Theresa Kurth

Effects of daily sodium intake and ANG II on cortical and medullary renal blood flow in conscious rats

Implanted optical fibers and laser-Doppler flow measurement techniques were used for the sequential measurement of regional renal blood flow in conscious rats to determine the effects of an increase of daily NaCl intake on the renal cortical blood flow and blood flow to the outer and inner medulla. Cortical blood flow was increased significantly (32%) by the second day when NaCl intake was increased from 1 to 7 meq/day and was increased further (50%) on the second day after a further elevation of NaCl intake to 13 meq/day. Blood flow to the outer and inner medulla was not changed as NaCl intake was elevated. The increase in renal cortical flow was closely associated with significant reductions in circulating concentrations of ANG II from 31 to 16 pg/ml. Rats given a continuous infusion of nonpressor doses of ANG II (5.0 ng ⋅ kg-1 ⋅ min-1) to maintain constant plasma concentrations of ANG II as sodium intake was increased exhibited no increase of cortical flow. We conclude that reductions of plasma ANG II associated with incremental increases of daily sodium intake result in a rise of renal cortical flow. The elevated blood flow to the renal cortex may enhance sodium excretion and contribute to long-term sodium homeostasis.Implanted optical fibers and laser-Doppler flow measurement techniques were used for the sequential measurement of regional renal blood flow in conscious rats to determine the effects of an increase of daily NaCl intake on the renal cortical blood flow and blood flow to the outer and inner medulla. Cortical blood flow was increased significantly (32%) by the second day when NaCl intake was increased from 1 to 7 meq/day and was increased further (50%) on the second day after a further elevation of NaCl intake to 13 meq/day. Blood flow to the outer and inner medulla was not changed as NaCl intake was elevated. The increase in renal cortical flow was closely associated with significant reductions in circulating concentrations of ANG II from 31 to 16 pg/ml. Rats given a continuous infusion of nonpressor does of ANG II (5.0 ng.kg(-1).min-1) to maintain constant plasma concentrations of ANG II as sodium intake was increased exhibited no increase of cortical flow. We conclude that reductions of plasma ANG II associated with incremental increases of daily sodium intake result in a rise of renal cortical flow. The elevated blood flow to the renal cortex may enhance sodium excretion and contribute to long-term sodium homeostasis.

Effects of long-term vasopressin receptor stimulation on medullary blood flow and arterial pressure

Studies were carried out using instrumented unanesthetized rats to determine the long-term effects of arginine vasopressin (AVP) and a specific vasopressin V1 receptor agonist (V1AG; [Phe2, Ile3, Orn8]- vasopressin) on the renal medullary blood flow and arterial blood pressure. It was hypothesized that the hypertension observed with chronic medullary infusion of a V1 receptor agonist may be associated with a sustained reduction of blood flow, whereas infusion of AVP may fail to produce a sustained reduction of blood flow and thereby be unable to produce hypertension. Uninephrectomized Sprague-Dawley rats were prepared with implanted renal cortical and medullary optical fibers for daily measurements of cortical and medullary blood flow using laser-Doppler flowmetry techniques. An implanted renal medullary interstitial infusion catheter delivered either AVP or a specific V1AG at a dose of 2 ng ⋅ kg-1 ⋅ min-1over a period of 5 days. The V1AG produced no change of cortical blood flow but a chronic 35% reduction of medullary blood flow ( P < 0.05) and mild hypertension (11 ± 4 mmHg, P < 0.05). AVP produced only an initial, nonsignificant 1- to 2-day reduction of medullary blood flow (-13%) and failed to raise arterial pressure significantly. We conclude that a sustained V1AG response is necessary to achieve a chronic reduction of medullary blood flow and hypertension. The present data are consistent with the idea that chronic stimulation of V2receptors by AVP offsets the vasoconstrictor and hypertension actions of AVP-induced stimulation of medullary V1 receptors.Studies were carried out using instrumented unanesthetized rats to determine the long-term effects of arginine vasopressin (AVP) and a specific vasopressin V1 receptor agonist (V1AG; [Phe2, Ile3, Orn8]- vasopressin) on the renal medullary blood flow and arterial blood pressure. It was hypothesized that the hypertension observed with chronic medullary infusion of a V1 receptor agonist may be associated with a sustained reduction of blood flow, whereas infusion of AVP may fail to produce a sustained reduction of blood flow and thereby be unable to produce hypertension. Uninephrectomized Sprague-Dawley rats were prepared with implanted renal cortical and medullary optical fibers for daily measurements of cortical and medullary blood flow using laser-Doppler flowmetry techniques. An implanted renal medullary interstitial infusion catheter delivered either AVP or a specific V1AG at a dose of 2 ng . kg-1 . min-1 over a period of 5 days. The V1AG produced no change of cortical blood flow but a chronic 35% reduction of medullary blood flow (P < 0.05) and mild hypertension (11 +/- 4 mmHg, P < 0.05). AVP produced only an initial, nonsignificant 1- to 2-day reduction of medullary blood flow (-13%) and failed to raise arterial pressure significantly. We conclude that a sustained V1AG response is necessary to achieve a chronic reduction of medullary blood flow and hypertension. The present data are consistent with the idea that chronic stimulation of V2 receptors by AVP offsets the vasoconstrictor and hypertension actions of AVP-induced stimulation of medullary V1 receptors.

Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SSNox4−/−) rats. SSNox4−/− rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SSNox4−/− rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67phox, and p22phox were found to be significantly lower (P<0.05) in SSNox4−/− compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SSNox4−/− rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SSNox4−/− rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

Null Mutation of the Nicotinamide Adenine Dinucleotide Phosphate–Oxidase Subunit p67phox Protects the Dahl-S Rat From Salt-Induced Reductions in Medullary Blood Flow and Glomerular Filtration Rate

Null mutations in the p67phox subunit of NADPH-oxidase confer protection from salt-sensitivity on Dahl salt-sensitive (SS) rats. Here we track the sequential changes in medullary blood flow, glomerular filtration rate, urinary protein and mean arterial pressure in SSp67phox null rats and wild-type littermates during 21-days of 4.0% NaCl (high-salt [HS]) diet. Optical fibers were implanted in the renal medulla and medullary blood flow measured in conscious rats by laser-Doppler flowmetry. Separate groups of rats were prepared with femoral venous catheters and glomerular filtration rate measured by the transcutaneous assessment of fluorescein isothiocyanate-sinistrin disappearance curves. Mean arterial blood pressure was measured by telemetry. In wild-type rats HS caused a rapid reduction in medullary blood flow which was significantly lower than control values by HS day-6. Reduced medullary blood flow was associated with a progressive increase in mean arterial pressure, averaging 170 ± 5 mmHg by HS salt day-21. A significant reduction in glomerular filtration rate was evident at day-14 HS, after the onset of hypertension and reduced medullary blood flow. In contrast, HS had no significant effect on medullary blood flow in SSp67phox null rats and the pressor response to sodium was blunted, averaging 150 ± 3 mmHg at day-21 HS. Glomerular filtration rate was maintained throughout the study and proteinuria was reduced. In summary, when p67phox is not functional in the SS rat HS does not cause reduced medullary blood flow and salt-sensitive hypertension is attenuated, consequently renal injury is reduced and glomerular filtration rate is maintained.Null mutations in the p67phox subunit of nicotinamide adenine dinucleotide phosphate–oxidase confer protection from salt sensitivity on Dahl salt-sensitive rats. Here, we track the sequential changes in medullary blood flow (MBF), glomerular filtration rate (GFR), urinary protein, and mean arterial pressure in SSp67phox null rats and wild-type littermates during 21 days of 4.0% NaCl high-salt (HS) diet. Optical fibers were implanted in the renal medulla and MBF was measured in conscious rats by laser Doppler flowmetry. Separate groups of rats were prepared with femoral venous catheters and GFR was measured by the transcutaneous assessment of fluorescein isothiocyanate-sinistrin disappearance curves. Mean arterial blood pressure was measured by telemetry. In wild-type rats, HS caused a rapid reduction in MBF, which was significantly lower than control values by HS day-6. Reduced MBF was associated with a progressive increase in mean arterial pressure, averaging 170±5 mm Hg by HS salt day-21. A significant reduction in GFR was evident on day-14 HS, after the onset of hypertension and reduced MBF. In contrast, HS had no significant effect on MBF in SSp67phox null rats and the pressor response to sodium was blunted, averaging 150±3 mm Hg on day-21 HS. GFR was maintained throughout the study and proteinuria was reduced. In summary, when p67phox is not functional in the salt-sensitive rats, HS does not cause reduced MBF and salt-sensitive hypertension is attenuated, and consequently renal injury is reduced and GFR is maintained.

Characterization of biological pathways associated with a 1.37 Mbp genomic region protective of hypertension in Dahl S rats

The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood-pressure salt sensitivity in Dahl salt-sensitive (SS) rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high-salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) a linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups and 2) a Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na(+) and K(+) electrochemical gradients and tubular Na(+) transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms.

PAPPA2 IS LINKED TO SALT-SENSITIVE HYPERTENSION IN DAHL S RATS

A 1.37 Mbp region of chromosome 13 previously identified by exclusion mapping was consistently associated with a reduction of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. This region contained five genes that were introgressed from the salt-insensitive Brown Norway (BN) rat. The goal of the present study was to further narrow that region to identify the gene(s) most likely to protect from salt-induced hypertension. The studies yielded a subcongenic SS rat strain containing a 0.71 Mbp insert from BN (26-P strain) in which salt-induced hypertension was reduced by 24 mmHg. The region contained two protein-coding genes (Astn1 and Pappa2) and a microRNA (miR-488). Pappa2 mRNA in the renal cortex of the protected 26-P was 6- to 10-fold greater than in SS fed a 0.4% NaCl diet but was reduced to levels observed in SS when fed 8.0% NaCl diet for 7 days. Compared with brain nuclei (NTS, RVLM, CVLM) and the adrenal gland, Pappa2 in the renal cortex was the only gene found to be differentially expressed between SS and 26-P and that responded to changes of salt diet. Immunohistochemistry studies found Pappa2 localized in the cytosol of the epithelial cells of the cortical thick ascending limbs. In more distal segments of the renal tubules, it was observed within tubular lumens and most notably bound to the apical membranes of the intercalated cells of collecting ducts. We conclude that we have identified a variant form of Pappa2 that can protect against salt-induced hypertension in the Dahl S rat.

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

Increased Perfusion Pressure Drives Renal T-Cell Infiltration in the Dahl Salt-Sensitive RatNovelty and Significance

Renal T-cell infiltration is a key component of salt-sensitive hypertension in Dahl salt-sensitive (SS) rats. Here, we use an electronic servo-control technique to determine the contribution of renal perfusion pressure to T-cell infiltration in the SS rat kidney. An aortic balloon occluder placed around the aorta between the renal arteries was used to maintain perfusion pressure to the left kidney at control levels, ≈128 mm Hg, during 7 days of salt-induced hypertension, whereas the right kidney was exposed to increased renal perfusion pressure that averaged 157±4 mm Hg by day 7 of high-salt diet. The number of infiltrating T cells was compared between the 2 kidneys. Renal T-cell infiltration was significantly blunted in the left servo-controlled kidney compared with the right uncontrolled kidney. The number of CD3+, CD3+CD4+, and CD3+CD8+ T cells were all significantly lower in the left servo-controlled kidney. This effect was not specific to T cells because CD45R+ (B cells) and CD11b/c+ (monocytes and macrophages) cell infiltrations were all exacerbated in the hypertensive kidneys. Increased renal perfusion pressure was also associated with augmented renal injury, with increased protein casts and glomerular damage in the hypertensive kidney. Levels of norepinephrine were comparable between the 2 kidneys, suggestive of equivalent sympathetic innervation. Renal infiltration of T cells was not reversed by the return of renal perfusion pressure to control levels after 7 days of salt-sensitive hypertension. We conclude that increased pressure contributes to the initiation of renal T-cell infiltration during the progression of salt-sensitive hypertension in SS rats.

Breaking the Cycle: Estrous Variation Does Not Require Increased Sample Size in the Study of Female Rats.

Despite the striking differences between male and female physiology, female physiology is understudied. In response, the National Institutes of Health is promulgating new policies to increase the use of female organisms in preclinical research. Females are commonly believed to have greater variability than males because of the estrous cycle, but recent studies call this belief into question. Effects of estrous cycling on mean arterial pressure were assessed in female Dahl S rats using telemetry and vaginal cytometry and found that estrous cycling did not affect mean arterial pressure magnitude or variance. Data from the PhysGen arm of the Program for Genomic Applications was used to compare male and female variance and coefficient of variation in 142 heart, lung, vascular, kidney, and blood phenotypes, each measured in hundreds to thousands of individual rats from over 50 inbred strains. Seventy-four of 142 phenotypes from this data set demonstrated a sex difference in variance; however, 59% of these phenotypes exhibited greater variance in male rats rather than female. Remarkably, a retrospective power analysis demonstrated that only 16 of 74 differentially variable phenotypes would be detected when using an experimental cohort large enough to detect a difference in magnitude. No overall difference in coefficient of variation between male and female rats was detected when analyzing these 142 phenotypes. We conclude that variability of 142 traits in male and female rats is similar, suggesting that differential treatment of males and females for the purposes of experimental design is unnecessary until proven otherwise, rather than the other way around.

Breaking the Cycle

Despite the striking differences between male and female physiology, female physiology is understudied. In response, the National Institutes of Health is promulgating new policies to increase the use of female organisms in preclinical research. Females are commonly believed to have greater variability than males because of the estrous cycle, but recent studies call this belief into question. Effects of estrous cycling on mean arterial pressure were assessed in female Dahl S rats using telemetry and vaginal cytometry and found that estrous cycling did not affect mean arterial pressure magnitude or variance. Data from the PhysGen arm of the Program for Genomic Applications was used to compare male and female variance and coefficient of variation in 142 heart, lung, vascular, kidney, and blood phenotypes, each measured in hundreds to thousands of individual rats from over 50 inbred strains. Seventy-four of 142 phenotypes from this data set demonstrated a sex difference in variance; however, 59% of these phenotypes exhibited greater variance in male rats rather than female. Remarkably, a retrospective power analysis demonstrated that only 16 of 74 differentially variable phenotypes would be detected when using an experimental cohort large enough to detect a difference in magnitude. No overall difference in coefficient of variation between male and female rats was detected when analyzing these 142 phenotypes. We conclude that variability of 142 traits in male and female rats is similar, suggesting that differential treatment of males and females for the purposes of experimental design is unnecessary until proven otherwise, rather than the other way around.