Juxtamedullary Nephrons Research Articles

A mathematical model of the rat nephron: glucose transport

Mathematical models of the proximal tubule (PT), loop of Henle (LOH), and distal nephron have been combined to simulate transport by rat renal tubules. The ensemble is composed of 24,000 superficial (SF) nephrons and 12,000 juxtamedullary (JM) nephrons in 5 classes (according to LOH length); all coalesce into 7,200 connecting tubules (CNT). Medullary interstitial solute concentrations are specified. The model equations require that each nephron glomerular filtration rate (GFR) satisfies a tubuloglomerular feedback (TGF) relationship, and each initial hydrostatic pressure yields a common CNT pressure; that common CNT pressure is determined from an overall distal hydraulic resistance to flow. By virtue of the greater GFR for JM nephrons, fluid delivery to SF and JM tubules is comparable. Glucose reabsorption is restricted to the PT, cotransported with one Na in the convoluted tubule (SGLT2), and two Na in the straight tubule (SGLT1). Increasing ambient glucose from 5 to 10 mM increases proximal Na reabsorption and decreases distal delivery. This is mitigated by a TGF-mediated increase in GFR, and may thus be an etiology for TGF-mediated glomerular hyperfiltration. With SGLT2 inhibition by 95%, the model predicts that under normoglycemic conditions about 60% of filtered glucose will still be reabsorbed, so that profound glycosuria is not to be expected. Compared with glucose-driven osmotic diuresis, SGLT2 inhibition provokes greater natriuresis. When hyperglycemia is superimposed on SGLT2 inhibition, the model suggests that natriuresis may be severe, reflecting synergy of a proximal diuretic and osmotic diuresis. In sum, the model captures TGF-mediated diabetic hyperfiltration and predicts glomerular protection with SGLT2 inhibition.

A mathematical model of rat proximal tubule and loop of Henle.

Proximal tubule and loop of Henle function are coupled, with proximal transport determining loop fluid composition, and loop transport modulating glomerular filtration via tubuloglomerular feedback (TGF). To examine this interaction, we begin with published models of the superficial rat proximal convoluted tubule (PCT; including flow-dependent transport in a compliant tubule), and the rat thick ascending Henle limb (AHL). Transport parameters for this PCT are scaled down to represent the proximal straight tubule (PST), which is connected to the thick AHL via a short descending limb. Transport parameters for superficial PCT and PST are scaled up for a juxtamedullary nephron, and connected to AHL via outer and inner medullary descending limbs, and inner medullary thin AHL. Medullary interstitial solute concentrations are specified. End-AHL hydrostatic pressure is determined by distal nephron flow resistance, and the TGF signal is represented as a linear function of end-AHL cytosolic Cl concentration. These two distal conditions required iterative solution of the model. Model calculations capture inner medullary countercurrent flux of urea, and also suggest the presence of an outer medullary countercurrent flux of ammonia, with reabsorption in AHL and secretion in PST. For a realistically strong TGF signal, there is the expected homeostatic impact on distal flows, and in addition, a homeostatic effect on proximal tubule pressure. The model glycosuria threshold is compatible with rat data, and predicted glucose excretion with selective 1Na(+):1glucose cotransporter (SGLT2) inhibition comports with observations in the mouse. Model calculations suggest that enhanced proximal tubule Na(+) reabsorption during hyperglycemia is sufficient to activate TGF and contribute to diabetic hyperfiltration.

Abstract 299: Blockade of Toll-like Receptor 4 During Acute Lipopolysaccharide Treatment Preserves Afferent Arteriolar Autoregulatory Behavior.

Lipopolysaccharide (LPS), a component of the bacterial wall of gram-negative bacteria is a ligand for toll-like receptor 4 (TLR4). Immune system activation and generation of reactive oxygen species (ROS) is linked to blunted renal autoregulatory behavior. Accordingly, we hypothesize that blockade of TLR4 during acute LPS treatment preserves afferent arteriolar autoregulatory behavior. Rats received a bolus injection of LPS (1 mg/kg i.p.) and were treated with either anti-TLR4 antibody (1μg; i.p.) or TLR4 receptor antagonist (LPS-RS; 5mg; i.p.). Kidneys were harvested 4 hours later for juxtamedullary nephron studies. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. In control kidneys, afferent arteriolar diameter decreased by 29 ± 5% when perfusion pressure was increased from 65 to 170 mmHg. LPS alone (4 hrs.) significantly blunted the autoregulatory response such that afferent arteriolar diameter remains essentially unchanged between 65 (12.4 ± 0.9 μm) and 170 mmHg (12.7 ± 1.2 μm). In kidneys co-treated with LPS and anti-TLR4 antibody, afferent arteriolar diameter decreased by 26 ± 6% (P < 0.05; n=6) during increments in perfusion pressure from 65 to 170mmHg. In kidneys co-treated with LPS and the TLR4 receptor antagonist, LPS-RS, afferent arteriolar diameter decreased by 24 ± 5% (P < 0.05; n=4) over the same pressure range, indicating preservation of pressure-induced vasoconstriction. Reactive oxygen species play a role in blunting rat afferent arteriolar autoregulatory resistance adjustments. Therefore, we assessed autoregulatory behavior in LPS treated kidneys during acute exposure to the superoxide dismutase mimetic, Tempol (10-6 mol/L). In these kidneys, afferent arteriolar diameter decreased by 28 ± 3% (P < 0.05; n=6) indicating restoration of autoregulatory capability. These data indicate that LPS treatment induces TLR4 activation, and stimulates ROS generation. Blocking TLR4 with anti-TLR4 antibody or LPS-RS or scavenging ROS with Tempol preserves and restores afferent arteriolar autoregulatory behavior suggesting that blockade of TLR4 may open novel therapeutic targets for inflammatory kidney disease.

Abstract 522: Contribution of Reactive Oxygen Species and Rho Kinase in Sphingosine-1-phosphate Mediated Afferent Arteriolar Vasoconstriction of Ischemia-Reperfusion Rats

Our recent studies indicate that sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of afferent arterioles (AA) and is enhanced in renal ischemia-reperfusion (IR) injury. We hypothesized that renal IR-induced increases in reactive oxygen species (ROS) and activation of the rho kinase pathway contribute to the enhanced S1P sensitivity of AA in IR rats. IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. Renal function was assessed by plasma creatinine (Cr) or glomerular filtration rate (GFR). The AA response to S1P was assessed using the in vitro blood-perfused juxtamedullary nephron preparation with and without superfusion with the ROS scavenger, Tempol, or the rho kinase inhibitor, Y27623. Plasma Cr was markedly increased in IR (3.86±0.20 vs. 0.93±0.07 mg/L in sham, n=5-7, P < 0.05). GFR, calculated from the half time (T 1/2 ) of sinistrin clearance, was significantly reduced in IR rats. The T 1/2 averaged 166±28 min in IR vs. 25±4 min in sham rats corresponding to GFRs of 0.20±0.02 and 1.33±0.23 ml/min (P < 0.05, n=3 each), respectively. The basal AA diameter was significantly decreased in IR compared to sham rats (10.2±0.5 vs. 15.0±0.4 μm, P < 0.05, n=9 each). Superfusion of Tempol (10 -4 M) did not alter the basal AA diameter in sham but increased diameter in IR by 8±1% (P < 0.05, n=6 each). S1P superfusion evoked concentration-dependent AA vasoconstriction in both groups, however, Tempol significantly shifted the S1P response curve to the right in IR. Increasing concentrations of S1P (10 -10 -10 -5 M) reduced AA diameter to 98±2, 96±2, 98±2, 94±3, 81±9 and 59±5% of control in IR rats similar to the response in Tempol-treated sham rats (99±1, 99±2, 96±2, 88±5, 65±6 and 48±4%, respectively, P > 0.05). In contrast, superfusion of Y27632 (10 -5 M) markedly increased the basal AA diameter in both groups. AA diameter increased by 81±17% from 10.6±1.3 to 19.2±3.3 μm in IR vs. a 53±21% increase in sham rats (P > 0.05, n=3 each). Y27632 also significantly blunted S1P-mediated AA vasoconstriction similarly in both groups. These results indicate a significant contribution of ROS and rho kinase to both the elevated afferent arteriolar tone and the enhanced S1P sensitivity of AA in IR induced acute kidney injury.

High-salt diet blunts renal autoregulation by a reactive oxygen species-dependent mechanism.

High dietary salt is common in Western countries and is an important contributor to increased cardiovascular disease. Autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) is an essential function of the renal microcirculation that could be affected by excessive dietary salt. High salt (HS) increases renal ROS generation partly by the enzyme NADPH oxidase. We hypothesized that a HS diet would impair autoregulation via NADPH oxidase-dependent ROS generation. The role of NADPH-dependent ROS production on the blunted autoregulatory response with a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. The increase in renal lipid peroxidation and p67(phox) expression induced by HS was prevented by apocynin treatment. Control afferent arterioles exhibited normal autoregulatory behavior in response to acute increases in renal perfusion pressure, whereas arterioles from HS rats exhibited a blunted response. Autoregulatory behavior in HS rats was restored in vitro by acute exposure to the NADPH oxidase inhibitor apocynin. At the whole kidney level, in vivo experiments showed that both RBF and GFR declined in HS rats when left kidney renal perfusion pressure was reduced from ambient to 95 mmHg, whereas control rats maintained stable GFR and RBF consistent with efficient autoregulatory behavior. Apocynin treatment improved in vivo autoregulatory behavior in HS rats and had no detectable effect in normal salt diet-fed rats. These data support the hypothesis that impaired renal autoregulatory behavior in rats fed a HS diet is mediated by NADPH oxidase-derived ROS.

A mathematical model of rat proximal tubule and loop of Henle (1137.9)

PT and LOH function are coupled, with proximal transport determining loop fluid composition, and loop transport modulating glomerular filtration via tubuloglomerular feedback (TGF). To examine this interaction, we began with published models of superficial rat proximal convoluted tubule (PCT) (including flow‐dependent transport in a compliant tubule), and rat thick ascending Henle limb (AHL). This PCT was scaled down to represent outer medullary proximal straight tubule (PST), which was connected to thick AHL via a short descending limb. Superficial PT was scaled up to a juxtamedullary nephron, and connected to thick AHL via outer and inner medullary descending limbs, and inner medullary thin AHL. Medullary interstitial solute concentrations were specified. End‐tubule hydrostatic pressure was required to conform to a distal nephron flow resistance. The TGF signal was represented as a linear function of end‐AHL cytosolic Cl concentration. These two distal conditions required iterative solution of the model. Calculations revealed Na, K, and HCO3 reabsorption in all segments, with thin limb transport driven by the increases in lumen concentrations produced by osmotic water reabsorption. PST was a critical locus for NH4 secretion, due to high interstitial NH4, and cellular uptake via the Na,K‐ATPase. Late AHL cytosolic Cl was a sigmoidal function of flow, and appears suitable to represent the TGF signal.Grant Funding Source: Supported by NIH RO1‐DK29857

Direct effect of glucagon‐like peptide‐1 on the autoregulatory response of the afferent arteriole (692.2)

It has become clear that glucagon‐like peptide‐1 (GLP‐1) has many effects and the GLP‐1 receptor is expressed in many tissues outside the pancreas. GLP‐1 increases renal blood flow (RBF), glomerular filtration rate and sodium and water excretion. The aim of this study was to examine expression of the GLP‐1 receptor in the renal vasculature and assess the effects of GLP‐1 in the afferent arteriole.The GLP‐1 receptor was localized using 125I‐GLP‐1 in kidney slices. In the isolated juxtamedullary nephron preparation, renal perfusion pressure was increased in steps and changes in afferent arteriolar diameter were measured before and after perfusion with GLP‐1. In vivo experiments were performed on rats monitoring blood pressure (BP), RBF and urine flow. GLP‐1 was infused directly into the kidney before and after infusion of the GLP‐1 receptor blocker Exendin9‐39 and indomethacin and L‐NAME.Strong binding of 125I‐GLP‐1 was found in the vascular smooth muscle cells in afferent arterioles. Perfusion with GLP‐1 in isolated kidneys completely abolished the autoregulatory response of the afferent arteriole. Intrarenal infusion of GLP‐1 increased BP, RBF and urine flow significantly in rats. Heart rate and plasma renin were unchanged. The effect on RBF was inhibited by Exendin9‐39 whereas the increase in BP and urine flow was still present. Infusion of indomethacin and L‐NAME did not change the GLP‐1 effect on BP, RBF or urine flow.

Superoxide dismutase mimetic, tempol, aggravates renal injury in advanced-stage stroke-prone spontaneously hypertensive rats

The aim of this study was to determine whether antioxidant therapy could relieve hypertension and retard the progression of renal damage in advanced-stage hypertensive rats. Twenty-four-week-old spontaneously hypertensive stroke-prone rats were treated for 8 weeks with the superoxide dismutase mimetic tempol, low-dose or high-dose candesartan (an angiotensin receptor blocker), or hydralazine, and blood pressure and renal damage were compared. Elevated blood pressure and renal damage with heterogeneity were present after 8 weeks, with greater glomerulosclerosis in the juxtamedullary glomeruli than in the superficial glomeruli. Although both tempol and candesartan effectively reduced reactive oxygen species production in the kidney, tempol did not decrease blood pressure and exacerbated urine protein and histological damage, such as glomerulosclerosis and interstitial fibrosis, particularly in juxtamedullary nephrons (tempol vs. untreated: glomerulosclerosis index, 2.0 vs. 1.5, P<0.01; fibrosis, 15 vs. 10%, P<0.001). In contrast, high-dose candesartan and hydralazine prevented these forms of renal damage with lowering blood pressure. Low-dose candesartan also prevented this renal damage without lowering blood pressure. Moreover, there were increased numbers of larger and smaller glomeruli in the juxtamedullary cortex of tempol-treated rats, suggesting that changes in glomerular hemodynamics may be responsible for the exacerbation of glomerulosclerosis. Both candesartan- and hydralazine-treated rats had glomeruli that were slightly decreased in size. These results suggest that single-antioxidant therapy starting at an advanced-stage may be ineffective for hypertension and rather exacerbate renal damage in nonsalt loaded SHRSP. Furthermore, lowering blood pressure and inhibiting the renin-angiotensin system could be critical for slowing the progression of hypertensive renal damage at an advanced stage.

Sphingosine-1-Phosphate Evokes Unique Segment-Specific Vasoconstriction of the Renal Microvasculature

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has been implicated in regulating vascular tone and participating in chronic and acute kidney injury. However, little is known about the role of S1P in the renal microcirculation. Here, we directly assessed the vasoresponsiveness of preglomerular and postglomerular microvascular segments to exogenous S1P using the in vitro blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (0.001-10 μM) evoked concentration-dependent vasoconstriction in preglomerular microvessels, predominantly afferent arterioles. After administration of 10 μM S1P, the diameter of afferent arterioles decreased to 35%±5% of the control diameter, whereas the diameters of interlobular and arcuate arteries declined to 50%±12% and 68%±6% of the control diameter, respectively. Notably, efferent arterioles did not respond to S1P. The S1P receptor agonists FTY720 and FTY720-phosphate and the specific S1P1 receptor agonist SEW2871 each evoked modest afferent arteriolar vasoconstriction. Conversely, S1P2 receptor inhibition with JTE-013 significantly attenuated S1P-mediated afferent arteriolar vasoconstriction. Moreover, blockade of L-type voltage-dependent calcium channels with diltiazem or nifedipine attenuated S1P-mediated vasoconstriction. Intravenous injection of S1P in anesthetized rats reduced renal blood flow dose dependently. Western blotting and immunofluorescence revealed S1P1 and S1P2 receptor expression in isolated preglomerular microvessels and microvascular smooth muscle cells. These data demonstrate that S1P evokes segmentally distinct preglomerular vasoconstriction via activation of S1P1 and/or S1P2 receptors, partially via L-type voltage-dependent calcium channels. Accordingly, S1P may have a novel function in regulating afferent arteriolar resistance under physiologic conditions.

Abstract 286: Acute Toll-like Receptors 4 Activiation by Lipopolysaccharide Attenuates Afferent Arteriolar Autoregulatory Behavior

Pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) activate toll like receptors (TLRs) that stimulate the innate immune system. Immune system activation is linked to blunted renal autoregulatory behavior. Lipopolysaccharide (LPS), a component of the bacterial wall of gram-negative bacteria, is a ligand for TLR4. Preliminary experiments indicates that TLR4 protein expression is increased approximately by 28% (n=2) in small intrarenal arteries after 4 hours of acute LPS treatment versus control. Accordingly, we hypothesize that acute LPS treatment blunts afferent arteriolar autoregulatory behavior through TLR4 activation. Rats received a bolus injection of LPS (1mg/kg I.P.) and kidneys were harvested 4 hours later for juxtamedullary nephron studies. At a perfusion pressure of 100 mmHg, baseline arteriolar diameters in LPS treated kidneys averaged 11.9 ± 0.5μm (n=15) compared to 14.5 ± 0.7μm (n=14) in untreated controls (P &lt; 0.05). Autoregulatory behavior was assessed in these same groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. In control kidneys, afferent arteriolar diameter decreased by 29 ± 5 % (n=6) as perfusion pressure increased from 65 to 170 mmHg. In LPS treated kidneys, afferent arteriolar diameter actually increased by 3 ± 8% (P &lt; 0.05; n=7) over the same pressure range, indicating marked blunting of pressure-induced vasoconstriction. P2 receptors have been implicated in autoregulatory resistance adjustments. Therefore, we assessed afferent arteriolar responsiveness to P2 receptor activation in vitro. In control kidneys, log concentrations of ATP (10 -8 - 10 -4 mol/L) reduced arteriolar diameter by 4 ± 1, 10 ± 2, 17 ± 4, 26 ± 5, and 32 ± 5% respectively (P &lt; 0.05; n=8). In LPS treated kidneys, ATP reduced arteriolar diameter similarly by 6 ± 1, 11 ± 2, 15 ± 3, 18 ± 4, and 21 ± 5% respectively (P &lt; 0.05; n=8). No significant difference in afferent arteriolar reactivity to ATP was found between the two groups. These data support the hypothesis that TLR4 activation by acute LPS treatment attenuates afferent arteriolar autoregulatory behavior. Accordingly, these data may open novel therapeutic approaches for prevention of inflammatory kidney disease.

Attenuated contractility in afferent arterioles during development of proteinuria in two‐kidney, one‐clip hypertensive rats

AimsWe wanted to investigate the contractile properties of afferent arterioles (AAs) from two‐kidney, one‐clip (2K1C) hypertensive rats before and during development of proteinuria, and whether these vessels behave differently in outer and inner renal cortex.MethodsWistar rats were subjected to either a 2K1C or sham operation. Blood pressure was measured and urine was sampled before the rats were killed after 18 ± 4 weeks. AAs were isolated from outer and inner renal cortex of the non‐clipped kidney of the 2K1C and from both kidneys of the sham rats with the agarose infusion technique. The AAs were stimulated with angiotensin II (AngII) in increasing concentrations (10−10 M to 10−7 M), and degree of contraction was recorded.ResultsThe preliminary results are based on AAs isolated from 9 rats (Sham: 3, 2K1C without proteinuria (2K1C Prot−): 2, and 2K1C with proteinuria (2K1C Prot+): 4). There was no difference in mean arterial pressure between the 2K1C with and without proteinuria (184 ± 6 vs. 178 ±8 mmHg, NS).We found a significantly attenuated response to AngII 10−7 M in the superficial AAs from both hypertensive groups compared to sham. However, in the AAs from the juxtamedullary nephrons there was a preserved response in the vessels from the 2K1C rats without proteinuria.ConclusionLoss of contractile properties of AAs in deep cortex may contribute to development of renal damage and proteinuria in this model.

Modeling the effects of medullary blood flow regulation on pressure natriuresis

The regulation of medullary blood flow by the vasoactive decending vasa recta may play an essential role in pressure natriuresis. While the regulatory mechanism remains incompletely understood, and quantities such as the absolute medullary blood flow remain experimentally inaccessible, many factors involved in that regulation have been identified. We present a whole‐kidney model of water and sodium balance that encompasses the renal vasculature and a population of superficial and juxtamedullary nephrons, each of which has a separate juxtaglomerular apparatus and autoregulates autonomously. Vasoconstriction of the vasculature and the epithelial transport are regulated by plasma hormone levels. Model predictions are consistent with acute renal function curves obtained from in vivo studies of the rat under a wide range of physiological conditions. We use this model to investigate the effects of vascular vasoconstriction and medullary blood flow regulation on the kidney's ability to produce concentrated urine at high arterial pressures. These findings are evaluated against the known and hypothesized actions of the known regulatory mechanisms. This research was supported in part by NIH grant DK‐89066.

Role of connexin 45 in the autoregulatory response of the afferent arteriole

Connexins expressed in renal arterioles are believed to influence the regulation of arteriolar tonus and renal blood flow. Connexin 45 (Cx45), which is primarily expressed in vascular smooth muscle cells of the renal afferent and efferent arterioles, has been suggested to play a role in the regulation of renin secretion. Also, Cx45 may be involved in the transmission of the signal initiating the tubuloglomerular feedback (TGF) response.In mice lacking Cx45 expression in vascular smooth muscle cells we examined the renal autoregulatory response by measuring changes in afferent arteriolar diameter in response to step increases in perfusion pressure. We used the isolated blood‐perfused juxtamedullary nephron preparation to visualize afferent arterioles. Cx45 deficient mice did not seem to have a higher mean arterial blood pressure than age‐matched litter‐mate wild type controls. The reduction in afferent arteriole diameter in response to step increases in renal perfusion pressure was significantly reduced in Cx45 deficient mice compared to the wild‐type litter‐mates. Removal of the TGF response did not seem to affect the ability to regulate afferent diameter in Cx45 deficient animals compared to wild‐type litter mates. These results suggest that Cx45 plays a major role in the myogenic component of the autoregulatory response of the afferent arteriole.

Renal ischemia‐reperfusion led to enhanced renal microvascular response to sphingosine‐1‐phosphate

Sphingosine‐1‐phosphate (S1P) potently vasoconstricts renal microvessels and participates in ischemia‐reperfusion (IR) induced acute kidney injury (AKI). We postulated that IR enhances renal vascular reactivity to S1P and contributes to AKI. Reactivity of juxtamedullary afferent arterioles (AA) to S1P was assessed in vitro in rats receiving 60 min bilateral ischemia followed by 24 hr reperfusion (B60IR) using the blood‐perfused juxtamedullary nephron technique (n=5–7/group). Plasma creatinine increased in B60IR (3.7±0.2 vs. 1.2±0.1 mg/dl in sham, p&lt;0.05). Baseline AA diameter decreased in B60IR (11.8±0.7 vs. 14.6±0.6 μm in shams, p&lt;0.05). S1P evoked concentration‐dependent AA vasoconstriction in both groups, however, the response was markedly enhanced in B60IR. S1P (10−10–10−5 M) reduced AA diameter to 93±1, 88±1, 76±1, 64±5, 46±5 and 35±4% of control in B60IR vs. 100±1, 94±2, 91±2, 81±4, 59±5 and 32±4%, respectively in shams. Conversely, AA responses to norepinephrine (NE) or KCl were unchanged. NE (10−8–10−6 M) reduced AA diameter to 92±2, 81±5 and 42±8% of control in B60IR compared to 92±2, 78±5 and 34±6% in shams. KCl (30, 60 and 90 mM) decreased AA diameter to 89±4, 51±6 and 40±7% in B60IR vs. 80±4, 38±4 and 32±4% in shams (p&gt;;0.05). These data reveal that IR enhances AA reactivity to S1P and may contribute importantly to IR‐AKI.Funding sources: AHA10SDG3770010, DK44628 and HL074167

Immunosuppression preserves renal autoregulatory function and microvascular P2X1 receptor reactivity in ANG II-hypertensive rats

Autoregulation is critical for protecting the kidney against arterial pressure elevation and is compromised in some forms of hypertension. Evidence indicates that activated lymphocytes contribute importantly to cardiovascular injury in hypertension. We hypothesized that activated lymphocytes contribute to renal vascular dysfunction by impairing autoregulation and P2X(1) receptor signaling in ANG II-infused hypertensive rats. Male Sprague-Dawley rats receiving ANG II infusion were treated with a lymphocyte proliferation inhibitor, mycophenolate mofetil (MMF) for 2 wk. Autoregulation was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. ANG II-treated rats exhibited impaired autoregulation. At the single vessel level, pressure-mediated afferent arteriolar vasoconstriction was significantly blunted (P < 0.05 vs. control rats). At the whole kidney level, renal blood flow passively decreased as renal perfusion pressure was reduced. MMF treatment did not alter the ANG II-induced hypertensive state; however, MMF did preserve autoregulation. The autoregulatory profiles in both in vitro or in vivo settings were similar to the responses from control rats despite persistent hypertension. Autoregulatory responses are linked to P2X(1) receptor activation. Accordingly, afferent arteriolar responses to ATP and the P2X(1) receptor agonist β,γ-methylene ATP were assessed. ATP- or β,γ-methylene ATP-induced vasoconstriction was significantly attenuated in ANG II-infused hypertensive rats but was normalized by MMF treatment. Moreover, MMF prevented elevation of plasma transforming growth factor-β1 concentration and lymphocyte and macrophage infiltration in ANG II-infused kidneys. These results suggest that anti-inflammatory treatment with MMF prevents lymphocyte infiltration and preserves autoregulation in ANG II-infused hypertensive rats, likely by normalizing P2X(1) receptor activation.

Abstract 491: Activation of Voltage-dependent L-type Calcium Channels and the Rho Kinase Pathway Are Involved in S1P-induced Vasoconstriction in Rat Juxtamedullary Afferent Arterioles

Sphingosine-1-phosphate (S1P) is an important regulator of resistance vessels and is implicated in pathological processes including diabetic nephropathy and acute kidney injury. Previous studies show that S1P causes marked vasoconstriction in preglomerular but not postglomerular microvessels, however, the intracellular signaling pathways underlying S1P-mediated vasoconstriction are undefined. We postulated that S1P-mediated afferent arteriolar (AA) vasoconstriction involves activation of voltage-dependent L-type Ca 2+ channels (L-VDCC) and the rho kinase pathway. Studies were conducted in vitro using the blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (n=6) evoked concentration-dependent AA vasoconstriction. Control diameter averaged 13.8±0.9 μm and declined to 95±2, 85±4, 75±6, 60±5 and 46±5% of control diameter in response to S1P (from 10 -9 to 10 -5 M), respectively. Superfusion with nifedipine (10 -6 M, n=6), a L-VDCC inhibitor, increased basal diameter by 39±18% (p&lt;0.05) and significantly attenuated the S1P-induced vasoconstriction. S1P reduced AA diameter to 99±1, 98±1, 90±3, 79±3 and 52±5% of control (p&lt;0.05 vs. S1P alone at 10 -8 to 10 -6 M) during nifedipine treatment. Superfusion with the rho kinase inhibitor, Y27632 (10 -5 M, n=6), increased diameter by 60±12% and inhibited S1P-induced vasoconstriction. AA diameter averaged 103±1, 101±1, 93±3, 72±9 and 56±10% of control in response to increasing concentrations of S1P (p&lt;0.05 vs. S1P alone at 10 -9 to 10 -7 M). In contrast, S1P-induced vasoconstriction was unaltered by Ca 2+ store depletion using the Ca 2+ -ATPase inhibitors, thapsigargin (Tg) and cyclopiazonic acid (CPA). Although Tg (10 -6 M, n=4) increased diameter by 38±16%, the S1P-induced vasoconstrictor profile was not different from that without Tg. AA declined to 99±3, 95±5, 85±10, 60±13 and 32±8% (p&gt;0.05), respectively. Similar results were obtained with CPA (10 -5 M, n=6). S1P reduced diameter to 97±2, 93±2, 75±5, 53±6 and 36±5% of the control (p&gt;0.05), respectively, suggesting that mobilization of intracellular Ca 2+ store is not required for S1P-induced vasoconstriction. Overall, these data indicate that both L-VDCC and rho kinase pathways contribute to S1P-mediated AA vasoconstriction.

Albuminuria indicates the pressure-associated injury of juxtamedullary nephrons and cerebral strain vessels in spontaneously hypertensive stroke-prone rats

Albuminuria is an indicator of renal injury and is closely linked with cardiovascular disease (CVD). However, the mechanism by which albumin is excreted in the urine remains unclear. As the juxtamedullary region of the kidney is highly susceptible to pressure increase, juxtamedullary injury is observed from an early phase in hypertensive rat models. Anatomical similarities are observed between the pre-glomerular vessels of the juxtamedullary nephron and the cerebral vasculature. We previously named these ‘strain vessels' for their high vascular tone and exposure to higher pressures. The current studies were designed to determine whether albuminuria is the result of juxtamedullary nephron injury, indicating the presence of pressure injury to the strain vessels in spontaneously hypertensive stroke-prone rats (SHR-SP) fed a high-salt diet. Albuminuria was associated with juxtamedullary nephron injury, and the enhanced expression of monocyte chemotactic protein-1 (MCP-1) and tumor growth factor-beta (TGF-β) in 12-week-old SHR-SP rats fed a 4% high-salt diet from the age of 6 weeks. The wall thickness of the pre-glomerular vessels of the juxtamedullary nephron was also associated with that of the perforating artery of the middle cerebral artery. Reducing the blood pressure with nifedipine reduced the degree of albuminuria and juxtamedullary nephron injury as well as MCP-1 and TGF-β expression in the SHR-SP rats fed an 8% high-salt diet from the age of 9 weeks. Nifedipine inhibited stroke events in these animals until they were 14 weeks old. These results indicate that albuminuria is a result of juxtamedullary nephron injury and a marker of pressure-induced injury of the strain vessels.

Role of specific T-type calcium channel blocker R(−) efonidipine in the regulation of renal medullary circulation

Blockade of the T-type calcium channel (TCC), which is expressed in the renal efferent arterioles of the juxtamedullary nephron and vasa recta, has been shown to protect against renal injury. Studies were designed to determine the effects of a specific TCC blocker, R(-) efonidipine [R(-)EFO], on the regulation of renal circulation. Renal medullary blood flux (MBF) and cortical blood flux (CBF) were simultaneously monitored using laser-Doppler flowmetry in Sprague-Dawley rats. Responses were also determined in rats with angiotensin II (AngII) induced renal ischemia. Intravenous (i.v.) or renal interstitial (r.i.) infusion of R(-)EFO (0.25 mg/h, i.v. or r.i.) significantly increased MBF by 24.0 ± 7.0 and 21.0 ± 4.4%, respectively, but without changing CBF or mean arterial pressure. The nitric oxide (NO) synthase inhibitor NG-nitro-L-argininemethylester (L-NAME, 1 μg/kg per min, i.v. or r.i.) significantly attenuated R(-)EFO-induced increase in MBF. R(-)EFO inhibited the AngII-mediated (50 ng/kg per min, i.v.) reduction of MBF (28.4 ± 1.7%), which was associated with increased urinary NO(2) + NO(3) excretion and decreased urinary hydrogen peroxide (H(2)O(2)) excretion. Intracellular H(2)O(2) fluorescence (real-time fluorescence imaging) in the epithelial cells of isolated medullary thick ascending limb (mTAL) significantly increased following AngII stimulation (1 μmol/L, 235 ± 52 units), which was significantly inhibited by pre and coincubation with R(-)EFO. R(-)EFO stimulation also increased the intracellular NO concentration in the epithelial cells of mTAL (220 ± 62 units). These results suggest that TCC blockade with R(-)EFO selectively increases MBF, an effect that appears to be mediated by changes in renal NO and oxidative stress balance, which may protect against ischemic renal injury in the renal medullary region.

Role of connexin40 in the autoregulatory response of the afferent arteriole

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.

Nebivolol-induced vasodilation of renal afferent arterioles involves β3-adrenergic receptor and nitric oxide synthase activation

Nebivolol is a β(1)-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a β(3)-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor N(ω)-nitro-l-arginine (l-NNA) or the β-blockers metoprolol (β(1)), butoxamine (β(2)), and SR59230A (β(3)). Nebivolol (100 μmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 μmol/l) or ODQ (10 μmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to β(3)-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of β(1)-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial β(3)-receptors.