CASE PRESENTATION
A 50-year-old woman with type 2 diabetes mellitus and hypertension was admitted to Brigham and Women's Hospital in Boston for entry into a clinical research protocol involving hemodynamic responses to angiotensin II (Ang II) receptor blockade. She presented to a local emergency room with severe headache at the age of 40. Her blood glucose at that time exceeded 800 mg/dL. After 1 year of failed therapy with oral hypoglycemic agents, insulin therapy was initiated. Her blood sugar control has been fair; she complains of twice nightly nocturia. She also has a burning and tingling neuropathy in her hands and feet, and she suffers from migraines related to stress.
The patient's mother and father both died from complications of type 2 diabetes mellitus. One of her five siblings has diabetes that is treated with oral medications. The patient has three children who are well.
The medical history is significant for hypertriglyceridemia and asthma; the most recent episode was 8 years ago. She also has angina. Thallium perfusion imaging was performed for chest pain 2 years prior to admission; both the stress and rest scan studies were normal. She had a tubal ligation at age 35, gastric stapling, and a cholecystectomy. Her current medications included lisinopril, 10 mg daily (held for 2 weeks prior to physiologic study); insulin, 46 units NPH, 8 regular every morning; and 32 NPH, 6 regular each evening; gemfibrozil, 1200 mg daily; nizatidine, 150 mg twice daily; amitriptyline, 100 mg at bedtime for neuropathy; and albuterol, 2 puffs as needed, but she hasn't required it in many months.
Physical examination revealed: height, 158 cm; weight, 106.2 kg. Her blood pressures while seated with a thigh cuff were: 146/94 mm Hg, 144/94 mm Hg, and 146/94 mm Hg. The standing blood pressure was 142/90 mm Hg; pulse, 120 beats/min; and she reported feeling slightly dizzy. Her heart rate was 100 beats/min. Examination of the head, eyes, ears, nose, and throat was normal, with no carotid bruits and no jugular venous distention. Cardiac examination disclosed tachycardia, a regular S1 and S2, and no murmurs. The patient was morbidly obese and her abdomen was soft; bowel sounds were present. Her lungs were clear, and there was no costovertebral angle tenderness. No edema was present in her extremities.
Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined under low-salt conditions (after 1 week of a 10 mmol Na+ diet) from the p-aminohippuric acid (PAH) (Merck Sharp & Dohme, West Point, PA, USA) and inulin clearances after achieving metabolic balance on the low-salt diet. An intravenous catheter was placed in each of the patient's arms, one for the infusion, the other for blood sampling. Basal PAH and inulin clearances were calculated from their plasma levels and infusion rates for each substance. Plasma samples reflecting the control clearances were obtained 60 minutes after the start of the PAH infusion, when a steady state had been achieved, and 90 minutes thereafter following treatment with the Ang II type 1 (AT1) receptor antagonist irbesartan.
Following 1 week on the low-salt diet, her basal plasma renin activity (PRA) was 2.8 ng/Ang I/mL/hour and her control arterial pressures during the equilibration phase averaged 123/72 mm Hg. Following treatment with 150 mg of irbesartan, her arterial pressure fell slightly to an average of 113/71 mm Hg. Basal RPF measured 596 mg mL/min/1.73 m2. Ninety minutes after receiving an acute dose of 150 mg irbesartan, the RPF was 786 mL/min/1.73 m2, for a response to the angiotensin receptor blocker of 190 mL/min/1.73 m2. The basal GFR was 122 mL/min/1.73 m2, and it increased to a peak GFR post irbesartan of 163 mL/min/1.73 m2, for an increase of 41 mL/min/1.73 m2.
DISCUSSION
DR. L. GABRIEL NAVAR (Professor and Chairman, Department of Physiology; and Co-Director, Hypertension and Renal Center of Excellence, Tulane University School of Medicine, New Orleans, Louisiana, USA): This patient is a dramatic example of a metabolic syndrome of hypertension, diabetes, and obesity associated with hypertriglyceridemia. Even though this woman does not have severe hypertension, even moderately elevated arterial pressure in patients with diabetes can pose serious long-term risks for target organ damage, including the kidney. Treatment with angiotensin-converting enzyme (ACE) inhibitors or AT1 receptor blockers, however, can markedly retard the rate of loss of renal function1,2. She was given a low-salt diet for 1 week. Then an acute dose of irbesartan, an Ang II AT1 receptor blocker (ARB), was administered to determine the renal responsiveness to acute blockade of AT1 receptors. We should note that this patient did not exhibit an unusually large increase in PRA in response to the low-salt diet. Thus, the circulating renin activity did not suggest a particularly marked activation of the renin-angiotensin system (RAS). Nevertheless, in response to acute treatment with the ARB, she did manifest dramatic increases both in renal plasma flow, measured by PAH clearance, and GFR, measured by inulin clearance. This patient reflects a group of individuals who might have marked activation of the intrarenal RAS even though it is apparent neither from the PRA data nor from the responses in systemic arterial pressure to AT1 receptor blockade3. After 1 week of a low-salt diet, this patient's arterial pressure had fallen to 123/72 mm Hg the day of the study and only decreased slightly in response to irbesartan treatment to 113/71 mm Hg. Nevertheless, RPF increased by 32%, and the GFR increased by 34%. Importantly, both the RPF and GFR increased proportionally to a similar extent, leaving the filtration fraction essentially unchanged (20% to 21%). Similar results have been reported using ACE inhibitors, ARBs, and renin inhibitors in normal subjects and in patients with essential hypertension4,5,6.
Renal hemodynamic responses to Ang II and Ang II blockade
On first impression, it would seem that this patient's response was paradoxical and at variance with the commonly held concept that high intrarenal Ang II levels primarily constrict efferent arterioles, while ACE inhibitors and ARBs primarily dilate efferent arterioles. This misconception resulted from the generalization of the effects of ACE inhibitors in patients with severe renal arterial lesions or stenosis or with long-standing structural damage of the preglomerular arteriolar vasculature. When the preglomerular vasculature loses its functional integrity, blockade of the RAS system can lead to predominant efferent arteriolar dilation and reductions in GFR. However, the bulk of the data both in patients and experimental animals indicates that Ang II elicits dose-dependent decreases in RPF but often with lesser falls in GFR, leading to increases in filtration fraction7,8,9. Likewise, blockade of Ang II receptors raises both RPF and GFR but decreases the filtration fraction3,7. Unfortunately, the Ang II-induced increases in filtration fraction and the Ang II blockade-induced decreases in filtration fraction frequently have been interpreted as evidence supporting a predominant effect of Ang II on efferent arterioles. Let me emphasize that this interpretation is faulty in that changes in filtration fraction often occur as a consequence of parallel changes in afferent and efferent resistances8,10. A variety of studies both in normal and hypertensive models have shown that Ang II elicits reductions in single-nephron GFR and glomerular plasma flow because Ang II increases both afferent and efferent arteriolar resistances; direct microcirculation studies have shown dose-dependent afferent and efferent arteriolar vasoconstriction elicited by Ang II11,12. This patient clearly demonstrated a robust increase in GFR as well as in RPF in response to irbesartan. These increases indicate that the preglomerular vasculature was highly responsive, not functionally impaired, and was under the substantial influence of high intrarenal levels of Ang II that elicited strong influences on the renal microvasculature. It is also possible that the increases in GFR were partly due to (1) the ability of AT1 receptor blockade to reverse Ang II-mediated decreases in the glomerular filtration coefficient, and (2) to a component of plasma flow dependency3. The estimated changes in the determinants of glomerular dynamics are shown in Figure 1. Although the magnitude of the increases observed is greater than the responses generally observed, increases in RPF and GFR have been reported often. Thus, one important objective of this Forum is to correct a commonly held view that angiotensin antagonists predominantly dilate the efferent arterioles.
Figure 1.
Representative values for glomerular and peritubular capillary dynamics in humans. The increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) in response to irbesartan observed in this patient can be explained through a combination of decreases in afferent as well as efferent arteriolar resistance such that glomerular pressure is increased along with increases in the glomerular filtration coefficient (arrows). Assuming the normal values shown in the figure and an increase of Kf of 20%, the decreases in afferent and efferent resistance would be estimated at 42% and 24%, respectively, to account for the observed changes in RPF and GFR (for further details on analysis, refer to10).
Full figure and legend (54K)The reason for the very robust responses to irbesartan in certain patients is not clear, but it is assumed that when intrarenal Ang II levels remain chronically elevated, various compensatory mechanisms such as vasodilator prostanoids and intrarenal nitric oxide activity are increased13. When the AT1 receptors were blocked acutely, the stimulated vasodilatory mechanisms were left unopposed and markedly raised RPF and GFR8. With continued treatment, the activity of these compensatory mechanisms would be expected to abate and renal hemodynamic function would return toward normal.
In general, elevated circulating renin levels in various hypertensive and diabetic conditions are certainly recognized as being indicative of a role for inappropriate activation of the RAS; but increased local activity of the RAS can occur even when the circulating indices are within the normal range. This increased activity could be due, in part, to increased expression of ACE on renal vascular endothelial cells in certain diseases, including diabetes and hypertension14. In some organs, particularly the kidneys and adrenal glands, intrarenal Ang II, expressed as content per mass of tissue, is much greater than what can be explained on the basis of passive equilibration with the circulating components15,16. In the case of the kidney, the level of complexity might be even greater than was previously thought in that there is specific compartmentalization of renin and angiotensin levels with distinct regulatory mechanisms predominating in the separate compartments17.
Studies in human subjects have provided support for kidney-specific augmentation of the intrarenal RAS. Indeed, some of the earliest reports of the effects of ACE inhibition on renal function in humans demonstrated that administration of the first ACE inhibitor known as teprotide or SQ20881 to patients with essential hypertension significantly increased GFR and sodium excretion despite the associated decreases in arterial pressure18. Sodium excretion increased in every patient, and neither the magnitude of the fall in blood pressure nor the change in creatinine clearance influenced the degree of natriuresis. Patients with essential hypertension treated with captropril had a similar or greater natriuretic response5. These early findings set the stage for many subsequent studies that led to the recognition that the natriuretic responses to ACE inhibitors cannot be explained solely on the basis of the hemodynamic response but might include direct changes in tubular sodium reabsorption. That the responses to Ang II blockade in these patients could not be correlated with plasma Ang II levels again suggested independent regulation of intrarenal Ang II.
Several experimental models of hypertension support an overactive RAS in the development and maintenance of hypertension7,19,20. In Ang II-dependent forms of hypertension, the inappropriate activation of the intrarenal RAS limits the kidney's ability to maintain sodium balance when perfused at normal arterial pressures7,21. Overactivation of the intrarenal RAS leads to alterations in hemodynamic and transport function that contribute to the development and maintenance of hypertension. Persistence of this overactivation leads to long-term consequences, including cellular proliferation and renal injury.
Intrarenal Ang II receptors
The complex and extensive actions of Ang II on renal function are mediated by the widespread distribution of Ang II receptors throughout the kidney in various nephron segments as well as in the vasculature and interstitium. The two major types of Ang II receptors are AT1 and AT2, but the hypertensinogenic actions of Ang II are primarily attributed to the AT1 receptor because of its multiple vascular and transport effects Figure 2. In addition to the vascular AT1 receptors, AT1 receptors have been localized to glomerular podocyte cells, proximal tubule brush border and basolateral membranes, interstitial cells, thick ascending limb epithelia, distal tubules, collecting ducts, and macula densa cells22,23,24. Because of the extensive localization of AT1 receptors in luminal as well as basolateral membranes of proximal and distal nephron segments, interest is growing in the relative roles of Ang II in the renal interstitium and the tubular network. Much less AT2 receptor immunostaining occurs in adult kidneys, but it has been found in proximal tubules, collecting ducts, and some of the vasculature23,24. Thus, it is now well recognized that Ang II has many other actions in addition to the more obvious hemodynamic responses. While some of these actions have obvious physiologic relevance, others, such as activation of cytokines and reactive oxygen species, occur primarily in a pathophysiologic setting and are linked to tissue injury, fibrosis, and proliferation25. In the final analysis, however, the local effects depend on the actual concentrations of the Ang II maintained within the corresponding compartment.
Figure 2.
Angiotensin II (Ang II) receptor subtypes and major renal actions attributed to activation of AT1 and AT2 receptors. Rodents appear to have two AT1 receptor subtypes, but humans seem to have only one. Abbreviations are: Na, sodium; TGF, tubular glomerular feedback; ET, endothelin; TxA2, thromboxane A2; ICAM1, intercellular adhesion molecule-1; MCP1, monocyte chemoattractant protein-1; IL-6, interleukin-6; TGF
, transforming growth factor beta; PAI-1, plasminogen-activated inhibitor-1; NF
B, nuclear factor kappaB; RANTES, regulated upon activation, normal T cell expressed and secreted.
Intrarenal levels of Ang II
Although direct measurements in human subjects are not available, experimental studies indicate that intrarenal Ang II tissue content is much higher than can be explained on the basis of non-specific equilibration between plasma Ang II concentrations and intrarenal extracellular fluid26,27,28. Variations in dietary sodium chloride intake are closely associated with changes in renal renin, Ang I, and Ang II content. However, the total renal levels of Ang I and Ang II remain higher than the corresponding plasma concentrations, so the intrarenal levels likely are not due to simple equilibration with circulating Ang II27,28,29,30.
Renal Ang II levels in several experimental models of angiotensin-dependent hypertension are much higher than the plasma concentrations. This has been demonstrated in 2-kidney, 1-clip (2K1C) Goldblatt hypertension, Ang II-infused animals with hypertension, Ren2 TGR hypertension, and Dahl salt-sensitive rats fed a high-salt diet29,30,31,32,33. In these models, the augmentation of intrarenal Ang II content is the consequence of several mechanisms. In addition to intrarenal formation of Ang II, the kidney accumulates Ang II from the circulation via an AT1 receptor-mediated process31,34. Sustained elevations in circulating Ang II cause progressive accumulation of intrarenal Ang II levels even in the presence of marked suppression of renin formation. Of clinical relevance is the finding that even non-clipped kidneys of 2K1C Goldblatt rats have elevated intrarenal Ang II levels. Increased Ang II levels occur in renin-depleted kidneys of 2K1C Goldblatt hypertensive rats29,30,35,36, Ang II-infused hypertensive rats29,31, and Ren2 transgenic rats33. In Ang II-infused rats, the augmentation of intrarenal Ang II is dependent on an AT1 receptor-mediated process, as it can be prevented by concomitant treatment with AT1 receptor blockers31,34. These data demonstrate that intrarenal accumulation of Ang II occurs via an AT1 receptor-mediated mechanism and suggest the presence of receptor-mediated endocytosis and the existence of an intracellular pool of Ang II [28].
Compartmentalization of intrarenal Ang II
The intrarenal content of Ang II is not distributed in a homogeneous fashion but is compartmentalized in both a regional and segmental manner17. Ang II levels in the deep medulla are much higher than cortical levels in normal rats and increase further in Ang II-infused hypertensive rats15,37. Ang II in the renal medulla might be particularly significant in regulating medullary hemodynamics38. Also, Ang II receptor density is much greater in the medulla than in the cortex39.
Within the cortex, Ang II is distributed in the interstitial and tubular fluid as well as within the cells. The interstitial fluid contributes to the disproportionately high total intrarenal Ang II levels. Studies using microdialysis probes implanted in the renal cortex have shown that Ang II and Ang I concentrations in interstitial fluid are much higher than plasma concentrations40,41. Renal interstitial fluid Ang II concentrations were 3 to 5 pmol/mL; Ang I concentrations were lower, in the range of 1 pmol/mL, but still much higher than the corresponding plasma Ang I concentrations. Interestingly, in rat studies, ACE inhibition was unable to lower the renal interstitial Ang II concentrations significantly. This failure suggested that much of interstitial Ang II is derived from sites not readily accessible to ACE inhibitors41. As Figure 3 illustrates, renal interstitial Ang II levels were significantly elevated by about twofold in Ang II-infused rats, and this increase was prevented by simultaneous treatment with an AT1 receptor blocker, candesartan42.
Figure 3.
Renal interstitial fluid (RIF) angiotensin II (Ang II) concentrations, renal Ang II levels, and plasma Ang II concentrations in control rats, rats infused with Ang II for 2 weeks, and rats infused with Ang II that also were treated with the AT1 receptor blocker candesartan. Ang II-infused rats had an almost twofold increase in RIF Ang II, but this increase was prevented by treatment with candesartan. The RIF Ang II concentrations paralleled the renal Ang II contents and were dissociated from the plasma Ang II concentrations, which increased further with candesartan (from42, with permission).
Full figure and legend (25K)It is likely that interstitial Ang II is more important in regulating hemodynamic and transport function than is circulating Ang II7. Perfusion of Ang I and Ang II into peritubular capillaries leads to infiltration into the surrounding interstitium, afferent and efferent arteriolar vasoconstriction, and decreases in single-nephron GFR. Ang II in interstitial fluid might be principally responsible for maintaining tone of the pre- and post-glomerular vessels and of influencing tubular transport function by acting on Ang II receptors on the basolateral membranes of the tubular cells. Interstitial Ang II also might be of particular importance in regulating the sensitivity of the tubuloglomerular feedback mechanism, which communicates signals from the macula densa cells to the afferent arterioles and which maintains a balance between filtered load and the reabsorptive capabilities of the tubules7,43. Thus, it is likely that in the patient being discussed, the Ang II-dependent influences on vascular tone were due primarily to the prevailing renal interstitial Ang II concentrations and not to the plasma concentrations.
Intratubular Ang II
As I mentioned, Ang II receptors are present on the luminal membranes of proximal and distal nephron cells. Activation of these receptors stimulates the activity and increases expression of the Na+/H+ exchanger in proximal and distal tubules, and also stimulates activity of the sodium channel in the collecting duct; blockade of these receptors reduces net sodium reabsorption7,44,45. Another intriguing finding is that proximal tubule fluid concentrations of Ang I and Ang II are in the range of 5–10 pmol/mL, much greater than the plasma concentrations16,46,47. The finding that Ang II concentrations in perfused tubules were similar to those measured in non-perfused tubules demonstrated that the tubular fluid Ang II concentrations are not derived from the glomerular filtrate47. Ang I and angiotensinogen (AGT), as well as Ang II, are present in proximal tubular fluid. Thus, intratubular Ang II could be formed from precursors secreted into the tubular lumen. The various potential pathways are depicted in Figure 4. It is interesting that tubular fluid Ang II concentrations are not reduced in several hypertensive models, including the non-clipped kidney of the Goldblatt hypertensive rat, the Ren2 transgenic rat, and the Ang II-infused hypertensive rat as compared to controls33,35,48. Considering that in all these cases, the kidneys are markedly renin depleted and exposed to elevated arterial pressures, the maintenance of high proximal tubular Ang II concentrations reflects inappropriate intrarenal Ang II formation that might exert sustained effects on tubular transport function mediating sodium reabsorption.
Figure 4.
Tubular and interstitial formation, secretion, and uptake of angiotensin II (Ang II). Ao, angiotensinogen. Summarized from data presented in15,16,35,37,41,42,43,44,47,48.
Full figure and legend (50K)The Ang II concentrations in tubular fluid from other segments of the nephron remain unknown due to technical difficulties of collecting sufficient volumes and to a lack of access to these segments. However, studies support an important role for luminal Ang II in regulating reabsorptive function in distal nephron and collecting duct segments, as well as in proximal tubule segments49,50. Ang II concentrations as low as 10 fmol/mL increase distal tubular sodium transport49,50. Studies by Peti-Peterdi, Warnock, and Bell51 have shown that Ang II at 1 pmol/mL stimulates sodium channel activity. Therefore, the distal nephron represents a potentially important site for further control of sodium transport regulation by Ang II.
Endosomal accumulation of Ang II
The augmentation of intrarenal Ang II that occurs during physiologic increases in angiotensin levels produced by salt restriction or chronic infusion of Ang II includes a component of AT1 receptor-mediated accumulation of Ang II into an intracellular compartment31,34,52. The resultant increases in intracellular Ang II can serve intracrine functions53. Haller et al54 showed that Ang II micro-injected into vascular smooth muscle cells stimulates inositol trisphosphate (IP3) production via an AT1 receptor-mediated action. In addition, endocytosis of the Ang II-AT1 receptor complex, required for the full expression of functional responses, is coupled to the activation of signal transduction pathways and increased sodium transport55,56. A role for intracellular Ang II is supported further by the demonstration that angiotensin peptides, ACE, and Ang II receptors are present in renal endosomes28. These results demonstrate that Ang II is either formed or trafficked through intracellular endosomal compartments. Zhuo et al57 demonstrated that cortical endosomes, harvested after 2 weeks of Ang II infusion in rats, exhibited marked increases in Ang II. Concurrent treatment with the ARB candesartan prevented the increases in endosomal, as well as total-kidney, Ang II levels57. These data demonstrate increased uptake and trafficking of Ang II into renal endosomes, mediated by AT1 receptors, in Ang II-dependent hypertension.
The roles of the internalized Ang II remain unclear. Several possibilities exist. As I said earlier, intracellular Ang II might exert cytosolic actions. Ang II also could be recycled and secreted to exert further actions by binding to Ang II receptors on the cell membrane. A particularly intriguing hypothesis is that Ang II might migrate to the nucleus to exert transcriptional effects58. Chen et al58 transfected Chinese hamster ovary cells with an AT1 receptor fused with green fluorescent protein, which allowed visualization of trafficking of the internalized ligand-receptor complex. Ang II increased co-localization of the green fluorescent protein with nuclear markers, thus suggesting migration of the receptor complex to the nucleus58. Recent studies demonstrated specific nuclear Ang II binding in renal cortical cell sites that is predominantly of the AT1 type59. Therefore, direct Ang II actions intracellularly might include regulation of nuclear signaling events.
Origins of intrarenal Ang II
The finding that renal interstitial fluid Ang II concentrations are much higher than plasma levels reflects substantial interstitial Ang II formation. This is easily explained, as all the components needed for Ang II generation are present within the renal interstitium7,60. Nevertheless, the mechanisms regulating renal interstitial fluid Ang I and Ang II concentrations remain unclear. Although it has generally been thought that interstitial AGT is of circulating origin, the finding of substantial AGT mRNA and protein levels in renal proximal tubule cells has raised the possibility that some of the interstitial Ang II is derived from locally formed AGT and might not be dependent on circulating Ang II or AGT61,62,63. Nishiyama, Seth, and I42 found that administering AT1 receptor blockers to Ang II-infused rats prevented not only the increase in total-kidney content but also the increase in renal interstitial Ang II concentration, even in the presence of elevated circulating levels. This finding suggests that a substantial fraction of the interstitial Ang II is formed from intrarenally produced AGT.
The localization of intrarenal AGT mRNA and protein in proximal tubule cells indicates that proximal tubule cells provide the substrate for intratubular and interstitial Ang I and Ang II64,65,66,67. Importantly, AGT mRNA levels and protein can be stimulated by Ang II. This paradoxical positive amplification mechanism by which local production of substrate is increased by its own product likely contributes to the progressive increases in intrarenal production of Ang II in hypertension61,62,68. The AGT produced in proximal tubule cells might be directly secreted into the tubular lumen in addition to producing its metabolites intracellularly and secreting them into the tubular lumen67,69. Proximal tubule AGT concentrations in anesthetized rats approximate 300 nM, greatly exceeding the free Ang I and Ang II tubular fluid concentrations15. Because AGT's molecular weight is 62,000 daltons, it seems unlikely that much of the plasma AGT filters across the glomerular membrane. Thus, the evidence indicates that AGT is secreted directly into the tubular lumen15,67,69,70,71. Ang I then is formed in the tubular lumen by renin or renin-like enzymes64,72. In addition, cultured proximal tubule cells produce renin and contain renin mRNA, so a low-level constitutive renin secretion likely exists in proximal tubule cells64,72,73. Once Ang I is formed, conversion to Ang II readily occurs by the substantial ACE in the proximal tubule brush border and in the tubular fluid74,75,76. However, it remains unclear how much of the peptides are formed intracellularly and how much are formed in the tubular fluid.
Augmentation of intrarenal AGT production
Although increased intrarenal uptake of Ang II contributes to the increased intrarenal Ang II in the Ang II-infused model of hypertension, total intrarenal Ang II levels are also due to additional intrarenal Ang II formation as a consequence of increased AGT production. In vivo and in vitro studies have shown that Ang II stimulates intrarenal AGT mRNA localized in proximal tubule cells61,63,68. Furthermore, intrarenal AGT protein as well as AGT mRNA levels increase in response to chronic Ang II infusions62. This positive feedback system could be responsible for sustained or increased generation of AGT, which leads to continued intrarenal production of Ang II. The intrarenally produced Ang II would be additive with the Ang II that is internalized by the AT1 receptors and lead to the overall increase in intrarenal Ang II contents. The presence of high concentrations of AGT in proximal tubule fluid indicates substantial secretion of AGT into the proximal tubule lumen. Furthermore, the finding of intact AGT in urine suggests its presence throughout the nephron and, to the extent that renin and ACE are available along the nephron, there might be continued Ang I generation and Ang II conversion in segments beyond the proximal tubule69,71,77.
Renin has been found on the luminal side of connecting tubule cells in mouse and human kidneys; thus, renin also might be secreted into distal tubular fluid69. AGT has been detected at low nanomolar concentrations in urine from mice and human volunteers, and mice given low dietary salt intake had increased urinary AGT levels69. Transgenic mice harboring the gene for human AGT fused to the kidney androgen-protein promoter also excreted human AGT in urine but it was not detected in the systemic circulation71. Ang II-infused rats exhibited an increase in AGT mRNA levels and intrarenal AGT protein62. As Figure 5 shows, chronic Ang II infusions also led to marked increases in urinary AGT excretion rates in association with the increases in systolic blood pressure78,79. Furthermore, there was a direct relationship between urinary AGT excretion rates and renal Ang II contents78,79. The increased urinary AGT was specifically associated with Ang II-dependent hypertension and was not simply due to hypertension-induced proteinuria. These data thus support the concept that angiotensin-dependent hypertension is accompanied by increased AGT secretion by the proximal tubule cells, which leads to spillover of intact AGT into distal nephron segments. Because distal tubule cells have available renin and ACE or other enzymes that can subserve similar functions, the expected result is increased distal tubular formation of Ang II and increased Ang II-dependent stimulation of the distal sodium reabsorption rate51. This increase in distal sodium reabsorption might help explain the markedly increased fractional sodium reabsorption and suppression of pressure natriuresis that occurs in Ang II-infused rats80. Thus it seems likely that urinary AGT concentrations or excretion rates reflect the distal nephron spillover of AGT and, accordingly, provide an index of the magnitude of the increased intrarenal AGT production in angiotensin-dependent hypertension.
Figure 5.
Urinary angiotensinogen (AGT) excretion rates and systolic blood pressures (BP) in control rats and rats infused with Ang II at two rates, 40 ng/min and 80 ng/min (from79, with permission).
Full figure (26K)From a functional perspective, the additive effects of Ang II working on distal nephron transport function coupled with the associated actions of elevated aldosterone levels markedly increase the sodium-retaining ability of the kidney which, when inappropriately sustained, contributes to the development and maintenance of hypertension. Furthermore, these sustained increases in intrarenal Ang II in a setting of hypertension produce progressive renal injury and cellular proliferation associated with activation of several major cytokines and growth factors25.
QUESTIONS AND ANSWERS
DR. NICOLAOS E. MADIAS (Dean ad Interim, Tufts University School of Medicine, Boston, Massachusetts): You described the effects of exogenous Ang II infusion on intrarenal AGT and Ang II. What about the effects of endogenous increases in systemic Ang II as a result of volume depletion or other states of secondary hyperaldosteronism (that is, congestive heart failure, cirrhosis)?
DR. NAVAR: We do not have data on hyperaldosteronism or volume depletion. However, there are data regarding changes during physiologic manipulation of the RAS, such as with differences in salt intake. In a setting like that, we and others have observed parallel changes in circulating as well as intrarenal renin levels. With a low-salt diet, there is a marked stimulation of plasma renin, plasma Ang II, and renal Ang II levels28,52. However, the renal Ang II levels expressed per gram of kidney weight are still much higher than the plasma concentrations. Under these conditions, the circulating and renal levels change in a parallel manner, and it's very hard to determine how much of the change that occurs in the kidney is due to intrarenal formation and how much is due to delivery from the plasma.
An important difference is that during physiologic stimulation of the RAS, neither hypertension nor renal injury occurs. This suggests that other factors associated with the development of hypertension facilitate the Ang II internalization as well as the injury that occurs.
DR. MADIAS: What do we know about the mechanism of entry of AGT from the proximal tubule cell into the lumen? Also, you showed a correlation between urinary AGT and intrarenal Ang II. What about measuring Ang II itself in the urine?
DR. NAVAR: Ang II is formed and degraded rapidly. Ang II in the urine might be derived from the circulation or the kidney, so it is not possible to determine the source of the Ang II. In contrast, AGT is a large molecule, so very little, if any, crosses the glomerular membrane70. Because practically none of the circulating AGT appears in the urine79, urinary AGT is probably all of intrarenal origin. That's why we use AGT as a reflection of intrarenal formation. In terms of the actual mechanism of secretion, it is an exocytotic mechanism, but we have not looked at specific, targeted mechanisms by which the AGT is secreted in the proximal tubule.
DR. MADIAS: Can you exclude the possibility that part of the AGT measured in the urine reflects degradation fragments of systemic AGT that were filtered at the glomerulus?
DR. NAVAR: We have used infusions of human AGT to determine how much circulating AGT appears in the urine. We assayed for human AGT in both plasma and urine in rats infused with human AGT79. We did not find human AGT either intact or in immunoreactive fragments in either normal control rats or Ang II-infused rats. That's the only study we have done, but I think that further studies are necessary in this area.
DR. RONALD PERRONE (Division of Nephrology, Tufts-New England Medical Center, Boston, Massachusetts): In Boston, we've been extensively exposed to the concept of efferent arteriolar vasoconstriction in progressive kidney disease. How would you integrate your findings into this model, that is, an increased intrarenal production of Ang II causing efferent arteriolar vasoconstriction? ACE inhibitors have slowed progression of renal disease in rat models and in human disease. What happens where there's intrarenal production of Ang II? Does that also lead to uptake and increased production of AGT? Are angiotensin receptor blockers more effective than ACE inhibitors in inhibiting Ang II in the kidney?
DR. NAVAR: Let me address your last question first. From what we know of the differences in actions between ACE inhibitors and Ang II receptor blockers, one would expect to see greater differences in the responses observed. However, experimental studies and clinical trials have failed to show any major differences in responses. The key lies in the differences between therapeutic doses as compared to the doses used experimentally. The doses used therapeutically just diminish the level of activity rather than completely block Ang II actions or circulating concentrations. Thus, it is difficult to discriminate between the effects of ACE inhibition and those of Ang II receptor blockade in patients.
With regard to the other point, it was originally thought that almost everything related to hypertension-induced renal injury could be explained on the basis of altered renal hemodynamics. We now know that Ang II stimulates many other factors, as highlighted recently25. Various cytokines that might or might not have effects on renal hemodynamics are activated. Some of these are involved in long-term injury and proliferative responses and have important roles.
With regard to the effects of Ang II on afferent and efferent arteriolar resistances, I emphasized that the often-stated concept that Ang II only constricts efferent arterioles is simply not true under most conditions. Ang II clearly exerts vasoconstrictor actions both on afferent and efferent arterioles. Thus, ACE inhibitors and Ang II receptor blockers do vasodilate afferent as well as efferent arterioles, but the important point is that the efferent action helps protect against unusually large increases in glomerular pressure under conditions of renal vascular dilation. Clearly, if you compare calcium channel blockers with either ACE inhibitors or Ang II receptor blockers, the calcium channel blockers primarily dilate afferent arterioles. For any given level of arterial pressure, the effects of calcium channel blockers would be associated with a much greater degree of glomerular hypertension than are the ACE inhibitors.
Unfortunately, the concept often transmitted to students is that Ang II only affects the efferent arterioles. If this were true, every patient taking an ACE inhibitor or a receptor blocker would go into acute renal failure. All you have to do is drop glomerular pressure a few millimeters of mercury to drastically reduce GFR. The important message to emphasize is that Ang II has combined effects on both pre- and post-glomerular arterioles. Because the structural integrity of efferent arterioles is protected in many kinds of injury mechanisms to a greater extent than is that of the pre-glomerular vasculature, in certain diseases associated with pre-glomerular vascular disease, the pre-glomerular arterioles become unresponsive. Thus, a patient with severe arteriolar damage might have unresponsive pre-glomerular arterioles that are no longer functionally intact. This can lead to a situation in which only the efferent arterioles respond to antagonism.
DR. D. PAK: You suggested that calcium channel blockers and ACE inhibitors have different actions on afferent and efferent arterioles. Would this combination be good for our patients?
DR. NAVAR: Some studies have shown that the combination of a calcium channel blocker with either an ACE inhibitor or an Ang II receptor blocker would be synergistic and could provide added benefit. Experimental studies have shown that L-type calcium channel blockers produce predominantly afferent vasodilation with negligible efferent effects and can elevate glomerular pressure. Thus it would be beneficial to combine a calcium channel blocker with an Ang II antagonist. Calcium channel blockers have a more powerful natriuretic effect and a more powerful ability to dilate vascular smooth muscle throughout the body, so a physician might choose to give a calcium channel blocker. However, the addition of an ACE inhibitor or Ang II receptor blocker would provide added benefit by directly blocking Ang II's effects on efferent arterioles and by inhibiting the effects of Ang II on tubular transport.
DR. MADIAS: What have we learned about AGT and systemic blood pressure from gene-targeting studies or genetic studies in humans?
DR. NAVAR: Studies in humans based on the genetic analysis of large populations and analysis of polymorphisms have provided mixed results70. As reviewed by Lalouel et al81, the results from studies of renin and ACE have been inconclusive. However, significant genetic linkage observed for AGT81 suggested that individual differences in the AGT gene might contribute to disease development. The studies done in gene-targeted mice, in particular by Oliver Smithies' group, clearly show that hypertension is a function of the level of expression of the AGT gene82. The four-copy AGT mice have much higher blood pressures than do the zero-copy animals. Smithies emphasized that, in contrast to the ACE gene, under- and overexpression of AGT clearly projects changes in blood pressure. What is needed now is more extensive studies of renal function in the mice overexpressing and underexpressing AGT.
DR. MADIAS: Are the changes in the intrarenal RAS also reflected in other tissue renal-angiotensin systems, for example, that of the heart or the adrenal gland?
DR. NAVAR: The only organs with clear indications of abundant local generation of Ang II are the kidneys and adrenal glands. The adrenal glands have very high Ang II and renin levels. Because the adrenal glands are so small, however, it is unlikely that adrenal renin contributes very much to the circulating renin. Nevertheless, as Dzau83 and others have emphasized for many years, Ang II is formed locally in many tissues, and components of the RAS are present in tissues throughout the body, including the brain, heart, and vasculature, especially under conditions where there is injury. However, the circulating renin levels are derived almost exclusively from the kidneys. John Laragh has emphasized this point many times. If the kidneys are removed, renin levels become undetectable within 48 hours.
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Acknowledgments
The Principal Discussant gratefully acknowledges assistance from Dr. Naomi Fisher at Brigham and Women's Hospital in Boston for providing the case discussed. He also is indebted to Debbie Olavarrieta for preparing the manuscript and to the many fellows and coworkers who contributed to the studies described. Research performed in the author's laboratory was supported by NHLBI grant HL26371, by the Millennium Health Excellence Fund of the Louisiana Board of Regents, and by an NIH COBRE grant, P20RR017659 from the Institutional Development Award (IDEA) Program of the National Center for Research Resources.
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