Afferent Arteriolar Responses Research Articles

Autoregulatory mission impossible: when afferent arterioles lose contractility

The myogenic response of afferent arterioles is a key autoregulatory mechanism that protects the glomeruli from barotrauma. Afferent arteriolar smooth muscle cells contract to increased intraluminal pressure through mechanosensitive cation channels and interactions between integrin and extracellular matrix that trigger calcium-dependent actomyosin contraction. The study by Feng etal. provides evidence supporting the concept that increased matrix metalloproteinase 9 in kidney microvessels of Dahl salt-sensitive rats interferes with integrin-matrix binding and promotes phenotypic transformation of afferent arterioles, causing loss of myogenic constriction and hypertensive nephropathy.

High-mobility group box 1 impairs renal microvascular autoregulatory responses in male and female rats

High-mobility group box 1 (HMGB1) is a nuclear protein that functions as a paracrine signaling molecule when released from the nucleus of stressed or injured cells into the interstitial fluid. Secreted HMGB1 can activate toll-like receptor 4 (TLR4) and we showed that activation of renal TLR4 causes autoregulatory dysfunction. In addition, microvascular function is seriously compromised in hypertension but the specific mechanisms involved are poorly defined. We tested the hypothesis that HMGB1 negatively influences renal microvascular autoregulatory capability by activating TLR4. Experiments were performed in male rats using the blood-perfused juxtamedullary nephron technique. Three groups were studied: control, HMGB1 (~1.25 μg/ml in the blood perfusate) and HMGB1+LPS-RS (TLR4 blocker; ~10 μg/ml in the blood perfusate). Afferent arteriole autoregulatory behavior was assessed in these groups by increasing perfusion pressure from 65 to 170 mmHg in 15 mmHg increments. Starting diameters of male arterioles, measured at 100 mmHg, were similar across all three groups (15.3±0.7; 15.1±0.2 and 15.3±0.1 μm, respectively). When perfusion pressure increased from 65 to 170 mmHg, control arteriole diameter decreased to 69±3% of baseline (P<0.05; n=6), exhibiting normal pressure-induced autoregulatory vasoconstriction. Afferent arteriole diameters in kidneys receiving HMGB1 remained unchanged at 97±1% over the same pressure range (n=9; P>0.05 vs. baseline; P<0.05 compared to control kidneys), indicating significant loss of autoregulatory behavior. In the HMGB1+LPS-RS group, afferent arteriole responses were similar to normal controls and decreased to 76±1% (n=6; P<0.05 vs baseline; P<0.05 vs. HMGB1) over the same pressure range, signifying normalization of autoregulatory behavior by TLR4 blockade. There are few published data on the response of female afferent arterioles to the change in perfusion pressure, so we conducted separate experiments using females in two groups of rats, (normal kidneys with or without HMGB1 in the perfusate blood). Baseline diameters of female arterioles (100 mmHg) were similar across both groups (14.7±0.4 and 14.2±0.3 μm, respectively) and were similar to baseline diameters in males (P>0.05). Increasing perfusion pressure from 65 to 170 mmHg decreased female control arteriole diameter to 78±1% (P<0.05 vs baseline; n=6), reflecting normal autoregulatory vasoconstriction resembling that observed in males. Afferent arteriole diameters in female kidneys receiving HMGB1 actually became slightly larger to 103±1% (P<0.05 vs baseline; P<0.05 vs control kidneys; n=6) by 170 mmHg, indicating significant loss of autoregulatory reactivity and that female renal microvessels respond to HMGB1 similarly to males. These data demonstrate that HMGB1 exposure can impair afferent arteriole autoregulatory capability in male and female rats and suggests that impairment involves HMGB1-dependent TLR4 activation. Study supported by NIH (DK044628). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys.

Renal autoregulation is critical in maintaining stable renal blood flow (RBF) and glomerular filtration rate (GFR). Renal ischemia-reperfusion (IR)-induced kidney injury is characterized by reduced RBF and GFR. The mechanisms contributing to renal microvascular dysfunction in IR have not been fully determined. We hypothesized that increased reactive oxygen species (ROS) contributed to impaired renal autoregulatory capability in IR rats. Afferent arteriolar autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. IR was induced by 60 min of bilateral renal artery occlusion followed by 24 h of reperfusion. Afferent arterioles from sham rats exhibited normal autoregulatory behavior. Stepwise increases in perfusion pressure caused pressure-dependent vasoconstriction to 65 ± 3% of baseline diameter (13.2 ± 0.4 μm) at 170 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in IR rats. Baseline diameter averaged 11.7 ± 0.5 µm and remained between 90% and 101% of baseline over 65-170 mmHg, indicating impaired autoregulatory function. Acute antioxidant administration (tempol or apocynin) to IR kidneys for 20 min increased baseline diameter and improved autoregulatory capability, such that the pressure-diameter profiles were indistinguishable from those of sham kidneys. Furthermore, the addition of polyethylene glycol superoxide dismutase or polyethylene glycol-catalase to the perfusate blood also restored afferent arteriolar autoregulatory responsiveness in IR rats, indicating the involvement of superoxide and/or hydrogen peroxide. IR elevated mRNA expression of NADPH oxidase subunits and monocyte chemoattractant protein-1 in renal tissue homogenates, and this was prevented by tempol pretreatment. These results suggest that ROS accumulation, likely involving superoxide and/or hydrogen peroxide, impairs renal autoregulation in IR rats in a reversible fashion.NEW & NOTEWORTHY Renal ischemia-reperfusion (IR) leads to renal microvascular dysfunction manifested by impaired afferent arteriolar autoregulatory efficiency. Acute administration of scavengers of reactive oxygen species, polyethylene glycol-superoxide dismutase, or polyethylene glycol-catalase following renal IR restored afferent arteriolar autoregulatory capability in IR rats, indicating that renal IR led to reversible impairment of afferent arteriolar autoregulatory capability. Intervention with antioxidant treatment following IR may improve outcomes in patients by preserving renovascular autoregulatory function and potentially preventing the progression to chronic kidney disease after acute kidney injury.

ADAMTS13 inhibits oxidative stress and ameliorates progressive chronic kidney disease following ischaemia/reperfusion injury.

Reduced A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif member 13 (ADAMTS13) levels are observed in kidney disease. We test whether recombinant human ADAMTS13 (rhADAMTS13) mitigates renal injury in chronic kidney disease (CKD) and the potential mechanisms. CKD was established 3months after ischaemia/reperfusion (IR). ADAMTS13 and von Willebrand factor (vWF) levels, renal function and morphological changes were analysed. Afferent arteriolar responses to angiotensin II (Ang II) and acetylcholine (ACh) were measured. Oxidative stress-related molecules were detected. Higher vWF and lower ADAMTS13 levels were observed in CKD mice, which were markedly attenuated by rhADAMTS13. rhADAMTS13 alleviated renal dysfunction, as documented by decreased blood urea nitrogen (BUN), serum creatinine, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) levels in CKD mice. Moreover, rhADAMTS13 attenuated transforming growth factor (TGF)-β1/Smad3 activation. Plasma vWF: ADAMTS13 ratio showed positive correlations with malondialdehyde (MDA), hydrogen peroxide (H2 O2 ) and proteinuria, and correlated inversely with superoxide dismutase (SOD) and catalase (CAT). Finally, rhADAMTS13 inhibited reactive oxygen species (ROS) levels and improved microvascular functional disorders, accompanied by the inhibition of glycogen synthase kinase (GSK) 3β hyperactivity and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Acute kidney injury (AKI) reduces the expression of ADAMTS13 that contributes to progressive CKD, microvascular dysfunction, oxidative stress, inhibition of Nrf2 activity and renal histopathological damage. All of which can be alleviated by administration of rhADAMTS13.

A Mutation in γ-Adducin Impairs Autoregulation of Renal Blood Flow and Promotes the Development of Kidney Disease.

The genes and mechanisms involved in the association between diabetes or hypertension and CKD risk are unclear. Previous studies have implicated a role for γ-adducin (ADD3), a cytoskeletal protein encoded by Add3. We investigated renal vascular function in vitro and in vivo and the susceptibility to CKD in rats with wild-type or mutated Add3 and in genetically modified rats with overexpression or knockout of ADD3. We also studied glomeruli and primary renal vascular smooth muscle cells isolated from these rats. This study identified a K572Q mutation in ADD3 in fawn-hooded hypertensive (FHH) rats-a mutation previously reported in Milan normotensive (MNS) rats that also develop kidney disease. Using molecular dynamic simulations, we found that this mutation destabilizes a critical ADD3-ACTIN binding site. A reduction of ADD3 expression in membrane fractions prepared from the kidney and renal vascular smooth muscle cells of FHH rats was associated with the disruption of the F-actin cytoskeleton. Compared with renal vascular smooth muscle cells from Add3 transgenic rats, those from FHH rats had elevated membrane expression of BKα and BK channel current. FHH and Add3 knockout rats exhibited impairments in the myogenic response of afferent arterioles and in renal blood flow autoregulation, which were rescued in Add3 transgenic rats. We confirmed these findings in a genetic complementation study that involved crossing FHH and MNS rats that share the ADD3 mutation. Add3 transgenic rats showed attenuation of proteinuria, glomerular injury, and kidney fibrosis with aging and mineralocorticoid-induced hypertension. This is the first report that a mutation in ADD3 that alters ACTIN binding causes renal vascular dysfunction and promotes the susceptibility to kidney disease.

Aging Impairs Renal Autoregulation in Mice.

Impaired renal autoregulation permits more transmission of disturbance in systemic blood pressure, which initiates barotrauma in intrarenal microvasculatures such as glomerular and tubulointerstitial capillaries, contributing to the development of kidney damage and deterioration in renal function, especially under the conditions with high blood pressure. Although it has been postulated that autoregulatory efficiency is attenuated in the aging kidney, direct evidence remains lacking. In the present study, we measured the autoregulation of renal blood flow, myogenic response of afferent arteriole (Af-Art), tubuloglomerular feedback in vivo with micropuncture, as well as tubuloglomerular feedback in vitro in isolated perfused juxtaglomerular apparatus in young and aged C57BL/6 mice. We found that renal blood flow was not significantly changed in response to a defined elevation of renal arterial pressure in young mice but significantly increased in aged mice. Additionally, myogenic response of Af-Art measured by microperfusion with a stepwise increase in perfusion pressure was significantly blunted in the aging kidney, which is associated with the attenuation of intraluminal pressure-induced intracellular calcium increases, as well as the reduced expression of integrin α5 (Itga5) in Af-Art. Moreover, both tubuloglomerular feedback in vivo and in vitro were nearly inactive in the aging kidney, which is associated with the significantly reduced expression of adenosine A1 receptor (A1AR) and suppressed vasoconstrictor response to adenosine in Af-Art. In conclusion, this study demonstrates that aging impairs renal autoregulation with blunted myogenic response and inhibited tubuloglomerular feedback response. The underlying mechanisms involve the downregulations of integrin α5 and A1AR in the Af-Art.

Avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) plays a role in renal microvascular pathophysiology in the Dahl salt-sensitive rat

Prior studies reported that haploinsufficiency of the transcription factor ETS-1 is renoprotective in Dahl salt-sensitive rats, but the mechanism is unclear. Here, we tested whether ETS-1 is involved in hypertension-induced renal microvascular pathology and autoregulatory impairment. Hypertension was induced in salt-sensitive rats and salt-sensitive rats that are heterozygous with 1 wild-type or reference allele of Ets1 (SSEts1+/-) by feeding a diet containing 4% sodium chloride for 1 week. Increases in blood pressure did not differ. However, phosphorylated ETS-1 increased in afferent arterioles of hypertensive salt-sensitive rats, but not in hypertensive SSEts1+/- rats. Afferent arterioles of hypertensive salt-sensitive rats showed increased monocyte chemotactic protein-1 expression and infiltration of CD68 positive monocytes/macrophages. Isolated kidney microvessels showed increased mRNA expression of vascular cell adhesion molecule, intercellular adhesion molecule, P-selectin, fibronectin, transforming growth factor-β, and collagen I in hypertensive salt-sensitive rats compared with hypertensive SSEts1+/- rats. Using the in vitro blood-perfused juxtamedullary nephron preparation, pressure-mediated afferent arteriolar responses were significantly blunted in hypertensive salt-sensitive rats compared to hypertensive SSEts1+/- rats. Over a 65-170 mmHg pressure range tested baseline arteriolar diameters averaged 15.1 μm and remained between 107% and 89% of baseline diameter in hypertensive salt-sensitive rats vs. 114% and 73% in hypertensive SSEts1+/- rats (significantly different). Thus, ETS-1 participates in renal arteriolar pathology and autoregulation and thereby is involved in hypertension-mediated kidney injury in salt-sensitive rats.

Afferent arteriole responsiveness to endothelin receptor activation: does sex matter?

BackgroundThe pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1.MethodsMale and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10−12 to 10−8 M) and sarafotoxin 6c (S6c, ETB agonist, 10−12 to 10−8 M) using the blood-perfused juxtamedullary nephron preparation.ResultsControl afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 μm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10−8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10−8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10−8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10−12 to 10−9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation.ConclusionThese data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

Inactivation of p66Shc Decreases Afferent Arteriolar KATP Channel Activity and Decreases Renal Damage in Diabetic Dahl SS Rats.

Increased expression of adaptor protein p66Shc has been associated with progression of diabetic nephropathy. Afferent arteriolar dilation and glomerular hyperfiltration in diabetes are due to increased KATP channel availability and activity. Hyperglycemia was induced in Dahl salt-sensitive (SS) rats in a model of diabetes induced by streptozotocin (STZ). Renal injury was evaluated in SS rats and genetically modified SS rats either lacking p66Shc (p66Shc knockout [p66ShcKO]) or expressing p66Shc mutant (p66Shc-S36A). Afferent arteriolar diameter responses during STZ-induced hyperfiltration were determined by using the juxtamedullary nephron technique. Albuminuria and glomerular injury were mitigated in p66ShcKO and p66Shc-S36A rats with STZ-induced diabetes. SS rats with STZ-induced diabetes had significantly increased afferent arteriolar diameter, whereas p66ShcKO and p66Shc-S36A rats did not. SS rats with STZ-induced diabetes, but not p66ShcKO or p66Shc-S36A rats with STZ-induced diabetes, had an increased vasodilator response to the KATP channel activator pinacidil. Likewise, the KATP inhibitor glibenclamide resulted in a greater decrease in afferent arteriolar diameter in SS rats with STZ-induced diabetes than in STZ-treated SS p66ShcKO and p66Shc-S36A rats. Using patch-clamp electrophysiology, we demonstrated that p66ShcKO decreases KATP channel activity. These results indicate that inactivation of the adaptor protein p66Shc decreases afferent arteriolar KATP channel activity and decreases renal damage in diabetic SS rats.

Lack of contribution of nitric oxide synthase to cholinergic vasodilation in murine renal afferent arterioles.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (-10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01-1 mM) or VNIO (0.01-1 μM) induced significant dose-dependent vasoconstrictions (-19.8 ± 4.0% 1 mM NLA; -7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.

Tempol Protects Against Acute Renal Injury by Regulating PI3K/Akt/mTOR and GSK3β Signaling Cascades and Afferent Arteriolar Activity

Background/Aims: Free radical scavenger tempol is a protective antioxidant against ischemic injury. Tubular epithelial apoptosis is one of the main changes in the renal ischemia/reperfusion (I/R) injury. Meanwhile some proteins related with apoptosis and inflammation are also involved in renal I/R injury. We tested the hypothesis that tempol protects against renal I/R injury by activating protein kinase B/mammalian target of rapamycin (PKB, Akt/mTOR) and glycogen synthase kinase 3β (GSK3β) pathways as well as the coordinating apoptosis and inflammation related proteins. Methods: The right renal pedicle of C57Bl/6 mouse was clamped for 30 minutes and the left kidney was removed in the study. The renal injury was assessed with serum parameters by an automatic chemistry analyzer. Renal expressions of Akt/mTOR and GSK3β pathways were measured by western blot in I/R mice treated with saline or tempol (50mg/kg) and compared with sham-operated mice. Results: The levels of blood urea nitrogen (BUN), creatinine and superoxide anion (O₂^.-) increased, and superoxide dismutase (SOD) and catalase (CAT) decreased significantly after renal I/R injury. However, tempol treatment prevented the changes. Besides, I/R injury reduced renal expression of p-Akt, p-GSK3β, p-mTOR, Bcl2 and increased NF-κB, p-JNK and p53 in kidney, tempol significantly normalized these changes. In addition, renal I/R injury reduced the response of afferent arteriole to Angiotensin II (Ang II), while tempol treatment improved the activity of afferent arteriole. Conclusion: Tempol attenuates renal I/R injury. The protective mechanisms seem to relate with activation of PI3K/Akt/mTOR and GSK3β pathways, inhibition of cellular damage markers and inflammation factors, as well as improvement of afferent arteriolar activity.

Abstract 068: Impact of Renal Outer Medullary Potassium Channel Inhibition on Afferent Arteriolar Tone in Rats With Type 1 Diabetes

The bee venom toxin tertiapin Q (TPQ) evokes tubuloglomerular feedback-independent afferent arteriolar constriction in kidneys from rats with streptozotocin-induced type 1 diabetes (STZ rats). Because TPQ inhibits both Kir1.1 and Kir3.x channels, the contribution of Kir1.1 (renal outer medullary potassium channel; ROMK) to afferent arteriolar tone in diabetes remains uncertain. To test the hypothesis that Kir1.1 exerts a tonic dilator influence on the afferent arteriole during diabetes, we compared afferent arteriolar responses to the novel, small molecule Kir1.1-selective inhibitor Compound C (CmpdC) to those evoked by TPQ. The ex vivo blood-perfused juxtamedullary nephron technique was used to monitor afferent arteriolar lumen diameter before and during exposure to TPQ (1-100 nM) or CmpdC (10 nM-10 μM). Neither agent altered afferent arteriolar diameter in kidneys from normal rats. In kidneys from STZ rats (blood glucose concentration = 465 ± 12 mg/dl), baseline afferent arteriolar diameter averaged 22.9 ± 0.6 μm ( n = 30). Both TPQ and CmpdC evoked concentration-dependent arteriolar constriction, with 100 nM TPQ decreasing diameter by 2.5 ± 0.6 μm ( n = 6; P < 0.001 vs baseline) and 100 nM CmpdC reducing diameter by 1.7 ± 0.6 μm ( n = 10; P = 0.004 vs baseline; P = 0.42 vs 100 nM TPQ). During subsequent exposure to 10 μM CmpdC, arteriolar diameter was 2.1 ± 0.8 μm below baseline. The similar effects of the Kir1.1/3.x inhibitor TPQ and the Kir1.1-selective inhibitor CmpdC on arteriolar diameter in kidneys from STZ rats support the contention that Kir1.1 exerts a tonic afferent arteriolar dilator influence during type 1 diabetes. In follow-up studies, the NKCC inhibitor furosemide (30-300 μM) administered via the bath did not alter afferent arteriolar diameter in kidneys from STZ rats; however, prior exposure to furosemide prevented the vasoconstrictor response to 100 nM CmpdC (Δ diameter = -0.4 ± 0.3 μm; n = 8) although the response to 100 nM TPQ remained intact (Δ diameter = -2.6 ± 1.3 μm; n = 6). The differential impact of furosemide pretreatment on arteriolar responses to TPQ and CmpdC suggests a complex interplay between the furosemide-sensitive vascular NKCC1 and tubular NKCC2, and the TPQ- and CmpdC-sensitive Kir1.1 expressed in both the vasculature and tubule.

Hypoxia/re‐oxygenation increases the response of afferent arterioles to angiotensin II ‐ Investigation in mouse kidney slices

Hypoxia/re‐oxygenation (H/R) plays an important role in the pathogenesis of acute kidney injury (AKI). However, the influence of H/R on cortical microvessel function, especially that of afferent arterioles, is sparsely investigated. We hypothesize that H/R increases the vasoreactivity to angiotensin II (Ang II), which may promote the development of kidney injury. To test the hypothesis, we applied an innovative in situ model of live kidney slices.Experiments were performed in acute kidney slices (200 μm) of C57BL/6 mice. The TUNEL assay was used to estimate viability of slices by determining the percentage of apoptotic cells. For functional experiments, kidney slices were situated in a chamber at 37°C. Bath solution was equilibrated with carbogen. The afferent arterioles were visualized by differential interference contrast applied to an inverted microscope. To characterize arteriolar reactivity in this model of kidney slices, Ang II, norepinephrine, endothelin‐1 and ATP were administered. For H/R experiments, the slices were transferred into a hypoxia chamber (1% O2) for 30, 60 or 90 minutes and re‐oxygenated for 10 or 20 minutes.H/R did not significantly influence the apoptotic cell portion in the slice compared to control conditions. Afferent arterioles responded to the bolus application of Ang II (10−7 mol/l) with a maximum constriction, to 44.5 ± 2.8 % of the initial diameter. Norepinephrine (10−5 mol/l), endothelin‐1 (10−5 mol/l) and ATP (10−5 mol/l) reduced the diameter under control conditions to 50.0 ± 2.2%, 45.3 ± 2.6% and 74.1 ± 1.8, respectively. Inhibition of the NO‐synthesis by L‐NAME (10−4 mol/l) significantly enhanced the vasoconstriction to Ang II (32.3 ± 2.3 %). H/R increased the Ang II response of afferent arterioles compared to control conditions. Longer periods of hypoxia were associated with greater arteriolar vasoconstriction to Ang II (30/20 vs. 90/20: 39.7 ± 4.1% vs. 25.9 ± 2.0 %.). Also, increasing the duration of re‐oxygenation resulted in increased vasoconstriction (90/10 vs. 90/20: 34.7 ± 3.5% vs. 25.9 ± 2.00%).TUNEL staining and functional testing with vasoactive substances indicates the usefulness of the in situ model for the investigation of afferent arteriolar function. The increased response to Ang II in H/R suggests this effect as one component in the multifactorial genesis of AKI.Support or Funding InformationGerman Research Foundation (DFG PA 479/10‐2)

Administration of Tempol Improves Renal Autoregulation in Ischemia‐reperfusion Induced Acute Kidney Injury

Renal autoregulation is critical for protecting the kidney from fluctuations in arterial pressure by regulating renal vascular resistance to maintain stable renal perfusion and glomerular filtration rate (GFR). Renal ischemia‐reperfusion (IR) induced acute kidney injury (IR‐AKI) is characterized by reduced renal blood flow and a decline in GFR. We hypothesized that increased reactive oxygen species (ROS) contribute to impairment of renal autoregulatory capability in IR‐AKI. Afferent arteriole autoregulatory behavior was assessed using the in vitro blood‐perfused juxtamedullary nephron preparation. IR‐AKI was induced by 60 minutes of bilateral renal artery occlusion followed by 24 hours of reperfusion. We found that plasma creatinine was increased in IR rats (3.86 ± 0.2 mg/dl) compared to 0.93 ± 0.07 mg/dl in sham rats (n=6–7, p<0.05). Baseline afferent arteriole diameter averaged 13.0 ± 0.4 μm (n=5) in kidneys from sham rats and these arterioles exhibited normal autoregulatory behavior. Decreasing renal perfusion pressure from 100 to 65 mmHg increased arteriole diameter from 13.0 ± 0.4 to 15.1 ± 0.8 μm, corresponding to 116 ± 5% of the baseline diameter. Subsequent stepwise increases in perfusion pressure (15 mmHg) caused pressure‐dependent afferent arteriole vasoconstriction to 66 ± 2% of baseline diameter at 170 mmHg. In contrast, pressure‐mediated afferent arteriole responses were markedly attenuated in IR‐AKI rats. Baseline diameter averaged 11.7 ± 0.6 μm and remained between 91 and 101% of baseline over the 65–170 mmHg pressure range tested, indicating a loss of autoregulatory function. In another set of IR‐AKI rats, acute administration of the ROS scavenger, tempol (10−3 M), to afferent arterioles increased baseline diameter by 10 ± 2% from 11.3 ± 0.8 to 12.2 ± 0.9 μm (n=5). Furthermore, scavenging ROS production improved autoregulatory behavior of afferent arterioles from IR‐AKI rats. During exposure to tempol, decreasing renal perfusion pressure from 100 to 65 mmHg resulted in a diameter increase to 110 ± 2 % of the control diameter while increasing perfusion pressure to 170 mmHg decreased arteriole diameter to 78 ± 1% of control (P<0.05 vs IR). In conclusion, these results suggest that renal ischemia‐reperfusion leads to impaired autoregulatory behavior of afferent arterioles. Acutely scavenging endogenous ROS with tempol improves afferent arteriole autoregulatory responsiveness, suggesting that excessive ROS production contributes to impaired renal autoregulation in IR‐AKI.Support or Funding InformationThis study is supported by a Grant‐in‐Aid (15GRNT25240015) from the American Heart Association for ZG and by grants from NIH (DK044628, HL074167, HL098135 and HL095499) for EWI.

Abstract P266: Thromboxane-prostanoid Receptors (tp-rs) Mediate Hypertension, H2o2 Generation and Impaired Renal Afferent Arteriolar Myogenic Responses Leading to Nephropathy in Doca/salt Mice

Background: DOCA/uninephrectomy/high salt (DOCA) is a model of hypertensive nephropathy. Afferent arteriolar myogenic responses prevent hypertensive renal barotrauma but myogenic tone is blocked by vascular generation of H 2 O 2 . Since thromboxane-prostanoid receptors (TP-Rs) generate H 2 O 2 , we tested the hypothesis that they mediate hypertensive nephropathy. Methods: DOCA and Sham TP-R +/+ and -/- mice (n=6/group) were studied at 2 weeks and myogenic responses recorded from the diameter of perfused single afferent arterioles studied in a bath preparation during increased perfusion pressure (40 to 80 mmHg). Results: DOCA treatment in TP-R +/+ mice increased (p<0.001) 24-hour excretion of H 2 O 2 (45 ± 3 vs 220 + 15 nmol) , TxB 2 (4 ± 2 vs 29 ± 4 pmol) and albumin (20 ± 5 vs 270 ± 20 mg) and increased MAP by 35 ± 5 mmHg. However, all effects of DOCA were prevented in TP-R -/- mice. Sham treatment had no effect in TPR +/+ or -/- mice. Myogenic responses were severely impaired in DOCA vs sham WT mice (Δ diameter: -4 ± 1 vs -8 ± 1%; p< 0.005). Myogenic responses also were reduced by incubation of arterioles with 10 -10 mol·l -1 of the TP-R mimetic, U-46,619 vs vehicle added to the bath for 10 minutes (Δ diameter: -7 ± 1 vs -10 ± 1%; p<0.01) and in WT mice infused for 3 days with U-46,619 (500 ng·kg -1 ·d -1 x 3) vs vehicle (Δ diameter: -3 ± 1 vs -10 ± 1%; p<0.005). Conclusion: Hypertensive nephropathy is dependent on TP-Rs that mediate the increase in H 2 O 2 and blood pressure and likely the impaired myogenic responses that expose the kidney to barotrauma

Increased hydrogen peroxide impairs angiotensin II contractions of afferent arterioles in mice after renal ischaemia-reperfusion injury.

Renal ischaemia-reperfusion injury (IRI) increases angiotensin II (Ang II) and reactive oxygen species (ROS) that are potent modulators of vascular function. However, the roles of individual ROS and their interaction with Ang II are not clear. Here we tested the hypothesis that IRI modulates renal afferent arteriolar responses to Ang II via increasing superoxide (O2-) or hydrogen peroxide (H2 O2 ). Renal afferent arterioles were isolated and perfused from C57BL/6 mice 24h after IRI or sham surgery. Responses to Ang II or noradrenaline were assessed by measuring arteriolar diameter. Production of H2 O2 and O2- was assessed in afferent arterioles and renal cortex. Activity of SOD and catalase, and mRNA expressions of Ang II receptors were assessed in pre-glomerular arterioles and renal cortex. Afferent arterioles from mice after IRI had a reduced maximal contraction to Ang II (-27±2 vs. -42±1%, P<0.001), but retained a normal contraction to noradrenaline. Arterioles after IRI had a 38% increase in H2 O2 (P<0.001) and a 45% decrease in catalase activity (P<0.01). Contractions were reduced in normal arterioles after incubation with H2 O2 (-22±2 vs. -42±1%, P<0.05) similar to the effects of IRI. However, the impaired contractions were normalized by incubation with PEG catalase despite a reduced AT1 R expression. Renal IRI in mice selectively impairs afferent arteriolar responses to Ang II because of H2 O2 accumulation that is caused by a reduced catalase activity. This could serve to buffer the effect of Ang II after IRI and may be a protective mechanism.

Radiation-induced afferent arteriolar endothelial-dependent dysfunction involves decreased epoxygenase metabolites.

Chronic kidney disease is a known complication of hematopoietic stem cell transplant (HSCT) and can be caused by irradiation at the time of the HSCT. In our rat model there is a 6- to 8-wk latent period after irradiation that leads to the development of proteinuria, azotemia, and hypertension. The current study tested the hypothesis that decreased endothelial-derived factors contribute to impaired afferent arteriolar function in rats exposed to total body irradiation (TBI). WAG/RijCmcr rats underwent 11 Gy TBI, and afferent arteriolar responses to acetylcholine were determined at 1, 3, and 6 wk. Blood pressure and blood urea nitrogen were not different between control and irradiated rats. Afferent arteriolar diameters were not altered in irradiated rats. Impaired endothelial-dependent responses to acetylcholine were evident at 3 and 6 wk following TBI. Nitric oxide synthase (NOS), cyclooxygenase (COX), and epoxygenase (EPOX) contribution to acetylcholine dilator responses were evaluated. NOS inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME) reduced acetylcholine responses by 50% in controls and 90% in 3-wk TBI rats. COX inhibition with indomethacin did not significantly alter the acetylcholine response in the presence or absence of l-NAME. EPOX inhibition with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide significantly decreased acetylcholine responses (35%) in controls but did not significantly alter acetylcholine responses (4%) in TBI rats. Biochemical analysis revealed decreased urinary EPOX metabolites but no change in COX, NOS, or reactive oxygen species at 3 wk TBI. Taken together, these results indicate that afferent arteriolar endothelial dysfunction involves a decrease in EPOX metabolites that precedes the development of proteinuria, azotemia, and hypertension in irradiated rats.

Abstract MP15: Afferent Arteriolar Endothelial-dependent Dilation is Impaired Prior to the Development of Radiation-induced Nephropathy and Hypertension

Chronic kidney disease (CKD) occurs in 15% of hematopoietic stem cell transplant (HSCT) patients, and has been clearly linked to irradiation at the time of the HSCT. CKD due to radiation also occurs after radionuclide therapy for cancer. Radiation nephropathy is expressed in rats and in humans as proteinuria, azotemia, and hypertension. There is a latent period of 6-8 weeks after irradiation to the development of proteinuria, azotemia, and hypertension in rats. This study tested the hypothesis that afferent arteriolar responses to the endothelial-dependent dilator acetylcholine are impaired prior to the development of proteinuria, azotemia, and hypertension in rats exposed to total body irradiation (TBI). Male WAG/RijCmcr rats were subjected to TBI (11 Gy) and afferent arteriolar responses to acetylcholine using the juxtamedullary nephron technique were determined at one, three, and six weeks. Systolic blood pressure (117 ± 6 vs. 119 ± 4 mmHg) and BUN (15.6 ± 1.4 vs. 15.8 ± 0.8 mg/dl) were not different between control and TBI groups at 6 weeks. Afferent arteriolar diameters averaged 22.5 ± 0.8 μm (n=30) in controls and 21.7 ± 0.7 μm (n=27) in TBI rats and were not different between control and TBI groups at 1, 3, or 6 weeks. Acetylcholine dilator responses were progressively attenuated from one to six weeks in TBI compared to control rats. During exposure to acetylcholine (0.01, 0.1, 1, and 10 μmol/L), afferent diameter increased by 8 ± 2%, 18 ± 3%, 27 ± 2% and 31 ± 3% in control rats (n=6), and 3 ± 2%, 9 ± 3%, 12 ± 3%, and 16 ± 3% in kidneys of 3 week TBI rats (n=8, p&lt;0.05). However, TBI did not change the norepinephrine-mediated vasoconstrictor responses or the nitroprusside vasodilator responses at 1, 3, or 6 weeks. Renal microvessels were isolated from control and TBI groups at 3 and 6 weeks for protein expression assessment of endothelial enzymatic pathways Cyp2C, COX, and NOS. We found that epoxygenase Cyp2C23 and Cyp2C11 protein expressions were significantly lower 3 and 6 weeks following TBI compared to control rats. Taken together, these results indicate that the impaired afferent arteriolar endothelial-dependent acetylcholine responses and decreased epoxygenase enzymes precede the development of proteinuria, azotemia, and hypertension in irradiated rats.

Abstract 060: POLDIP-2/NOX-4 Generates Hydrogen Peroxide that Impairs Myogenic Response of Afferent Arterioles from Mice with the Reduced Renal Mass Model of Chronic Kidney Disease

Background: Because we have found that myogenic contractions are stimulated by superoxide (O 2 .- ) but inhibited by hydrogen peroxide (H 2 O 2 ), we tested the hypothesis that H 2 O 2 is the cause of the impaired myogenic responses of afferent arterioles from mice with the reduced renal mass (RRM) model of chronic kidney disease (CKD). Methods: Mice were subjected to 5/6 surgical nephrectomy or sham operations and fed 6% salt for 3 months. Single afferent arterioles were perfused, their diameter measured directly and O 2 .- and H 2 O 2 measured by fluorescence microscopy. Results: The perfusion pressure of isolated afferent arterioles was increased from 40 to 80 mmHg to study myogenic responses. Arterioles from mice with RRM (vs sham) had a greater increase in O 2 .- (21.2 ± 1.9 versus 11.3 ± 2.5%; p &lt; 0.01) and especially H 2 O 2 (28.7 ± 3.7 versus 4.2 ± 0.4%, P&lt;0.005), but a reduction in myogenic contraction (-1.7 ± 4.3 versus -14.4 ± 3.6%; p &lt; 0.005) . Myogenic contractions were paradoxically reversed in afferent arterioles from mice with RRM after reduction in O 2 .- by PEG-SOD (+3.3 ± 1.5 versus -1.7 ± 4.3%, P&lt;0.05) or deletion of p47 phox (+2.5 ± 1.4 versus -1.7 ± 4.3%, P&lt;0.05). In contrast, myogenic responses were increased even above the levels of shams in arterioles from mice with RRM after reduction in H 2 O 2 by PEG-catalase (-19.1 ± 1.6 versus -1.7 ± 4.3%, P&lt;0.005) or transgenic overexpression of catalase in smooth muscle cells (-10.7 ± 1.3 versus -1.7 ± 4.3%, P&lt;0.01). Gene expression for NOX-4 and POLDIP-2 (main source of H 2 O 2 ) was increased 40-50% (P&lt;0.05) in individual afferent arterioles from mice with RRM. Moreover, the myogenic contractions in the arterioles from POLDIP-2+/- mice with RRM were similar to POLDIP-2+/- with sham operations (-7.7 ± 0.9 versus -8.0 ± 0.6, P=NS). Conclusions: Afferent arterioles from mice with RRM had severely impaired myogenic responses that were attributed to increased H 2 O 2 generation from POLDIP-2/NOX-4 that may therefore be novel targets to maintain autoregulation and protect kidneys from barotrauma in CKD.

Activation of GLP-1 receptors on vascular smooth muscle cells reduces the autoregulatory response in afferent arterioles and increases renal blood flow.

Glucagon-like peptide (GLP)-1 has a range of extrapancreatic effects, including renal effects. The mechanisms are poorly understood, but GLP-1 receptors have been identified in the kidney. However, the exact cellular localization of the renal receptors is poorly described. The aim of the present study was to localize renal GLP-1 receptors and describe GLP-1-mediated effects on the renal vasculature. We hypothesized that renal GLP-1 receptors are located in the renal microcirculation and that activation of these affects renal autoregulation and increases renal blood flow. In vivo autoradiography using (125)I-labeled GLP-1, (125)I-labeled exendin-4 (GLP-1 analog), and (125)I-labeled exendin 9-39 (GLP-1 receptor antagonist) was performed in rodents to localize specific GLP-1 receptor binding. GLP-1-mediated effects on blood pressure, renal blood flow (RBF), heart rate, renin secretion, urinary flow rate, and Na(+) and K(+) excretion were investigated in anesthetized rats. Effects of GLP-1 on afferent arterioles were investigated in isolated mouse kidneys. Specific binding of (125)I-labeled GLP-1, (125)I-labeled exendin-4, and (125)I-labeled exendin 9-39 was observed in the renal vasculature, including afferent arterioles. Infusion of GLP-1 increased blood pressure, RBF, and urinary flow rate significantly in rats. Heart rate and plasma renin concentrations were unchanged. Exendin 9-39 inhibited the increase in RBF. In isolated murine kidneys, GLP-1 and exendin-4 significantly reduced the autoregulatory response of afferent arterioles in response to stepwise increases in pressure. We conclude that GLP-1 receptors are located in the renal vasculature, including afferent arterioles. Activation of these receptors reduces the autoregulatory response of afferent arterioles to acute pressure increases and increases RBF in normotensive rats.