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Essential hypertension is the leading cause of premature death worldwide. However, hypertension’s cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg−1·day−1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid’s (4-PBA’s) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC50 values. Of the chaperones, 4-PBA and DHA provided the greatest reduction of ER stress and did so at relatively low concentrations. Upon analyzing the efficiency of these compounds and their corresponding structures, it was determined that chaperones with a localized hydrophilic, polar end followed by a long hydrophobic chain, such as 4-PBA and DHA, were most effective at reducing ER stress. This study provides some insight into the use of low molecular weight chemical chaperones and may serve as the first step towards developing new chaperones of greater potency thereby providing potential treatments for diseases caused by protein aggregation.

Proteinuria is one of the primary risk factors for the progression of chronic kidney disease (CKD) and has been implicated in the induction of endoplasmic reticulum (ER) stress. We hypothesized that the suppression of ER stress with a low molecular weight chemical chaperone, 4-phenylbutyric acid (4-PBA), would reduce the severity of CKD and proteinuria in the Dahl salt-sensitive (SS) hypertensive rat. To induce hypertension and CKD, 12-wk-old male rats were placed on a high-salt (HS) diet for 4 wk with or without 4-PBA treatment. We assessed blood pressure and markers of CKD, including proteinuria, albuminuria, and renal pathology. Furthermore, we determined if HS feeding resulted in an impaired myogenic response, subsequent to ER stress. 4-PBA treatment reduced salt-induced hypertension, proteinuria, and albuminuria and preserved myogenic constriction. Furthermore, renal pathology was reduced with 4-PBA treatment, as indicated by lowered expression of profibrotic markers and fewer intratubular protein casts. In addition, ER stress in the glomerulus was reduced, and the integrity of the glomerular filtration barrier was preserved. These results suggest that 4-PBA treatment protects against proteinuria in the SS rat by preserving the myogenic response and by preventing ER stress, which led to a breakdown in the glomerular filtration barrier. As such, alleviating ER stress serves as a viable therapeutic strategy to preserve kidney function and to delay the progression of CKD in the animal model under study.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population. CKD leads to end stage renal disease and increases the risk of cardiovascular disease. As such, it is important to study the mechanisms underlying CKD progression. To this end, an animal model was developed to allow the testing of new treatment strategies or molecular targets for CKD prevention. Many underlying risk factors result in CKD but the disease itself has common features, including renal interstitial fibrosis, tubular epithelial cell loss through apoptosis, glomerular damage, and renal inflammation. Further, CKD shows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD. We sought to develop and characterize an animal model with these common features of human CKD in the C57BL/6 mouse. Mice of this genetic background have been used to produce transgenic strains that are commercially available. Thus, a CKD model in this strain would allow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost. This paper describes such a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers.

Background— Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H 2 S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H 2 S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H 2 S in atherosclerosis development. Methods and Results— Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N -acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. Conclusions— Endogenously synthesized H 2 S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H 2 S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H 2 S pathway is an important therapeutic target for protection against atherosclerosis.

Synthesis of transmembrane and secretory proteins occurs within the endoplasmic reticulum (ER) and is extremely important in the normal functioning of both the heart and kidney. The dysregulation of protein synthesis/processing within the ER causes the accumulation of unfolded proteins, thereby leading to ER stress and the activation of the unfolded protein response. Sarcoplasmic reticulum/ER Ca 2+ disequilibrium can lead to cardiac hypertrophy via cytosolic Ca 2+ elevation and stimulation of the Ca 2+ /calmodulin, calcineurin, NF-AT3 pathway. Although cardiac hypertrophy may be initially adaptive, prolonged or severe ER stress resulting from the increased protein synthesis associated with cardiac hypertrophy can lead to apoptosis of cardiac myocytes and result in reduced cardiac output and chronic heart failure. The failing heart has a dramatic effect on renal function because of inadequate perfusion and stimulates the release of many neurohumoral factors that may lead to further ER stress within the heart, including angiotensin II and arginine–vasopressin. Renal failure attributable to proteinuria and uremia also induces ER stress within the kidney, which contributes to the transformation of tubular epithelial cells to a fibroblast-like phenotype, fibrosis, and tubular cell apoptosis, further diminishing renal function. As a consequence, cardiorenal syndrome may develop into a vicious circle with poor prognosis. New therapeutic modalities to alleviate ER stress through stimulation of the cytoprotective components of the unfolded protein response, including GRP78 upregulation and eukaryotic initiation factor 2α phosphorylation, may hold promise to reduce the high morbidity and mortality associated with cardiorenal syndrome.

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

A causal relationship between diet-induced hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE(-/-)) mice. However, it is not known whether the proatherogenic effect of HHcy in apoE(-/-) mice is independent of hyperlipidemia and/or deficiency of apoE. In this study, a comprehensive dietary approach using C57BL/6J mice was used to investigate whether HHcy is an independent risk factor for accelerated atherosclerosis or dependent on additional dietary factors that increase plasma lipids and/or inflammation. C57BL/6J mice at 4 wk of age were divided into 6 dietary groups: chow diet (C), chow diet + methionine (C+M), western-type diet (W), western-type diet + methionine (W+M), atherogenic diet (A), or atherogenic diet + methionine (A+M). After 2, 10, 20, or 40 wk on the diets, mice were sacrificed, and the levels of total plasma homocysteine, cysteine, and glutathione, as well as total plasma cholesterol and triglycerides were analyzed. Aortic root sections were examined for atherosclerotic lesions. HHcy was induced in all groups supplemented with methionine, compared to diet-matched control groups. Plasma total cholesterol was significantly increased in mice fed the W or A diet. However, the W diet increased LDL/IDL and HDL levels, while the A diet significantly elevated plasma VLDL and LDL/IDL levels without increasing HDL. No differences in plasma total cholesterol levels or lipid profiles were observed between methionine-supplemented groups and the diet-matched control groups. Early atherosclerotic lesions containing macrophage foam cells were only observed in mice fed the A or A + M diet. Furthermore, lesion size was significantly larger in the A + M group compared to the A group at 10 and 20 wk; however, mature lesions were never observed even after 40 wk on these diets. The presence of lymphocytes, increased hyaluronan staining, and the expression of endoplasmic reticulum (ER) stress markers were also increased in atherosclerotic lesions from the A + M group. Taken together, these results suggest that HHcy does not independently cause atherosclerosis in C57BL/6J mice even in the presence of increased total plasma lipids induced by the W diet. However, HHcy can accelerate atherosclerotic lesion development under dietary conditions that increase plasma VLDL levels and/or inflammation.

Endoplasmic reticulum stress has been identified as a feature of chronic kidney disease and appears to be associated with proteinuria. We have found the endoplasmic reticulum stress inhibitor, 4-ph...

Large mesenteric arteries from 3- to 4-wk-old spontaneously hypertensive rats (SHR) showed medial hypertrophy and an increased contractile response to various agonists before significant blood pressure increase. Here we determined the cellular nature of this vascular hypertrophy. Large mesenteric arteries from SHR and Wistar-Kyoto (WKY) rats were fixed at maximal relaxation either with an in situ perfusion fixation or an in vitro fixation method. With the use of morphometric protocols and confocal microscopy, the volume of the medial wall and lumen, numerical density of smooth muscle cell nuclei in the medial layer, and smooth muscle cell and nuclear length were measured. Both methods of fixation yielded similar results, showing significant medial volume expansion in SHR than WKY without lumen change. Numerical density of medial smooth muscle cells was significantly less in SHR than WKY, and their total number per 100 μm length were similar between the strains. Average smooth muscle nuclear and cell length from SHR was significantly longer than that of WKY. Regression analysis showed that the increase in smooth muscle cell length explained 80% of the medial volume increase. We concluded that increased smooth muscle cell length in prehypertensive SHR is responsible for increased medial volume in the mesenteric arteries.

Acute coronary syndrome (ACS) represents a series of indications of sudden cardiac ischemia that involve a variety of causes, including acute myocardial infarction and unstable angina.1 ACS usually is associated with thrombus formation in the coronary artery or coronary artery vasospasm, resulting in myocardial ischemia. 2 Thrombus formation may involve platelet adhesion and degranulation on damaged or dysfunctional endothelium overlying the intact or ruptured atherosclerotic plaque, as well as microthrombi.3 Atherosclerotic plaque rupture represents a series of deleterious events linked to the breakdown of the fibrous cap. This process may occur as a result of the increased secretion of matrix metalloproteinases by lesion resident macrophages or enhanced apoptotic cell death within the fibrous cap. However, viable smooth muscle cells (SMCs) within the fibrous cap synthesize interstitial collagen fibers that enhance the structural integrity of the plaque and prevent its rupture.4

The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or "proteostasis," prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases.

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine β-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by β-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine β-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

Studies were designed to determine the source of NO responsible for buffering of the angiotensin II (Ang II)–mediated decrease of blood flow in the renal medulla. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) and NO production ([NO] i ) of pericytes and endothelium of the vasa recta were independently measured with the use of fura 2-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), respectively, in microtissue strips of the vascular bundles of the outer medullary vasa recta. Disruption of the endothelium of the vasa recta by perfusion with latex microspheres enabled imaging of the pericytes. Ang II (1 μmol/L) produced an increase of [NO] i of 19±6 U in pericytes of the vasa recta when the vessels were adjacent to medullary thick ascending limbs (mTALs). Pericytes of isolated vasa recta without surrounding mTALs showed a rapid peak increase in [Ca 2+ ] i of 248±107 nmol/L, with a sustained elevation of 107±75 nmol/L, but did not show an increase in [NO] i to either Ang II (1 μmol/L) or the Ca 2+ ionophore 4-bromo-A23187 (5 μmol/L). These observations indicated the lack of Ang II and Ca 2+ -sensitive NO production in pericytes of the vasa recta. In isolated vasa recta with intact endothelium, Ang II reduced [Ca 2+ ] i from 128±28 to 62±13 nmol/L and failed to increase [NO] i . However, the Ca 2+ ionophore did increase [NO] i in the endothelium (47±8 U), indicating the presence of Ca 2+ -sensitive NO production. Significant increases of [NO] i were observed in single isolated mTALs in response to both Ang II (33±6 U) and the Ca 2+ ionophore (51±18 U). We conclude that Ang II increases [Ca 2+ ] i in pericytes of the descending vasa recta as part of its constrictor action and that this vasoconstriction is buffered by the NO from the surrounding tubular elements, such as mTALs.

The course of hypertension development in young spontaneously hypertensive rats (SHR) was studied by the measurement of changes in systolic blood pressure (BP), body weight, and heart rate (HR) at 2, 3, 4, and 6 wk of age. To achieve this, we compared inbreeding lines of SHR and Wistar-Kyoto rats (WKY) to determine if differences in BP, body weight, or HR were present among inbreeding lines of the same strain or between strains. The effect of these differences on the eventual level of BP was then assessed. We found that BP began to diverge between SHR and WKY at 4 wk of age. Significant differences in systolic BP (24 mmHg) between SHR inbreeding lines at 4 wk of age did not affect the BP at 8 wk (172 vs. 170 mmHg). Pulse pressure was significantly higher in SHR than in WKY at 4 wk of age. HR was elevated in SHR over age-matched WKY at 3 wk of age and positively correlated to the level of BP attained by individual animals at 6 wk ( P = 0.037). Moreover, WKY inbreeding lines showing elevated HR developed higher BP (145 vs. 127 mmHg) at 10–12 and 20 wk of age. The prehypertensive tachycardia in SHR was investigated further and found to result from an increased intrinsic HR. Because HR at 3 wk is a genetic trait that can be partitioned into inbreeding lines, and inbreeding lines most expressive of this trait showed the highest eventual BP, we conclude that prehypertensive tachycardia may be an important first step during hypertension development in SHR. Moreover, early elevations in HR are highly predictive ( r = 0.41) of hypertension occurrence in the animal population studied.