon injection of kidney extracts into rabbits.3... 

Milk Coagulation. The first step in cheese manufacture is the coagulation of milk. Traditionally, this coagulation step is catalyzed by the enzyme rennet. Rennet is a saline extract of the 4th stomach of calves, usually slaughtered before they are 30 days old. The principal protease in rennet is rennin. In an attempt to avoid confusion with the hormone peptide renin, the International Enzyme Nomenclature Committee has assigned the name chymosin to the protease in calf rennet. During the growth of calves, chymosin is replaced by pepsin, the acid protease of the mature stomach. 

Figure 6. Stereoview of the bound conformation of a phenyl tethered analog (upper, black lines) and a naphthyl tethered analog (lower, black lines) overlaid with Smo-Phe-Ets-ACDMH (gray lines) extracted from the endothiapepsin X-ray crystal structure [17] and oriented in the renin model active site.

Figure 8. Stereoview of renin inhibitors extracted from the X-ray crystal structures of three different aspartic proteases. The yellow inhibitor, 20, is from an HIV-1 protease structure (unpublished), the cyan inhibitor is from an endothiapepsin structure [17] and the green inhibitor is from a renin structure [43],...

By extracting water from intracellular compartments, osmotic diuretics expand the extracellular fluid volume, decrease blood viscosity, and inhibit renin release. These effects increase RBF, and the increase in renal medullary blood flow removes NaCl and urea from the renal medulla, thus reducing medullary tonicity. Under some circumstances, prostaglandins may contribute to the renal vasodilation and medullary washout induced by osmotic diuretics. A reduction in medullary tonicity causes a decrease in the extraction of water from the DTL, which limits the concentration of NaCl in the tubular fluid entering the ATL. This latter effect diminishes the passive reabsorption of NaCl in the ATL. In addition, osmotic diuretics may also interfere with transport processes in the TAL. 

A method for the extraction of papaverine from aqueous solution by toluene and organic acids has been described, as have methods for the determination of the alkaloid in blood by gas chromatography. The metabolism of papaverine has been studied, as have its effects on the release of renin in vivo and in... 

As we mentioned, the purification of renal renin had presented insurmountable difficulties in the past (8-17). Recently, several special techniques have been devised and applied to this difficult problem, including affinity chromatography using synthetic inhibitor peptides of renin as affinity ligands (17,20). Burton, et al. synthesized specific renin inhibitors for this purpose (21). These synthetic peptides, however, did not possess sufficiently strong affinity for renin to sequester it from a crude kidney extract. 

Corvol a. (27) and we (28,29) prepared pepstatin-aminoalkyl-agarose gels (Fig.3). This affinity column was highly effective in selectively absorbing renin present in minute quantity in crude kidney extracts containing vast amounts of non-renin proteins. 

There was another serious obstacle to the purification of renin. The reported instability of renin during its purification (12), particularly in its purer state, has been the major deterrent to the numerous attempts of renin purification in the past. We thought it likely that this instability was due to proteolytic destruction by contaminating proteases. Since elimination of these contaminants by physical separation methods was extremely difficult, they were selectively inactivated chemically by treating crude renal extracts with a cocktail of protease inactivators, diisopropyl-phosphoro-... 

Recently a number of investigators have reported the presence of multiple forms of renin in crude extracts of kidney and plasma (48-62). In addition to the enzyme with a molecular weight of 40,000, renins with higher molecular weight have been observed. 

Extracts of human kidney were assayed to see if they also contained inactive renin. However, the extracts showed peptidase activity that was not inhibited by the EDTA and o-phenanthroline buffer. To measure renin, the kidney extracts were chromatographed on DEAE-cellulose to remove excess peptidases (angiotensinases), before being assayed. Kidneys were ground up with sand and phosphate buffer, pH 7.4, in a ratio of 5 ml per gram of cortex (6). 

Active and inactive renin in extracts from eight kidneys after DEAE-cellulose chromatography... 

The inactive renin in humans may be a material similar to the inactive renin of rabbit kidney extracts. 

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