Crude Kidney Extract Research Articles

Aliskiren and Dual Therapy in Type 2 Diabetes Mellitus

The discovery 110 years ago that crude kidney extract elicited a pressor reaction provided the first evidence of the existence of the renin–angiotensin–aldosterone system, a major vasoactive system,1 and the complex actions and interactions of this important cascade are still unfolding. Over the past few decades, various components of the renin−angiotensin−aldosterone system have been elucidated more completely, and previously unknown members have been discovered.2 Drugs that specifically block various components of the renin−angiotensin−aldosterone system have been developed (Figure 1). Angiotensin-converting–enzyme (ACE) inhibitors block the conversion of angiotensin I to angiotensin II, and clinically available angiotensin-receptor blockers block the angiotensin II . . .

The Kallikrein–Kinin and Renin–Angiotensin Systems in the Kidney of an African Lungfish,Protopterus annectens

Renin-like activity (RLA), angiotensin I converting enzyme-like (ACELA), and kallikrein-like activity (KLA), activities of the key enzymes of renin–angiotensin and kallikrein–kinin systems, were sought in the kidney of the African lungfishProtopterus annectensduring the aquatic phase. RLA, examined by RIA (using porcine angiotensinogen as substrate), was 0.38 ± 0.05 ng angiotensin I/mg protein/hr. ACELA and KLA were investigated in assays spectrophotometrically. ACELA, measured at 37 and at 20°, was, respectively, 1.55 ± 0.55 and 0.61 ± 0.23 nmol hippurate/min/mg protein. KLA was 7.34 ± 0.93 mU/mg protein in the crude kidney extract and 31.05 ± 7.50 mU/mg protein after electrophoretic purification. Renal kininogenase activity was inhibited by 100% byD-Phe-Phe-Arg-chloromethyl ketone (10 μM), 98% by phenylmethylsulfonyl fluoride (2 nM), and 91% by aprotinin (1000 kIU). The apparent molecular weight of the renal kininogenase on SDS–PAGE was 27,000 Da. Both the renal enzyme and the purified glandular kallikrein, used as a control, have the same mobility on polyacrylamide gel electrophoresis. Immunoreactivities toward angiotensin II and bradykinin were localized by double immunostaining in the same cells of the proximal tubules. Putative angiotensin II receptors were demonstrated immunohistochemically, in the supranuclear region of proximal tubular cells, using an antibody to the sequence between amino acids 225 and 237 of the mammalian AT1receptor.

The effect of picrylsulphonic acid on In vitro conversion of cyanide to thiocyanate by 3-mercaptopyruvate sulphurtransferase and rhodanese

This study indicates that 3-mercaptopyruvate sulphurtransferase (MPST; EC 2.8.1.2) activity may serve as a useful in vitro indicator for the analysis of cyanide detoxification to thiocyanate. The time course and capacity of MPST to detoxify cyanide was equal to or exceeded that of rhodanese. Picrylsulphonic acid strongly inhibited purified rhodanese, but in the presence of mercaptopyruvate, it could increase the formation of thiocyanate catalysed by MPST. Formation of thiocyanate by MPST followed a linear time course and had a linear relation to enzyme level. However, substrate dependence did not produce linear Lineweaver-Burk plots when either mercaptopyruvate or cyanide concentration was varied. The differential effect of picrylsulphonic acid on the activities of these two enzymes was confirmed by using a crude kidney extract as the source of both enzymes. Picrylsulphonic acid may provide a useful scientific tool to examine which sulphurtransferase is most responsible for the detoxification of cyanide.

Historical perspectives on the renin-angiotensin-aldosterone system and angiotensin blockade

Advances leading to recognition of the relation of the renin-angiotensin system to aldosterone include: (1) development of analytic techniques for measuring aldosterone, (2) discovery of an aldosterone-stimulating factor in circulating plasma, (3) the finding that a potent aldosterone-stimulating factor is secreted by the kidney, (4) evidence that synthetic angiotensin II increases aldosterone secretion, (5) fractionation of crude kidney extracts and the finding that aldosterone-stimulating factor is renin, (6) the observation that high plasma renin activity occurs in secondary alldosteronism, and (7) recognition that the renin-angiotensin-aldosterone system occurs in congestive heart failure and in renovascular and malignant hypertension. The early use of blocking agents for the renin-angiotensin system is described along with the landmarks of progress. These include the observations that: (1) arterial pressure decreases in experimental renovascular hypertension in response to angiotensin blockade, (2) angiotensin provides important support for arterial pressure in low cardiac output states including congestive heart failure, (3) the kidney participates in this important compensatory mechanism, and (4) cellular receptors for angiotensin are present in the two inner zones of the adrenal cortex.

Purification of a completely inactive renin from hog kidney and identification as renin zymogen

Hog renal inactive renin was separated from active renin and completely purified to an electrophoretically homogeneous state by using a new procedure which consisted of affinity chromatography on pepstatin-Sepharose, octyl-Sepharose, Affil-Gel blue and Con A-Sepharose columns, ion exchange chromatography and gel filtration. By this method a 3,000,000-fold purification was obtained with a 6% recovery from a crude kidney extract. This pure preparation was totally inactive and underwent marked activation by trypsin. It is a glycoprotein as judged by affinity to concanavalin A and has an apparent molecular weight of 50,000 as determined by gel filtration on Sephadex G-100. Treatment of the inactive renin with guanidine, urea and Triton X-100 did not cause activation indicating that the inactive renin isolated in the present study is not a product of renin-inhibitor complex.

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.

Purification of human renin

Human renin was purified 2,800-fold from a partially purified preparation to an electrophoretically homogeneous state by a series of three different types of affinity chromatography and two additional conventional chromatographic steps at a yield of 9.7%. This amounts to a 420,000-fold purification from a crude kidney extract. This pure human renin preparation has a specific activity of 830 Goldblatt unit/mg and is stable at pH 6.2 and 4°C at least for 3 weeks.

Role of enzyme interactions in the regulation of glycolysis and gluconeogenesis. Purification and properties of the phospho- and dephospho-forms of glycogen phosphorylase from swine kidney.

Dephosphophosphorylase was isolated from swine kidney by a procedure involving precipitation with acetone and ethanol in the presence of glycogen, ammonium sulfate fractionation and chromatography on DEAE‐cellulose. The enzyme was purified about 15000‐fold to a final specific activity of 30 μmol glucose‐1‐phosphate formed from glycogen and inorganic phosphate per min per mg protein at 26°C. Yields of 20% of the activity present in crude extracts were obtained by this method. The purified enzyme could be completely converted to a phosphorylated form by incubation with ATP, magnesium ion and a partially purified soluble protein kinase which was isolated from the same crude kidney extracts. The final specific activity of phosphophosphorylase isolated by the same procedure was about 70 μmol glucose‐1‐phosphate formed from glycogen and inorganic phosphate per min per mg protein at 26°C.Ultracentrifugation and polyacrylamide‐gel electrophoresis studies with the purified enzymes indicated that the preparations were homogeneous. Molecular weight determination by Sephadex G‐200 and Sepharose 6‐B column chromatography revealed V0/Ve ratios which corresponded to a molecular weight of 190000 ± 5000 for both of the enzyme preparations. Sucrose‐density centrifugation further confirmed that both forms of kidney phosphorylase had similar molecular weights of about 190000 ± 10000 and s20,w values of from 8.1 to 8.3 S. The s20,w values obtained in ultracentrifugation studies, 8.3 to 8.4 S, agreed reasonably well with those obtained by sucrose density centrifugation. The molecular weights of the two forms of the enzyme calculated from this data and a diffusion coefficient of 0.4 μm2/s which was determined by exclusion chromatography and a partial specific volume of 0.735 cm3/g was 195000 ± 5000 in each case. When the enzymes were subjected to polyacrylamide‐gel electrophoresis in the presence of sodium dodecyl‐sulfate a single sharp protein band was observed. These studies showed that both forms of the enzyme contained two identical subunits with molecular weights of 95000.Some of the kinetic and allosteric properties of the phospho and dephospho‐forms of purified kidney phosphorylase were examined and compared. The Km of the phosphorylated form of the enzyme for Pi was 2.8 mM compared to 100 mM for the dephospho‐form in the presence of 0.2 mg glycogen and 2 mM AMP. The corresponding values for glycogen at about 4 mM Pi were 1.4 mM and 20 mM for the phospho and dephospho‐forms, respectively. The apparent Km values for Pi or glycogen varied as a function of the other substrate for the dephospho‐enzyme. This effect was not observed with the phospho‐enzyme. The dephospho‐form of kidney phosphorylase required AMP for activity and protamine also stimulated the activity of this form of the enzyme. Glucose (0.5 mM) inhibited the activity of kidney dephosphophosphorylase.A protein kinase isolated from crude kidney extracts converted the dephospho‐form of the enzyme to a phosphorylated form in the presence of ATP and magnesium. About two equivalents of phosphate were transferred from ATP to the phosphorylated form of the enzyme during this conversion.

Studies on a “renin-angiotensin” system in the normal and hypophysectomized pigeon ( Columba livia)

The extraction of unincubated bird plasma, according to the methods described in mammalian studies, yielded an angiotensin-like substance. This substance when assayed in the nephrectomized rat elicited an increase in arterial blood pressure similar to that of 5-valine angiotensin II. Following incubation, the amount of pressor substance present in the plasma was increased 20-fold. By analogy with the mammalian system, this increment represented the “renin” activity of the plasma. The pressor activity of all plasma extracts was destroyed by carboxypeptidase A and trypsin. Crude kidney extracts did not elicit a pressor response in the nephrectomized rat. However, after incubation of the kidney extract with nephrectomized rat plasma, a pressor substance was produced. Thus, a substance having enzymatic activity similar to that of renin was present in kidney tissue of the duck and the pigeon. Acute hemorrhage significantly increased the plasma “renin” activity in the pigeon, and the plasma renin activity of each sample was linearly proportional to the amount of blood withdrawn up to the time of sampling. After chronic hypophysectomy of the pigeon, the mean arterial blood pressure decreased. At the same time the plasma renin activity increased, although the concentration of renin substrate decreased. When the chronically hypophysectomized birds were treated for 1 week with ACTH, the renin plasma substrate concentration and the plasma renin activity returned to normal. The mean arterial blood pressure, however, was not restored to normal.

The effect of the renin-angiotensin system on mucosal water and sodium transfer in everted sacs of rat jejunum.

1. Mucosal water and sodium transfer were measured in everted sacs of rat jejunum.2. Rats maintained on a high sodium diet did not show consistently reduced mucosal water transfer and aldosterone also failed to stimulate transfer unless the control untreated values were low.3. Adrenalectomy produced little reduction in mucosal water transfer and aldosterone was without effect on water transfer in sacs from adrenalectomized rats.4. Vasopressin had no effect on mucosal transfer.5. Levels of water transfer were not reduced after hypophysectomy nor did aldosterone injection increase transfer.6. Mucosal water transfer in sacs from adrenalectomized-nephrectomized rats was low and was significantly increased by aldosterone injection or by injection of crude kidney extract.7. A significant increased mucosal water and sodium transfer was observed when sacs from adrenalectomized-nephrectomized rats were incubated with angiotensin at a concentration of 10(-10)g/ml.8. It is suggested that the renin-angiotensin system may be involved in the maintenance of sodium homoeostasis by a direct action on sodium transporting mechanisms.

Increased cardiovascular reactivity caused by depletion of endogenous renin in dogs.

Increase in cardiovascular reactivity following bilateral nephrectomy has previously been found to be independent of the presence of the central nervous system, mechanical exclusion of the renal vascular bed, or loss of renal excretory function. It was considered possible that increased responsiveness results from removal of endogenous stores of renin. In nephrectomized dogs treated with crude kidney extracts, or infused continuously with synthetic angiotensin for 48 hours, enhancement of response to angiotensin and renin was inhibited while that to norepinephrine was not; response to tyramine was augmented as after infusion of angiotensin into intact dogs. Treatment with liver extracts alone did not inhibit response to renin or angiotensin; neither did continuous infusion of norepinephrine. When hog renin was added to liver extracts, the combination had the same inhibitory effect as kidney extracts. The data are in accord with the concept that depletion of endogenous renin contributes to the enhanced pressor responsiveness that occurs following bilateral nephrectomy.

Reversible inactivation of d-fructose 1,6-diphosphatase by adenosine triphosphate and cyclic 3′, 5′-adenosine monophosphate

An analytical method for the quantitative recovery and purification of fructose 1,6-diphosphatase from incubation mixtures containing crude kidney extracts has been described. Utilizing this procedure it has been demonstrated that renal fructose 1,6-diphosphatase was inactivated on incubation with ATP and cyclic-3′,5′-AMP. Fructose 1,6-diphosphatase present in crude kidney extracts was inactivated by ATP and Mg ++ alone, and the rate of this inactivation was increased in the presence of cyclic 3′,5′-AMP. Purified enzyme was inactivated by ATP only in the presence of crude kidney extracts. Fructose 1,6-diphosphatase was also inactivated when kidney slices were incubated in the presence of epinephrine. When fructose 1,6-diphosphatase was incubated in the presence of an AT 32P generating system in crude extracts, 32P was incorporated into the enzyme protein with a concomitant decrease in enzyme activity. Very little or no 32P i was incorporated in the absence of adenosine nucleotide. The inactivated enzyme was reactivated on incubation with crude kidney extracts. The possible relationship of these effects to those obtained with three other enzymes involved in the synthesis and degradation of glycogen and the mechanism of action of epinephrine is discussed.

Purification and some properties of carnosinase of swine kidney

1. 1. A purification of carnosinase from swine kidney is described. The method involves heating, ammonium sulfate and acetone fractionation, and, as final steps, zone electrophoresis on cellulose-stabilized columns. 2. 2. The product has a proteolytic coefficient ( C 0) of 600, representing an apparent purification of 400 when compared to the crude kidney extract and approximately 150 when compared to the values achieved previously. 3. 3. The protein material has been investigated by ultracentrifugation and zone electrophoresis, and under the conditions employed it appears homogeneous. In addition, the enzymic homogeneity has been investigated and discussed. 4. 4. Some observations on the stability and solubility, together with ultraviolet and visible spectra, are presented.