Laragh's lessons in pathophysiology and clinical pearls for treating hypertension

Fig. 1 shows the concentrations of various hormones compared with the concentrations of glucose, urea, and cholesterol that occur in human blood. It can be seen at a glance that even the most abundant hormone, cortisol, is present in the blood in only 1/10,000th of the amount of glucose, cholesterol, or glucose. But cortisol in turn is a million times more abundant than is renin and 100,000 times more abundant than is angiotensin in the blood. Thus, renin occurs in the blood in one-ten billionth, and angiotensin in one billionth of the amounts of either glucose, urea, or cholesterol. To better visualize these relationships, consider that there are more than six billion individuals on earth. If each person on earth were to be represented by a cholesterol (or a glucose or urea molecule) in the blood, then, to keep the normal relationship, only one person on earth would be a renin molecule. And if renin were to double its concentration on the earth then only two of the six billion people would be renin molecules. Yet this still incredibly low concentration of renin would be enough to sharply increase your blood pressure and if renin were increased by three to fivefold (ie, to 5 people out of 6 billion), this could be enough to quickly cause a devastating stroke. Such is the incredible pathophysiologic power of circulating hormones such as renin, or angiotensin, or insulin. These tiny amounts are continuously changing their concentrations in the blood in response to everyday influences to do wonderful things to keep us healthy. But the tiny amounts of renin in our blood also create staggering problems for those trying to measure renin accurately. In fact, even the most sensitive antibody-based immunochemical techniques are not up to the task. Thus, contrary to a popular belief, highly immunoreactive monoclonal antibodies to renin cannot detect and quantify the lower portions of the range of plasma renin or angiotensin levels that occur everyday in human hypertension. This is why Dr. Sealey of our laboratory designed a modification of the kinetic assay in which renin is routinely incubated in its own plasma for 3 h. But for detection and definition of the lower range of plasma renin activity (PRA) <0.65, besides our routine 3 h, we incubate for 18 h to generate thousands of copies of angiotensin I, creating amounts well above the “blank” values to enable the precise quantitation of even the lowest naturally occurring plasma renin values and allowing us to establish, with great confidence, in individual patients the true absence and the lowest true presence of renin in blood samples collected. Only with this type of methodology can we define which hypertensive patient really has or does not have an active and pathogenic renin factor in the hypertension. This information is crucial for planning mechanistically specific targeted drug treatments by correctly giving an anti-sodium volume V drug as opposed to an anti-renin R drug, to those who lack, as opposed to those who exhibit, a demonstrable circulating renin factor. Plasma renin testing for the diagnosis and treatment of hypertensive patients has a checkered history as indicated in the previous lesson. This is because renin is so incredibly potent that it circulates in tiny amounts (<10−12 mol/L). These incredibly low concentrations of renin in blood, which create inherent difficulties in detecting it, caused the development of time-consuming stimulatory protocols to try to increase the endogenous renin level. This effort dampened the enthusiasm of the clinician and complicated the interpretation of results. For many years we insisted that the renin test be measured under special conditions of posture, diet, or stimulation, and that all hypertensive patients had to be removed from antihypertensive drugs for at least 3 weeks. This greatly reduced the number of physicians willing to perform the test because of the time and space that had to be invested and also because they were often reluctant to stop antihypertensive medications. Gradually we learned that these perceptions were incorrect and that renin is, in fact, best measured under ordinary ambulatory clinic conditions. With our sensitive assay this reveals the true level of renin in the blood under usual everyday conditions in which the hypertensive state is being maintained. To achieve this we had to readdress the methodologic problem of accurately measuring plasma renin. Over 8 years we developed a test that allows us to measure precisely even the smallest amounts of renin occuring in any blood sample, without the need to impose any artificial conditions for blood collection. But even before the above two simplified conditions were achieved it became apparent that there were several frequently used methodologic errors and differences in processing of samples among laboratories doing renin testing, which grossly changed the apparently “correct” renin answer. This situation led to internal disputes among experts as to the facts about renin's true role in hypertension and this in turn confused the practicing clinician who wanted to use the renin test to characterize and treat patients. Accordingly, by the early 1970s we realized that the methods for measuring plasma renin were grossly inadequate. These methods were used by investigators who strongly disagreed with our idea that plasma renin was causing most human hypertension. There were two main reasons for this disagreement: First, investigators in the US, new to the field, set up methods for measuring renin that were insensitive and inaccurate. One such method proselytized as the way of the future by Ed Haber at Harvard, was widely adopted. We investigated this method and found that renin was actually destroyed during this assay procedure by an uncontrolled and rising pH. This, plus a short incubation time, created a test that lacked any diagnostic power except for detecting very high values. Because of its poor sensitivity and lack of discriminatory power it finally died out. The second methodologic problem with the renin assay occurred because of an unforeseen flaw in renin methods designed by scientists in the UK and Australia (Robertson, Lever et al, and Skinner et al) in which the plasma samples were routinely strongly acidified before the assay. It was only learned later (in 1975) that an inactive prorenin exists in all human plasma at concentrations 2 to 200 times those of active renin1–3 and that the routine acid treatment used1–4 converted some, or all, of this large inactive flotilla ex vivo into active renin. This artifactually created varying but often large increments of measured active renin in the plasma sample thereby wiping out the low end of the true renin spectrum that occurs in hypertensive patients. Furthermore, this variable conversion of prorenin to renin unpredictably scrambled the true rank order throughout the entire spectrum of individual values in hypertensive patients. Consequently, investigators using these methods routinely could neither reveal nor characterize those hypertensive syndromes that had truly low renin values, such as low renin essential hypertension or primary aldosteronism. Moreover, their assays, to our surprise, also incorrectly reported the occurrence of considerable amounts of active renin in anephric humans, who, in fact, lack any renin but do have prorenin in their plasma. In anephric persons, active plasma renin levels are truly zero.5 But even bigger problems were created for us by this same unrecognized error caused by routine acidification of plasma samples. Thus, investigators using the acidification step could not show6,7 that β-blockers powerfully and persistently reduce plasma renin levels by about 75%. This led them to the false conclusion that β-blockers are not antirenin system drugs and led them to ridicule the idea that lowering the blood pressure with a β-blocker identified the presence of renin-dependent hypertension. To the credit of Dutch workers4 and Australian scientists,8 when they became aware of the tragic error caused by routine acid activation of plasma prorenin, to active renin, they published corrections, as did Tony Amery9 in Belgium, who retracted his own work and corrected it, stating that β-blockers really did consistently reduce renin levels. This work vindicated our methodology and validated our findings, but this confusion did a lot of damage that is only now being mended. Sadly, this situation led to an overall discouragement of many clinicians who had previously been intrigued by the idea of using renin testing to guide treatment of the hypertensive patient. The procedures for collecting blood for a plasma renin test are essentially the same as those for almost every other routine test3: 1) Collect 7 mL, or less (3 mL), blood into an EDTA Vacutainer (lavender top) from a quietly seated ambulatory patient in the clinic setting. Do not study patients in bed: the renin system is “asleep”. Fasting or special diets are not required. 2) After collecting the blood, avoid chilling on ice or during centrifugation because chilling to temperature below 6°C, but not freezing, like acid treatment (see Lesson XIX) by cryoactivation in vitro converts plasma prorenin to active renin making the results of the PRA test falsely high. 3) Accordingly, deliver the blood sample in the sealed Vacutainer at room temperature to the laboratory within 24 h. The plasma should be separated at room temperature and then stored frozen, if the assay is not to be performed that day. Send the blood sample for a renin test to a clinical laboratory that is able to routinely extend the Ang I generation step to 18 h for those samples incubated for 3 h that exhibit PRA <0.65 ng/mL/h. This is important for an accurate discrimination of low renin patients. This prolongation of sample incubation at 37°C allows the renin in the patient's plasma to generate thousands of copies of Ang I, which are then detected by radioimmunoassay, enabling greater sensitivity and specificity. Only when low renin patients are accurately separated out, can one be sure which patients are low, and then determine who in fact truly have medium and high values. Beware of so-called direct assays of renin in plasma such as direct immunoassays like the immunoradiometric assay (IRMA). They are neither sensitive enough nor specific enough for the detection and discrimination of low renin patients, an essential requirement of the Laragh Treatment Method. You can readily tell which type of assay is used by examining the reporting units. The preferred more sensitive and accurate enzyme kinetic assay for renin is reported as a rate of angiotensin generation (in nanograms per milliliter per hour [ng/mL/h]). The direct assay is reported as a direct concentration of renin (usually in picograms per milliliter or units per milliliter [pg/mL or U/mL]). These direct assays are only good for detecting the high renin values, which comprise <10% of the hypertensive population. Such assays also report their lower values, but they are inherently inaccurate because the test is not sensitive enough to discriminate among low and medium values and their differences above or below the “blank.” If you cannot identify and separate out the truly low and medium values you will never be able to say who really has renin in the blood and who does not among your patients. Thus, you cannot address the key pathophysiology and therapeutic question, that is, does the patient have a renin factor? If you follow all of these guidelines and the laboratory that serves you still finds renin in anephric human plasma, get your money back! To perform the renin test so that it can be used in the Laragh Treatment Method, it must have the sensitivity required to enable the detection and definition of subnormal plasma renin levels (ie, <0.65 ng/mL/h). Such low levels are encountered in some 30% of all hypertensive patients for whom, in the Laragh Treatment Method, the initial approach to antihypertensive drug therapy is quite different (see Lessons XVI and XVII). Maximum sensitivity is achieved in the samples from these patients shown to have PRA <0.65 ng/mL/h by prolonging the enzyme kinetic step in which Ang I is generated at 37°C from 3 h to 18 h. This does not mean that the sample must be prepared twice. It merely requires division of the prepared sample into two portions. One portion is incubated for 3 h and the other is frozen. Only if the PRA is <0.65 ng/mL/h, then the second portion is incubated overnight at 37°C. Otherwise, it is discarded. This option for the 18-h incubation allows generation by the patient's plasma renin of thousands of copies of Ang I, which then can be quantified by radioimmunoassay as accurately as if the sample had a medium or high renin level. Without this large multiplication factor, the more recently developed direct IRMA of plasma renin are not nearly sensitive enough to accurately quantify and discriminate among the low values and should be avoided. The intrinsic problem is the fact that the concentration of renin in plasma is an order of magnitude lower that for any other circulating hormone (see Lesson XIX and Fig. 1). Accordingly, as already indicated 1) the enzyme kinetic assay should be performed under conditions that enable Ang I to be generated under stable conditions for both 3 and 18 h. The longer time period is only needed for those 3-h samples with PRA <0.65 ng/mL/h. In these samples, the 18-h procedure generates much larger amounts of Ang I, which can then be detected with accuracy because any nonspecific plasma interferences (blank) with the radioimmunoassay become very small relative to the amounts of Ang I generated and can be ignored. Thus, this prolonged incubation creates the additional advantage of eliminating the need for measuring the endogenous “Ang I” in plasma, often called the blank. 2) The Ang I generated at 37°C must be protected from degradation as it is formed. That can be done most effectively at pH levels close to 5.7 because plasma angiotensinases are most easily blocked at this pH. An added plus of pH 5.7 is that Ang I is generated more rapidly at this pH than at pH 7.4 and in turn, this augments the sensitivity of the assay. 3) Before the Ang I generation step is carried out the plasma must be protected from temperatures between 6° and −6°C (ie, from temperatures that are cold but not so cold that the plasma freezes). This is because cold, but not freezing, temperatures “cryoactivate” the comparatively large amounts of prorenin in plasma into active renin, thereby artifactually causing, ex vivo, the higher renin values. Interpretation of the plasma renin test becomes easy once you understand that the normal function of the renin system is to make sure that the kidneys always have sufficient perfusion pressure to filter the plasma appropriately, but not so great that it results in inappropriate hyperfiltration. This means that the activity of the renin system (ie, kidney renin release) is suppressed as renal arterial perfusion pressure but when renal arterial pressure kidney renin is so that the PRA level Moreover, renal perfusion pressure is on arterial blood pressure it in with arterial The PRA when and it when In this renin system when or kidney perfusion pressure is low, renin is in greater amounts from the kidneys into the in an to arterial and renal perfusion That means that for normal the plasma renin levels in all of us are encountered under conditions of the lowest (ie, in those who are or as in or in or or for in response to In the very lowest plasma renin levels occur in those hypertensive by a volume such as low renin essential hypertension or primary aldosteronism. Accordingly, in the normal human our plasma renin levels occur at at which our is lower than at any other time in as during normal and plasma renin levels or so our plasma renin levels the lowest levels that can occur in a population. the lowest and truly subnormal plasma renin levels are encountered in low renin hypertensive patients who are a response of the kidney renin system to their high which is being by a volume this means that in all of us the plasma renin level must be in the of the level. First, as in Fig. a PRA level that is high when is normal to low is quite as it is to the normal and it from to even lower levels. a PRA that is high when is also high is quite a in the renal of this crucial system so that the high renin level is now the hypertension. a PRA level that is low when the is normal that the be volume In this the of renin is the from Accordingly, a PRA level that is low when is high an response of the renin system to the a medium PRA level in a patient with normal that the renin system is the amount of Ang to keep at a normal level. a medium PRA level in a patient with high is a in this crucial system in which the kidneys to turn renin in the of hypertension these relationships, the idea that a renin level is high and is in a hypertensive patient is to for people who do not like (ie, people who are not used to about there are many in of apparently normal hormone levels causing For a normal level is or low in a patient with an blood And a normal level is and can cause in a patient with It is the of this Lesson to consider a normal plasma renin level can and hypertension in a patient but it can and does (see Lesson that it is to the fact that renin is not by one the but by the of a million different individual per each with their own individual and within each of these with the of essential hypertension can become time, a we is then continuously and from these so that the kidney can longer its normal to keep normal by renin on or in response to in or Fig. 3 the clinical reporting guidelines for routinely the PRA values that occur in either or ambulatory hypertensive patients. These guidelines the facts that hypertension is by either a primary volume values <0.65 or by a plasma values in the by a relative of the volume factor relative to the renin level or This and of patients is because the primary drug therapy is quite different for each First, of hypertension to the drugs drug and not to the antirenin R drug those patients with PRA ng/mL/h best to the antirenin system R drug and not to the V Accordingly, a good response or a lack of response to the therapy then also the of a second drug type (see Lessons XVI and XVII). the same time, renin levels the of a renin or renin. these levels are also as a test for out of volume hypertension primary PRA <0.65 or hypertension ng/mL/h). it is apparent that this of patients for their diagnosis and treatment is not a sensitive type of kinetic assay is used with a to the plasma incubation to large amounts of angiotensin in patients with lower values. This routinely of the subnormal range to thereby those who do not from those who do have a true renin factor. Plasma renin testing is in patients with Ang renal or because the pathophysiologic by hypertension strongly plasma Ang and also in the of or renal to a or kidney In this plasma renin testing is to have diagnostic in the above Thus, it is now well that a but not all, patients an exhibit higher plasma renin levels than do those in the diagnosis is not patients with often exhibit high plasma renin often by or a of patients exhibit low or medium renin An important study by et that if patients are amounts of their renin levels to the subnormal range to even those occurring in low renin essential hypertension or in primary aldosteronism. This that renin system in not be primary but is to Thus, as in hypertensive patients the renin level either a volume or a renin of the blood pressure levels. Accordingly, in patients with low renin levels can be to a volume and it by V drug high renin levels the need to the R drug well be in both and renal patients. of these renal patients have never or renin This is a it is that an antirenin drug them these only to a with most patients testing could patients into and a for new therapeutic for the patients. The same to of the or or With and of specific R and V renin testing in these syndromes could have great for more specific The Laragh be used in an an or for and patients with hypertension or and renal Such an be to patient information such as clinical and laboratory values. This of the Laragh can be to almost diagnostic and treatment on and renin levels. The of such a allows much of the of patients and the of clinical to be carried out by or by other the from these time-consuming and the overall of of an of the Laragh is the of this to patient Such be in diagnosis and treatment and a specific of an a a for patient such as previous and PRA laboratory values. The then this information and a of treatment on the Laragh Method. The is in a on a or and of ambulatory plasma renin levels and of the various plasma renin response to of antihypertensive drugs the to the most drug, or drug for each patient. primary is to use one drug of or two of for the that hypertension Thus, the Laragh that a time be at the to work out the best drug for each which can have in and For the pathophysiologic treatment can often not to the and patient by their in a treatment The Laragh the to determine the and best drug treatment for each patient. This system a for new hypertensive patients as well as the hypertensive patient who is already on but is either not or should be for clinical or because the of the has led to physicians or in the will understand that some of the of these can be to still the method and its results.