Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound ... more Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammato...
Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with ... more Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with age. Estrogen plays a protective role against hypertension and end-organ damage by modulating the depressor actions of the AT 2 R (angiotensin type 2 receptor). Our aim was to determine whether estrogen replacement in aged female mice can lower arterial pressure, improve endothelial function, and reduce organ fibrosis via an AT 2 R-mediated mechanism. Mean arterial pressure was measured via radiotelemetry in ovary-intact adult (3–4-month-old), aged (16–18-month-old; reproductively senescent) and aged–17β-estradiol (E 2 )–treated (3 µg/day SC) female mice, which were administered vehicle, Ang II (angiotensin II; 600 ng/[kg·min] SC) or Ang II+PD123319 (AT 2 R antagonist; 3 mg/[kg·day SC). On day 21 of treatment, aortic endothelium-dependent relaxation and cardiac and renal tissue (fibrosis and gene expression) were analyzed. Basal mean arterial pressure was lower in E 2 -treated aged mice (89±1 mm Hg, n=20) relative to aged controls (94±1 mm Hg; n=18, P =0.002). The Ang II pressor response was enhanced by ≈20 mm Hg in aged compared with adult females ( P =0.01). E 2 -treatment reduced the Ang II pressor response in aged females ( P =0.002), an effect that was reversed by PD123319 in the aged E 2 –Ang II group ( P =0.0009). E 2 -treatment increased renal AT 2 R (≈6-fold; P <0.0001) and MasR (Mas oncoreceptor; 2–3-fold, P <0.05) gene expression in aged females. However, neither Ang II–induced endothelial dysfunction nor the age-related increase in renal and cardiac fibrosis was restored by E 2 -treatment in aged female mice. In conclusion, estrogen replacement in aged females may reduce arterial pressure to levels observed in adult females, via an AT 2 R-mediated renal mechanism.
Canadian Journal of Physiology and Pharmacology, Aug 1, 1987
In renal artery stenosis severe enough to cause hypertension, angiotensin II maintains glomerular... more In renal artery stenosis severe enough to cause hypertension, angiotensin II maintains glomerular filtration rate (GFR) both in the initial high renin phase of hypertension and later when plasma levels are normal. Angiotensin II also maintains GFR in less severe stenosis, which does not cause hypertension. This homeostatic action of angiotensin II to maintain GFR has minimal effects on blood flow. In renal-wrap hypertension, plasma renin levels are elevated for longer than after renal artery stenosis, but in other respects this initial phase of the hypertension is similar to that after renal artery stenosis. GFR is reduced, the rate of development of hypertension is accelerated by angiotensin II, and angiotensin II maintains the glomerular filtration fraction. Renal resistance is markedly increased owing to both compression of the kidney by the hypertrophying renal capsule and to angiotensin II. Thus angiotensin II apparently plays a primarily homeostatic role in renovascular hypertension to maintain glomerular ultrafiltration. It is suggested that the angiotensin II may be formed intrarenally and may act on sites other than resistance blood vessels.
Objective To determine whether 6 weeks continuous treatment with an angiotensin converting enzyme... more Objective To determine whether 6 weeks continuous treatment with an angiotensin converting enzyme (ACE) inhibitor reduced renal vascular responsiveness in vivo, since this treatment results in extensive phenotypic conversion of afferent arteriolar cells from contractile to endocrine-like, renin secretory cells. Methods Enalapril (10 ìg/kg per h s.c.) was delivered continuously for 6 weeks. In anaesthetized rabbits (treated or sham), arterial blood pressure and renal blood¯ow were measured and renal responsiveness tested by constructing dose±response curves to bolus doses of phenylephrine, angiotensin II and acetylcholine delivered directly into the renal artery. Results ACE inhibition resulted in a signi®cant shift to the left in the renal vascular conductance responses to acetylcholine (P , 0.005) and angiotensin II (P , 0.05), indicating enhanced, not reduced, responsiveness to these agents. There were no signi®cant effects of chronic ACE inhibition on the conductance responses to phenylephrine. Conclusions Contrary to our hypothesis, 6 weeks ACE inhibition did not reduce renal vascular responsiveness to three vasoactive agents, suggesting that the phenotypic changes observed in the afferent arterioles and to a lesser extent the interlobular arteries, were either insigni®cant or compensated for by other changes in renal circulatory control.
Bryna S. Man Chow, Martina Kocan, Matthew Shen, Yan Wang, Lei Han, Jacqueline Y. Chew, Chao Wang,... more Bryna S. Man Chow, Martina Kocan, Matthew Shen, Yan Wang, Lei Han, Jacqueline Y. Chew, Chao Wang, Sanja Bosnyak, Katrina M. Mirabito-Colafella, Giannie Barsha, Belinda Wigg, Elizabeth K.M. Johnstone, Mohammed A. Hossain, Kevin D.G. Pfleger, Kate M. Denton, Robert E. Widdop, Roger J. Summers, Ross A.D. Bathgate, Tim D. Hewitson, and Chrishan S. Samuel Cardiovascular Disease Program, Biomedicine Discovery Institute and Departments of Pharmacology and Physiology, Monash University, Clayton Victoria 3800; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052; Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052 Australia; Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria 3050; Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western ...
Transcutaneous assessment of fluorescein isothiocyanate (FITC)-sinistrin clearance using a small ... more Transcutaneous assessment of fluorescein isothiocyanate (FITC)-sinistrin clearance using a small animal imager has recently been validated as an accurate method for the measurement of glomerular filtration rate (GFR) in freely moving rats. This technique involves a brief, light period of anesthesia during which the imager is adhered to the rat and FITC-sinistrin is administered. The rat is then moved to an experimental chamber where it is housed unrestrained for a 2-h data collection period. This study assessed the impact of the experimental protocol on mean arterial pressure (MAP), heart rate, and locomotor activity in adult Sprague Dawley rats using radiotelemetry given that anesthesia and stress are known to affect arterial pressure and kidney function. These data were compared with time-equivalent measurements made in the same rats at rest. MAP was low following anesthesia, but increased within 15 min and remained stable thereafter. Heart rate was not affected by the GFR protocol. Locomotor activity increased following anesthesia before decreasing to relatively low levels during the final 75-min, the approximate period from which GFR is calculated. Moreover, MAP, heart rate, and locomotor activity during the final 75-min of the data collection period were not different to that observed during an equivalent time period at baseline. Taken together, our findings suggest that this recently developed minimally invasive procedure for the measurement of GFR in unanesthetized rats does not negatively impact arterial pressure, heart rate, or locomotor activity. Thus, it is likely to be a valuable implement for acquiring serial measurements of GFR in unanesthetized rats.
In May 2014, the National Institutes of Health (NIH) stated its intent to “require applicants to ... more In May 2014, the National Institutes of Health (NIH) stated its intent to “require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells.” Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sex and gender are used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the “observer effect”) and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in additio...
Background Sex differences play a critical role in the incidence and severity of cardiovascular d... more Background Sex differences play a critical role in the incidence and severity of cardiovascular diseases, whereby men are at a higher risk of developing cardiovascular disease compared to age-matched premenopausal women. Marked sex differences at the cellular and tissue level may contribute to susceptibility to cardiovascular disease and end-organ damage. In this study, we have performed an in-depth histological analysis of sex differences in hypertensive cardiac and renal injury in middle-aged stroke-prone spontaneously hypertensive rats (SHRSPs) to determine the interaction between age, sex and cell senescence. Methods Kidneys, hearts and urine samples were collected from 6.5- and 8-month-old (Mo) male and female SHRSPs. Urine samples were assayed for albumin and creatinine content. Kidneys and hearts were screened for a suite of cellular senescence markers (senescence-associated β-galactosidase, p16INK4a, p21, γH2AX). Renal and cardiac fibrosis was quantified using Masson’s trich...
Significance Statement Children born with a solitary functioning kidney (SFK) can develop kidney ... more Significance Statement Children born with a solitary functioning kidney (SFK) can develop kidney injury as a consequence of glomerular hyperfiltration. Angiotensin-converting enzyme inhibitors (ACEis) reduce BP and are renoprotective in adults. Our study demonstrates that treatment with ACEi early in life (between weeks 4 and 8 after birth) in sheep born with a SFK prevents albuminuria and reduces glomerular hyperfiltration, thus maintaining renal functional reserve, 6 months after treatment withdrawal. Further, improvements in kidney function were associated with increased nitric oxide bioavailability. This study suggests ACEi for 1 month early in life may improve the trajectory for the development of kidney disease in individuals born with SFK. Background Children born with a solitary functioning kidney (SFK) are predisposed to develop hypertension and kidney injury. Glomerular hyperfiltration and hypertrophy contribute to the pathophysiology of kidney injury. Angiotensin-converti...
Recently, we designed a group of peptides by sequential substitution of the naturally occurring α... more Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding β-amino acid. β-Amino acid substitution at the proline residue of Ang III (β-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of β-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of β-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and β-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of β-Pro7-Ang III. In both male and female rats, acute sys...
A glomerular layer electrolesioned kidney model in rabbits produces a stable moderate elevation i... more A glomerular layer electrolesioned kidney model in rabbits produces a stable moderate elevation in plasma creatinine and increased kidney profibrotic, proinflammatory, and oxidative stress markers. Blood pressure and renal sympathetic nerve activity were elevated, but global sympathetic activity was reduced. These findings suggest that targeting renal nerves with denervation may be suitable for chronic kidney disease, but treatments that target general reductions in sympathetic activity should be used with caution.
Strong evidence implicates dysfunction of the sympathetic nervous system in the pathogenesis of h... more Strong evidence implicates dysfunction of the sympathetic nervous system in the pathogenesis of hypertension. In patients, with borderline and established hypertension, indexes of sympathetic activity (muscle sympathetic nerve activity, plasma noradrenaline levels, organ noradrenaline spillover) positively correlate with hypertension severity. In particular, evidence indicates that renal and cardiac sympathetic activation is disproportionately elevated. However, this evidence was garnered, in large part, from cross-sectional studies disallowing assignment of causation. In addition, each study used indirect methods to assess sympathetic activation with inherent strengths and weaknesses, which need to be considered when interpreting the data. For example, muscle sympathetic nerve activity recording does not provide information about nerve activity to the heart or kidney, the release of neurotransmitter at the nerve terminal, and the peroneal nerve used in such recordings, contains a mixed population of nerves, and so is not solely an index of sympathetic activity. Animal studies, in which more invasive studies can be performed, have underpinned and supported the clinical studies. Currently, it is widely held that the renal nerves (afferent and efferent) play a causative role in the development of hypertension, though many questions remain. To provide direct evidence for the involvement of increased renal sympathetic nerve activity (RSNA) in the development of hypertension, preclinical studies, in which it is possible to directly determine RSNA via the implantation of recording electrodes around the main renal nerves, have been performed. Early studies were limited by the durability of the nerve recordings and were often performed under anesthesia. However, advancements in these methodologies, though technically challenging, meant that long-term RSNA recordings for periods of many weeks became possible. In this issue of Hypertension, Miki et al report, in a tour de force, the simultaneous continuous recording of arterial pressure, RSNA, and lumbar sympathetic nerve activity via telemetry in unrestrained rats over a period of 3 weeks. The hypothesis of this study was that an increase in RSNA would precede the increase in arterial pressure after high-salt feeding in Dahl salt-sensitive (Dahl-S) rats. The authors highlight the importance of this study by laying out in detail the conflicting evidence available in the literature for the involvement of the renal nerves in the development of salt-sensitive hypertension. The results, contrary to the hypothesis, clearly showed no marked change in RSNA before or during the salt-driven increase in arterial pressure in the Dahl-S rats. It was concluded that salt-sensitive hypertension in the Dahl-S rat is not caused by increased RSNA. Yet, previous studies have shown that renal denervation in Dahl-S rats attenuates renal injury, in response to a high-salt diet, indicating a role for the renal nerves. How then to bring these disparate findings together? Possible explanations include (Figure 1): 1. Renal nerve anatomy: a limitation of nerve recording is that it is not possible to know that all nerves have been placed around the recording electrode. This makes it all but impossible to determine absolute nerve activity and therefore to make comparisons between groups. Perhaps, the innervation of the Dahl-S rat is more diffuse. For example, we have observed in the spontaneously hypertensive rat as compared with the Sprague Dawley rat that nerve fibers arrive at the renal hilus from a much broader direction and need to be found and place on the stimulating electrode to reduce renal blood flow to zero in response to maximal electrical stimulation (unpublished observation). Leaving the possibility that apples and oranges are being compared, particularly in terms of baseline activity. 2. Decoding the information encoded in the nerve traffic: (1) the recordings are made from a multifiber bundle with both afferent and efferent activity being summated in the recording. Perhaps, a change in one of these negates an increase in the other, resulting in a null effect in total nerve activity? Furthermore, an increase in afferent nerve activity has been associated with hypertension in the DOCA-salt model. Perhaps, small but functionally significant changes in renal afferent nerve activity might not be discernible in multifiber recordings. (2) In addition, evidence exists that there are subpopulations of renal nerves, based on chemical coding of neurotransmitters. Perhaps, these subpopulations are being differentially activated. 3. Neuroeffector structure function: nerve activity may not have increased but differences in neuroeffector properties might contribute to an enhancement of the response to nerve activation. (1) Anatomic differences may The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Cardiovascular…
The Protective Arm of the Renin Angiotensin System (RAS), 2015
This chapter discusses the effects of both endogenous and synthetic peptides in the context of us... more This chapter discusses the effects of both endogenous and synthetic peptides in the context of use as AT2R agonists. Historically, peptides have often been disregarded for therapeutic development due to their poor in vivo stability and lack of receptor subtype selectivity. However, recent developments in drug delivery technologies and advanced knowledge of chemical modifications to alter pharmacological characteristics have reinvigorated the field of peptide therapeutics. Indeed, peptides have been frequently utilized as the basis of drug design due to beneficial properties such as relatively cheap and simple synthesis, high-affinity binding at cognate receptors, and potential for further chemical additions and substitutions. Moreover, considering the still unresolved role of AT2R function in many contexts, peptide agonists are also likely to remain invaluable experimental tools for the field of AT2R research well into the future.
The Protective Arm of the Renin Angiotensin System (RAS), 2015
This chapter summarizes the predominantly vasodilator effects of AT 2 R in the vasculature based ... more This chapter summarizes the predominantly vasodilator effects of AT 2 R in the vasculature based on both in vitro and in vivo preclinical studies, before describing the effects of chronic AT 2 R stimulation on blood pressure. Generally, AT 2 R agonists are not considered to be antihypertensive in preclinical models despite the fact that vascular AT 2 Rs are functionally active in preclinical studies and in a number of acute human studies ( ex vivo and in vivo ). Possible mechanisms are explored to explain the apparent lack of effect of AT 2 R on blood pressure in chronic pharmacological studies that would not have been predicted from the large body of evidence documenting AT 2 R-mediated vasodilator function.
We examined whether renal denervation (RDN) reduced blood pressure (BP), improved glomerular filt... more We examined whether renal denervation (RDN) reduced blood pressure (BP), improved glomerular filtration rate, albuminuria, and left ventricular mass in sheep with hypertensive chronic kidney disease (CKD). To examine whether renal nerve function returned in the long term, we examined vascular contraction to nerve stimulation in renal arteries and determined nerve regrowth by assessing renal TH (tyrosine hydroxylase), CGRP (calcitonin gene-related peptide), and norepinephrine levels in kidneys at 30 months after RDN. RDN normalized BP in hypertensive CKD sheep such that BP was similar to that of the normotensive sheep with intact nerves. Glomerular filtration rate decreased by ≈22% in CKD sheep with intact nerves but increased ≈26% in hypertensive CKD-RDN sheep by 30 months. At 30 months, urinary albumin was ≈127% and left ventricular mass was ≈41% greater in CKD sheep with intact nerves than control. However, urinary albumin was ≈60% less and left ventricular mass was ≈40% less in t...
Clinical and Experimental Pharmacology and Physiology, 1990
1. The effect of two doses of endothelin, 10 and 50 ng/kg per min, i.v., on glomerular filtration... more 1. The effect of two doses of endothelin, 10 and 50 ng/kg per min, i.v., on glomerular filtration rate (GFR), tubular stop flow pressure and pre- and postglomerular vascular resistance have been studied in anaesthetized rabbits.2. Blood pressure did not change significantly in response to 10 ng/kg per min endothelin or vehicle infusion, but rose steadily during infusion of 50 ng/kg per min endothelin, increasing 11.8±2.7 mmHg by 90 min of infusion.3. Glomerular filtration fraction (3H-inulin extraction ratio) rose and remained elevated throughout the endothelin infusion at 50 ng/kg per min. GFR did not change significantly until 70–90 min of the infusion (50 ng/kg per min) when it decreased by about 35%. No significant changes were seen at 10 ng/kg per min endothelin.4. Sodium excretion rate rose in response to the lower dose, due to an increase in fractional sodium excretion. No changes in sodium excretion were seen at the higher dose of endothelin.5. Glomerular capillary pressure rose significantly in response to endothelin infusion (50 ng/kg per min).6. Renal blood flow fell progressively in response to endothelin (50 ng/kg per min), to about one-third of the pre-infusion value.7. Renal vascular resistance increased progressively with both doses of endothelin, by about 35% at 10 ng/kg per min and about 400% at 50 ng/kg per min after 70–90 min. Preglomerular resistance increased from 1.0±0.1 to 5.0±1.9 mmHg/mL per min in response to endothelin 50 ng/kg per min. Postglomerular resistance rose from 1.0±0.1 to 5.6±2.17 mmHg/mL per min.8. Thus endothelin infusion caused progressive renal vasoconstriction with similar magnitude increases in both pre- and postglomerular vessels. The vasoconstriction of the kidney caused by endothelin occurred at a dose which did not effect systemic blood pressure.
Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound ... more Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammato...
Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with ... more Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with age. Estrogen plays a protective role against hypertension and end-organ damage by modulating the depressor actions of the AT 2 R (angiotensin type 2 receptor). Our aim was to determine whether estrogen replacement in aged female mice can lower arterial pressure, improve endothelial function, and reduce organ fibrosis via an AT 2 R-mediated mechanism. Mean arterial pressure was measured via radiotelemetry in ovary-intact adult (3–4-month-old), aged (16–18-month-old; reproductively senescent) and aged–17β-estradiol (E 2 )–treated (3 µg/day SC) female mice, which were administered vehicle, Ang II (angiotensin II; 600 ng/[kg·min] SC) or Ang II+PD123319 (AT 2 R antagonist; 3 mg/[kg·day SC). On day 21 of treatment, aortic endothelium-dependent relaxation and cardiac and renal tissue (fibrosis and gene expression) were analyzed. Basal mean arterial pressure was lower in E 2 -treated aged mice (89±1 mm Hg, n=20) relative to aged controls (94±1 mm Hg; n=18, P =0.002). The Ang II pressor response was enhanced by ≈20 mm Hg in aged compared with adult females ( P =0.01). E 2 -treatment reduced the Ang II pressor response in aged females ( P =0.002), an effect that was reversed by PD123319 in the aged E 2 –Ang II group ( P =0.0009). E 2 -treatment increased renal AT 2 R (≈6-fold; P <0.0001) and MasR (Mas oncoreceptor; 2–3-fold, P <0.05) gene expression in aged females. However, neither Ang II–induced endothelial dysfunction nor the age-related increase in renal and cardiac fibrosis was restored by E 2 -treatment in aged female mice. In conclusion, estrogen replacement in aged females may reduce arterial pressure to levels observed in adult females, via an AT 2 R-mediated renal mechanism.
Canadian Journal of Physiology and Pharmacology, Aug 1, 1987
In renal artery stenosis severe enough to cause hypertension, angiotensin II maintains glomerular... more In renal artery stenosis severe enough to cause hypertension, angiotensin II maintains glomerular filtration rate (GFR) both in the initial high renin phase of hypertension and later when plasma levels are normal. Angiotensin II also maintains GFR in less severe stenosis, which does not cause hypertension. This homeostatic action of angiotensin II to maintain GFR has minimal effects on blood flow. In renal-wrap hypertension, plasma renin levels are elevated for longer than after renal artery stenosis, but in other respects this initial phase of the hypertension is similar to that after renal artery stenosis. GFR is reduced, the rate of development of hypertension is accelerated by angiotensin II, and angiotensin II maintains the glomerular filtration fraction. Renal resistance is markedly increased owing to both compression of the kidney by the hypertrophying renal capsule and to angiotensin II. Thus angiotensin II apparently plays a primarily homeostatic role in renovascular hypertension to maintain glomerular ultrafiltration. It is suggested that the angiotensin II may be formed intrarenally and may act on sites other than resistance blood vessels.
Objective To determine whether 6 weeks continuous treatment with an angiotensin converting enzyme... more Objective To determine whether 6 weeks continuous treatment with an angiotensin converting enzyme (ACE) inhibitor reduced renal vascular responsiveness in vivo, since this treatment results in extensive phenotypic conversion of afferent arteriolar cells from contractile to endocrine-like, renin secretory cells. Methods Enalapril (10 ìg/kg per h s.c.) was delivered continuously for 6 weeks. In anaesthetized rabbits (treated or sham), arterial blood pressure and renal blood¯ow were measured and renal responsiveness tested by constructing dose±response curves to bolus doses of phenylephrine, angiotensin II and acetylcholine delivered directly into the renal artery. Results ACE inhibition resulted in a signi®cant shift to the left in the renal vascular conductance responses to acetylcholine (P , 0.005) and angiotensin II (P , 0.05), indicating enhanced, not reduced, responsiveness to these agents. There were no signi®cant effects of chronic ACE inhibition on the conductance responses to phenylephrine. Conclusions Contrary to our hypothesis, 6 weeks ACE inhibition did not reduce renal vascular responsiveness to three vasoactive agents, suggesting that the phenotypic changes observed in the afferent arterioles and to a lesser extent the interlobular arteries, were either insigni®cant or compensated for by other changes in renal circulatory control.
Bryna S. Man Chow, Martina Kocan, Matthew Shen, Yan Wang, Lei Han, Jacqueline Y. Chew, Chao Wang,... more Bryna S. Man Chow, Martina Kocan, Matthew Shen, Yan Wang, Lei Han, Jacqueline Y. Chew, Chao Wang, Sanja Bosnyak, Katrina M. Mirabito-Colafella, Giannie Barsha, Belinda Wigg, Elizabeth K.M. Johnstone, Mohammed A. Hossain, Kevin D.G. Pfleger, Kate M. Denton, Robert E. Widdop, Roger J. Summers, Ross A.D. Bathgate, Tim D. Hewitson, and Chrishan S. Samuel Cardiovascular Disease Program, Biomedicine Discovery Institute and Departments of Pharmacology and Physiology, Monash University, Clayton Victoria 3800; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052; Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052 Australia; Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria 3050; Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western ...
Transcutaneous assessment of fluorescein isothiocyanate (FITC)-sinistrin clearance using a small ... more Transcutaneous assessment of fluorescein isothiocyanate (FITC)-sinistrin clearance using a small animal imager has recently been validated as an accurate method for the measurement of glomerular filtration rate (GFR) in freely moving rats. This technique involves a brief, light period of anesthesia during which the imager is adhered to the rat and FITC-sinistrin is administered. The rat is then moved to an experimental chamber where it is housed unrestrained for a 2-h data collection period. This study assessed the impact of the experimental protocol on mean arterial pressure (MAP), heart rate, and locomotor activity in adult Sprague Dawley rats using radiotelemetry given that anesthesia and stress are known to affect arterial pressure and kidney function. These data were compared with time-equivalent measurements made in the same rats at rest. MAP was low following anesthesia, but increased within 15 min and remained stable thereafter. Heart rate was not affected by the GFR protocol. Locomotor activity increased following anesthesia before decreasing to relatively low levels during the final 75-min, the approximate period from which GFR is calculated. Moreover, MAP, heart rate, and locomotor activity during the final 75-min of the data collection period were not different to that observed during an equivalent time period at baseline. Taken together, our findings suggest that this recently developed minimally invasive procedure for the measurement of GFR in unanesthetized rats does not negatively impact arterial pressure, heart rate, or locomotor activity. Thus, it is likely to be a valuable implement for acquiring serial measurements of GFR in unanesthetized rats.
In May 2014, the National Institutes of Health (NIH) stated its intent to “require applicants to ... more In May 2014, the National Institutes of Health (NIH) stated its intent to “require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells.” Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sex and gender are used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the “observer effect”) and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in additio...
Background Sex differences play a critical role in the incidence and severity of cardiovascular d... more Background Sex differences play a critical role in the incidence and severity of cardiovascular diseases, whereby men are at a higher risk of developing cardiovascular disease compared to age-matched premenopausal women. Marked sex differences at the cellular and tissue level may contribute to susceptibility to cardiovascular disease and end-organ damage. In this study, we have performed an in-depth histological analysis of sex differences in hypertensive cardiac and renal injury in middle-aged stroke-prone spontaneously hypertensive rats (SHRSPs) to determine the interaction between age, sex and cell senescence. Methods Kidneys, hearts and urine samples were collected from 6.5- and 8-month-old (Mo) male and female SHRSPs. Urine samples were assayed for albumin and creatinine content. Kidneys and hearts were screened for a suite of cellular senescence markers (senescence-associated β-galactosidase, p16INK4a, p21, γH2AX). Renal and cardiac fibrosis was quantified using Masson’s trich...
Significance Statement Children born with a solitary functioning kidney (SFK) can develop kidney ... more Significance Statement Children born with a solitary functioning kidney (SFK) can develop kidney injury as a consequence of glomerular hyperfiltration. Angiotensin-converting enzyme inhibitors (ACEis) reduce BP and are renoprotective in adults. Our study demonstrates that treatment with ACEi early in life (between weeks 4 and 8 after birth) in sheep born with a SFK prevents albuminuria and reduces glomerular hyperfiltration, thus maintaining renal functional reserve, 6 months after treatment withdrawal. Further, improvements in kidney function were associated with increased nitric oxide bioavailability. This study suggests ACEi for 1 month early in life may improve the trajectory for the development of kidney disease in individuals born with SFK. Background Children born with a solitary functioning kidney (SFK) are predisposed to develop hypertension and kidney injury. Glomerular hyperfiltration and hypertrophy contribute to the pathophysiology of kidney injury. Angiotensin-converti...
Recently, we designed a group of peptides by sequential substitution of the naturally occurring α... more Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding β-amino acid. β-Amino acid substitution at the proline residue of Ang III (β-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of β-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of β-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and β-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of β-Pro7-Ang III. In both male and female rats, acute sys...
A glomerular layer electrolesioned kidney model in rabbits produces a stable moderate elevation i... more A glomerular layer electrolesioned kidney model in rabbits produces a stable moderate elevation in plasma creatinine and increased kidney profibrotic, proinflammatory, and oxidative stress markers. Blood pressure and renal sympathetic nerve activity were elevated, but global sympathetic activity was reduced. These findings suggest that targeting renal nerves with denervation may be suitable for chronic kidney disease, but treatments that target general reductions in sympathetic activity should be used with caution.
Strong evidence implicates dysfunction of the sympathetic nervous system in the pathogenesis of h... more Strong evidence implicates dysfunction of the sympathetic nervous system in the pathogenesis of hypertension. In patients, with borderline and established hypertension, indexes of sympathetic activity (muscle sympathetic nerve activity, plasma noradrenaline levels, organ noradrenaline spillover) positively correlate with hypertension severity. In particular, evidence indicates that renal and cardiac sympathetic activation is disproportionately elevated. However, this evidence was garnered, in large part, from cross-sectional studies disallowing assignment of causation. In addition, each study used indirect methods to assess sympathetic activation with inherent strengths and weaknesses, which need to be considered when interpreting the data. For example, muscle sympathetic nerve activity recording does not provide information about nerve activity to the heart or kidney, the release of neurotransmitter at the nerve terminal, and the peroneal nerve used in such recordings, contains a mixed population of nerves, and so is not solely an index of sympathetic activity. Animal studies, in which more invasive studies can be performed, have underpinned and supported the clinical studies. Currently, it is widely held that the renal nerves (afferent and efferent) play a causative role in the development of hypertension, though many questions remain. To provide direct evidence for the involvement of increased renal sympathetic nerve activity (RSNA) in the development of hypertension, preclinical studies, in which it is possible to directly determine RSNA via the implantation of recording electrodes around the main renal nerves, have been performed. Early studies were limited by the durability of the nerve recordings and were often performed under anesthesia. However, advancements in these methodologies, though technically challenging, meant that long-term RSNA recordings for periods of many weeks became possible. In this issue of Hypertension, Miki et al report, in a tour de force, the simultaneous continuous recording of arterial pressure, RSNA, and lumbar sympathetic nerve activity via telemetry in unrestrained rats over a period of 3 weeks. The hypothesis of this study was that an increase in RSNA would precede the increase in arterial pressure after high-salt feeding in Dahl salt-sensitive (Dahl-S) rats. The authors highlight the importance of this study by laying out in detail the conflicting evidence available in the literature for the involvement of the renal nerves in the development of salt-sensitive hypertension. The results, contrary to the hypothesis, clearly showed no marked change in RSNA before or during the salt-driven increase in arterial pressure in the Dahl-S rats. It was concluded that salt-sensitive hypertension in the Dahl-S rat is not caused by increased RSNA. Yet, previous studies have shown that renal denervation in Dahl-S rats attenuates renal injury, in response to a high-salt diet, indicating a role for the renal nerves. How then to bring these disparate findings together? Possible explanations include (Figure 1): 1. Renal nerve anatomy: a limitation of nerve recording is that it is not possible to know that all nerves have been placed around the recording electrode. This makes it all but impossible to determine absolute nerve activity and therefore to make comparisons between groups. Perhaps, the innervation of the Dahl-S rat is more diffuse. For example, we have observed in the spontaneously hypertensive rat as compared with the Sprague Dawley rat that nerve fibers arrive at the renal hilus from a much broader direction and need to be found and place on the stimulating electrode to reduce renal blood flow to zero in response to maximal electrical stimulation (unpublished observation). Leaving the possibility that apples and oranges are being compared, particularly in terms of baseline activity. 2. Decoding the information encoded in the nerve traffic: (1) the recordings are made from a multifiber bundle with both afferent and efferent activity being summated in the recording. Perhaps, a change in one of these negates an increase in the other, resulting in a null effect in total nerve activity? Furthermore, an increase in afferent nerve activity has been associated with hypertension in the DOCA-salt model. Perhaps, small but functionally significant changes in renal afferent nerve activity might not be discernible in multifiber recordings. (2) In addition, evidence exists that there are subpopulations of renal nerves, based on chemical coding of neurotransmitters. Perhaps, these subpopulations are being differentially activated. 3. Neuroeffector structure function: nerve activity may not have increased but differences in neuroeffector properties might contribute to an enhancement of the response to nerve activation. (1) Anatomic differences may The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Cardiovascular…
The Protective Arm of the Renin Angiotensin System (RAS), 2015
This chapter discusses the effects of both endogenous and synthetic peptides in the context of us... more This chapter discusses the effects of both endogenous and synthetic peptides in the context of use as AT2R agonists. Historically, peptides have often been disregarded for therapeutic development due to their poor in vivo stability and lack of receptor subtype selectivity. However, recent developments in drug delivery technologies and advanced knowledge of chemical modifications to alter pharmacological characteristics have reinvigorated the field of peptide therapeutics. Indeed, peptides have been frequently utilized as the basis of drug design due to beneficial properties such as relatively cheap and simple synthesis, high-affinity binding at cognate receptors, and potential for further chemical additions and substitutions. Moreover, considering the still unresolved role of AT2R function in many contexts, peptide agonists are also likely to remain invaluable experimental tools for the field of AT2R research well into the future.
The Protective Arm of the Renin Angiotensin System (RAS), 2015
This chapter summarizes the predominantly vasodilator effects of AT 2 R in the vasculature based ... more This chapter summarizes the predominantly vasodilator effects of AT 2 R in the vasculature based on both in vitro and in vivo preclinical studies, before describing the effects of chronic AT 2 R stimulation on blood pressure. Generally, AT 2 R agonists are not considered to be antihypertensive in preclinical models despite the fact that vascular AT 2 Rs are functionally active in preclinical studies and in a number of acute human studies ( ex vivo and in vivo ). Possible mechanisms are explored to explain the apparent lack of effect of AT 2 R on blood pressure in chronic pharmacological studies that would not have been predicted from the large body of evidence documenting AT 2 R-mediated vasodilator function.
We examined whether renal denervation (RDN) reduced blood pressure (BP), improved glomerular filt... more We examined whether renal denervation (RDN) reduced blood pressure (BP), improved glomerular filtration rate, albuminuria, and left ventricular mass in sheep with hypertensive chronic kidney disease (CKD). To examine whether renal nerve function returned in the long term, we examined vascular contraction to nerve stimulation in renal arteries and determined nerve regrowth by assessing renal TH (tyrosine hydroxylase), CGRP (calcitonin gene-related peptide), and norepinephrine levels in kidneys at 30 months after RDN. RDN normalized BP in hypertensive CKD sheep such that BP was similar to that of the normotensive sheep with intact nerves. Glomerular filtration rate decreased by ≈22% in CKD sheep with intact nerves but increased ≈26% in hypertensive CKD-RDN sheep by 30 months. At 30 months, urinary albumin was ≈127% and left ventricular mass was ≈41% greater in CKD sheep with intact nerves than control. However, urinary albumin was ≈60% less and left ventricular mass was ≈40% less in t...
Clinical and Experimental Pharmacology and Physiology, 1990
1. The effect of two doses of endothelin, 10 and 50 ng/kg per min, i.v., on glomerular filtration... more 1. The effect of two doses of endothelin, 10 and 50 ng/kg per min, i.v., on glomerular filtration rate (GFR), tubular stop flow pressure and pre- and postglomerular vascular resistance have been studied in anaesthetized rabbits.2. Blood pressure did not change significantly in response to 10 ng/kg per min endothelin or vehicle infusion, but rose steadily during infusion of 50 ng/kg per min endothelin, increasing 11.8±2.7 mmHg by 90 min of infusion.3. Glomerular filtration fraction (3H-inulin extraction ratio) rose and remained elevated throughout the endothelin infusion at 50 ng/kg per min. GFR did not change significantly until 70–90 min of the infusion (50 ng/kg per min) when it decreased by about 35%. No significant changes were seen at 10 ng/kg per min endothelin.4. Sodium excretion rate rose in response to the lower dose, due to an increase in fractional sodium excretion. No changes in sodium excretion were seen at the higher dose of endothelin.5. Glomerular capillary pressure rose significantly in response to endothelin infusion (50 ng/kg per min).6. Renal blood flow fell progressively in response to endothelin (50 ng/kg per min), to about one-third of the pre-infusion value.7. Renal vascular resistance increased progressively with both doses of endothelin, by about 35% at 10 ng/kg per min and about 400% at 50 ng/kg per min after 70–90 min. Preglomerular resistance increased from 1.0±0.1 to 5.0±1.9 mmHg/mL per min in response to endothelin 50 ng/kg per min. Postglomerular resistance rose from 1.0±0.1 to 5.6±2.17 mmHg/mL per min.8. Thus endothelin infusion caused progressive renal vasoconstriction with similar magnitude increases in both pre- and postglomerular vessels. The vasoconstriction of the kidney caused by endothelin occurred at a dose which did not effect systemic blood pressure.
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