Afferent Arteriolar Vasoconstriction Research Articles

Are SGLT2 Inhibitors Ready for Prime Time for CKD?

Sodium-glucose cotransporter-2 (SGLT2) is a sodium-dependent glucose transport protein, and its inhibitors (SGLT2i - gliflozins) are indicated as an adjunct therapy to diet and exercise for improving glycemic control in type 2 diabetes mellitus (DM2). In fact, SGLT2i usage has been associated with a

Mechanisms of sphingosine-1-phosphate-mediated vasoconstriction of rat afferent arterioles.

Sphingosine-1-phosphate (S1P) influences resistance vessel function and is implicated in renal pathological processes. Previous studies revealed that S1P evoked potent vasoconstriction of the pre-glomerular microvasculature, but the underlying mechanisms remain incompletely defined. We postulated that S1P-mediated pre-glomerular microvascular vasoconstriction involves activation of voltage-dependent L-type calcium channels (L-VDCC) and the rho/rho kinase pathway. Afferent arteriolar reactivity was assessed in vitro using the blood-perfused rat juxtamedullary nephron preparation, and diameter was measured during exposure to physiological and pharmacological agents. Exogenous S1P (10-9 -10-5 mol L-1 ) evoked concentration-dependent vasoconstriction of afferent arterioles. Superfusion with nifedipine, a L-VDCC blocker, increased arteriolar diameter by 39 ± 18% of baseline and significantly attenuated the S1P-induced vasoconstriction. Superfusion with the rho kinase inhibitor, Y-27632, increased diameter by 60 ± 12% of baseline and also significantly blunted vasoconstriction by S1P. Combined nifedipine and Y-27632 treatment significantly inhibited S1P-induced vasoconstriction over the entire concentration range tested. In contrast, depletion of intracellular Ca2+ stores with the Ca2+ -ATPase inhibitors, thapsigargin or cyclopiazonic acid, did not alter the S1P-mediated vasoconstrictor profile. Scavenging reactive oxygen species (ROS) or inhibition of nicotinamide adenine dinucleotide phosphate oxidase activity significantly attenuated S1P-mediated vasoconstriction. Exogenous S1P elicits potent vasoconstriction of rat afferent arterioles. These data also demonstrate that S1P-mediated pre-glomerular vasoconstriction involves activation of L-VDCC, the rho/rho kinase pathway and ROS. Mobilization of Ca2+ from intracellular stores is not required for S1P-mediated vasoconstriction. These studies reveal a potential role for S1P in the modulation of renal microvascular tone.

SGLT-2 Inhibitors in Heart Failure: Implications for the Kidneys.

This review aims to summarize the renal effects of sodium-glucose transporter-2 (SGLT-2) inhibitors and their potential implications in heart failure pathophysiology. In patients with diabetes and established atherosclerosis, the SGLT-2 inhibitor empagliflozin versus placebo significantly reduced the rate of heart failure admissions with 35%. Moreover, empagliflozin slowed kidney disease progression and reduced the need for renal replacement therapy. SGLT-2 inhibitors inhibit proximal tubular sodium and chloride reabsorption, leading to increased nephron flux throughout the distal renal tubules, most notably at the level of the macula densa. Afferent arteriolar vasoconstriction is promoted through tubulo-glomerular feedback and reduces glomerular capillary hydrostatic pressure, relieving podocyte stress and explaining renal preservation. Further, plasma volume is contracted and natriuresis promoted without inducing neurohumoral activation. Finally, SGLT-2 inhibitors may improve endothelial function and energy metabolism efficiency. Together, these promising features place them as a potential novel treatment for heart failure.

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.

Effect of nitric oxide on renal autoregulation during hypothermia in the rat

Hypothermia-induced reduction of metabolic rate is accompanied by depression of both glomerular perfusion and filtration. The present study investigated whether these changes are linked to changes in renal autoregulation and nitric oxide (NO) signalling. During hypothermia, renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced and urine production was increased, and this was linked with reduced plasma cGMP levels and increased renal vascular resistance. Although stimulation of NO production decreased vascular resistance, blood pressure and urine flow, intravenous infusion of the NO precursor L-arginine or the NO donor sodium nitroprusside did not alter RBF or GFR. In contrast, inhibition of NO synthesis by Nw-nitro-L-arginine led to a further decline in both parameters. Functional renal autoregulation was apparent at both temperatures. Below the autoregulatory range, RBF in both cases increased in proportion to the perfusion ±pressure, although, the slope of the first ascending limb of the pressure-flow relationship was lower during hypothermia. The main difference was rather that the curves obtained during hypothermia levelled off already at a RBF of 3.9 ± 0.3 mL/min then remained stable throughout the autoregulatory pressure range, compared to 7.6 ± 0.3 mL/min during normothermia. This was found to be due to a threefold increase in, primarily, the afferent arteriolar resistance from 2.6 to 7.5 mmHg min mL−1. Infusion of sodium nitroprusside did not significantly affect RBF during hypothermia, although a small increase at pressures below the autoregulatory range was observed. In conclusion, cold-induced rise in renal vascular resistance results from afferent arteriolar vasoconstriction by the autoregulatory mechanism, setting RBF and GFR in proportion to the metabolic rate, which cannot be explained by reduced NO production alone.

Myoglobin facilitates angiotensin II-induced constriction of renal afferent arterioles.

Vasoconstriction plays an important role in the development of acute kidney injury in rhabdomyolysis. We hypothesized that myoglobin enhances the angiotensin II (ANG II) response in afferent arterioles by increasing superoxide and reducing nitric oxide (NO) bioavailability. Afferent arterioles of C57Bl6 mice were isolated perfused, and vasoreactivity was analyzed using video microscopy. NO bioavailability, superoxide concentration in the vessel wall, and changes in cytosolic calcium were measured using fluorescence techniques. Myoglobin treatment (10-5 M) did not change the basal arteriolar diameter during a 20-min period compared with control conditions. NG-nitro-l-arginine methyl ester (l-NAME, 10-4 M) and l-NAME + myoglobin reduced diameters to 94.7 and 97.9% of the initial diameter, respectively. Myoglobin or l-NAME enhanced the ANG II-induced constriction of arterioles compared with control (36.6 and 34.2%, respectively, vs. 65.9%). Norepinephrine responses were not influenced by myoglobin. Combined application of myoglobin and l-NAME further facilitated the ANG II response (7.0%). Myoglobin or l-NAME decreased the NO-related fluorescence in arterioles similarly. Myoglobin enhanced the superoxide-related fluorescence, and tempol prevented this enhancement. Tempol also partly prevented the myoglobin effect on the ANG II response. Myoglobin increased the fura 2 fluorescence ratio (cytosolic calcium) during ANG II application (10-12 to 10-6 M). The results suggest that the enhanced afferent arteriolar reactivity to ANG II is mainly due to a myoglobin-induced increase in superoxide and associated reduction in the NO bioavailability. Signaling pathways for the augmented ANG II response include enhanced cytosolic calcium transients. In conclusion, myoglobin may contribute to the afferent arteriolar vasoconstriction in this rhabdomyolysis model.

SGLT2 Inhibitors and the Diabetic Kidney.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide. Blood glucose and blood pressure control reduce the risk of developing this complication; however, once DN is established, it is only possible to slow progression. Sodium-glucose cotransporter 2 (SGLT2) inhibitors, the most recent glucose-lowering oral agents, may have the potential to exert nephroprotection not only through improving glycemic control but also through glucose-independent effects, such as blood pressure-lowering and direct renal effects. It is important to consider, however, that in patients with impaired renal function, given their mode of action, SGLT2 inhibitors are less effective in lowering blood glucose. In patients with high cardiovascular risk, the SGLT2 inhibitor empagliflozin lowered the rate of cardiovascular events, especially cardiovascular death, and substantially reduced important renal outcomes. Such benefits on DN could derive from effects beyond glycemia. Glomerular hyperfiltration is a potential risk factor for DN. In addition to the activation of the renin-angiotensin-aldosterone system, renal tubular factors, including SGLT2, contribute to glomerular hyperfiltration in diabetes. SGLT2 inhibitors reduce sodium reabsorption in the proximal tubule, causing, through tubuloglomerular feedback, afferent arteriole vasoconstriction and reduction in hyperfiltration. Experimental studies showed that SGLT2 inhibitors reduced hyperfiltration and decreased inflammatory and fibrotic responses of proximal tubular cells. SGLT2 inhibitors reduced glomerular hyperfiltration in patients with type 1 diabetes, and in patients with type 2 diabetes, they caused transient acute reductions in glomerular filtration rate, followed by a progressive recovery and stabilization of renal function. Interestingly, recent studies consistently demonstrated a reduction in albuminuria. Although these data are promising, only dedicated renal outcome trials will clarify whether SGLT2 inhibitors, in addition to their glycemic and blood pressure benefits, may provide nephroprotective effects.

Hypertensive emergency in kidney transplant recipient with autosomal dominant polycystic kidney disease

Background: Aldosterone agonist is used for calcineurin inhibitor (CNI)-induced hyperkalemia in kidney transplant recipient; however, it potentially causes uncontrolled blood pressure (BP). We report a hypertensive kidney transplant recipient with hyperkalemia resolved by aldosterone agonist. Case Description: A 65-year-old man with ESRD from type 2 diabetes mellitus underwent uneventful kidney transplantation. Tacrolimus and prophylaxis trimethoprim/sulfamethoxazole (TMP/SMZ) was started. He had persistent hyperkalemia with serum potassium (K) of 5.1-5.7 mmol/L and non-anion gap metabolic acidosis (NAGMA). BP was uncontrolled with average SBP and DBP of 180 and 60 mmHg, respectively (Figure 1). On posttransplant day 11, K increased up to 6.4 mmol/L even good urine output (UOP) of 1.6-2.2 L/day and serum creatinine (SCr) of 2.3 mg/dL from the pretransplant SCr of 10.31 mg/dL. In addition to low K diet, he was treated with sodium polystyrene sulfonate with several bowel movements; however, K still elevated. Therefore, fludrocortisone was started and K was normalized. BP medications were adjusted. Discussion: Tacrolimus causes distal tubular defect mimicking type 4 renal tubular acidosis (T4RTA) by inhibiting basolateral Na-K ATPase at the collecting duct causing impaired transepithelial K secretion from decreased sodium uptake. It also causes hypertension (HTN) from renal afferent arteriolar vasoconstriction and activation of Na-Cl cotransporter (NCC) in the distal collecting tubule (DCT) and subsequently HTN. Hyperkalemia in our patient is from tacrolimus since he had posttransplant hyperkalemia even with improving renal allograft function. TMP/SMX could contribute to hyperkalemia. Aldosterone agonist corrects hyperkalemia but causes uncontrolled BP. Conclusions: Uncontrolled HTN limits the utility of aldosterone agonist for the treatment of CNI-induced hyperkalemia. Since tacrolimus stimulates NCC, thiazide diuretics could potentially be used for BP control once renal allograft function is stable.

Renal autoregulation in health and disease.

Intrarenal autoregulatory mechanisms maintain renal blood flow (RBF) and glomerular filtration rate (GFR) independent of renal perfusion pressure (RPP) over a defined range (80-180 mmHg). Such autoregulation is mediated largely by the myogenic and the macula densa-tubuloglomerular feedback (MD-TGF) responses that regulate preglomerular vasomotor tone primarily of the afferent arteriole. Differences in response times allow separation of these mechanisms in the time and frequency domains. Mechanotransduction initiating the myogenic response requires a sensing mechanism activated by stretch of vascular smooth muscle cells (VSMCs) and coupled to intracellular signaling pathways eliciting plasma membrane depolarization and a rise in cytosolic free calcium concentration ([Ca(2+)]i). Proposed mechanosensors include epithelial sodium channels (ENaC), integrins, and/or transient receptor potential (TRP) channels. Increased [Ca(2+)]i occurs predominantly by Ca(2+) influx through L-type voltage-operated Ca(2+) channels (VOCC). Increased [Ca(2+)]i activates inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) to mobilize Ca(2+) from sarcoplasmic reticular stores. Myogenic vasoconstriction is sustained by increased Ca(2+) sensitivity, mediated by protein kinase C and Rho/Rho-kinase that favors a positive balance between myosin light-chain kinase and phosphatase. Increased RPP activates MD-TGF by transducing a signal of epithelial MD salt reabsorption to adjust afferent arteriolar vasoconstriction. A combination of vascular and tubular mechanisms, novel to the kidney, provides for high autoregulatory efficiency that maintains RBF and GFR, stabilizes sodium excretion, and buffers transmission of RPP to sensitive glomerular capillaries, thereby protecting against hypertensive barotrauma. A unique aspect of the myogenic response in the renal vasculature is modulation of its strength and speed by the MD-TGF and by a connecting tubule glomerular feedback (CT-GF) mechanism. Reactive oxygen species and nitric oxide are modulators of myogenic and MD-TGF mechanisms. Attenuated renal autoregulation contributes to renal damage in many, but not all, models of renal, diabetic, and hypertensive diseases. This review provides a summary of our current knowledge regarding underlying mechanisms enabling renal autoregulation in health and disease and methods used for its study.

Endothelin-1, but not angiotensin II, induces afferent arteriolar myosin diphosphorylation as a potential contributor to prolonged vasoconstriction

Bolus administration of endothelin-1 elicits long-lasting renal afferent arteriolar vasoconstriction, in contrast to transient constriction induced by angiotensin II. Vasoconstriction is generally evoked by myosin regulatory light chain (LC20) phosphorylation at Ser19 by myosin light chain kinase (MLCK), which is enhanced by Rho-associated kinase (ROCK)-mediated inhibition of myosin light chain phosphatase (MLCP). LC20 can be diphosphorylated at Ser19 and Thr18, resulting in reduced rates of dephosphorylation and relaxation. Here we tested whether LC20 diphosphorylation contributes to sustained endothelin-1 but not transient angiotensin II-induced vasoconstriction. Endothelin-1 treatment of isolated arterioles elicited a concentration- and time-dependent increase in LC20 diphosphorylation at Thr18 and Ser19. Inhibition of MLCK or ROCK reduced endothelin-1-evoked LC20 mono- and diphosphorylation. Pretreatment with an ETB but not an ETA receptor antagonist abolished LC20 diphosphorylation, and an ETB receptor agonist induced LC20 diphosphorylation. In contrast, angiotensin II caused phosphorylation exclusively at Ser19. Thus, endothelin-1 and angiotensin II induce afferent arteriolar constriction via LC20 phosphorylation at Ser19 due to calcium activation of MLCK and ROCK-mediated inhibition of MLCP. Endothelin-1, but not angiotensin II, induces phosphorylation of LC20 at Thr18. This could contribute to the prolonged vasoconstrictor response to endothelin-1.

Role of Interstitial Angiotensin II and ATP in Mediating Renal Injury Induced by Recurrent Insulin Induced Hypoglycemia

Aim: The present study hypothesizes that recurrent insulin induced hypoglycemia (RIIH) elevates renal interstitial ATP levels which in turn enhances AngII production. This interrupts the normal tubuloglomerular feedback (TGF) mechanism by stimulating afferent arteriolar vasoconstriction resulting in hypertension, which augments oxidative stress and could promote renal damage. Study Design: In the present study we adopted a microdialysis technique, which is a minimally invasive tool for monitoring chronic changes in renal interstitial fluid. Place and Duration of Study: Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe between September 2012 – October 2013. Methodology: Eight male Sprague Dawley rats (200-225 g) were anesthetized and microdialysis probes were inserted into their renal cortex. Post-surgery rats were treated with insulin (7U/kg body Original Research Article Prathipati et al.; ARRB, 6(5): 328-336, 2015; Article no.ARRB.2015.092 329 weight) for 2 weeks. Food and water intake were monitored daily. Physiological saline was perfused through the probe and dialysate was collected daily after insulin dosing and analyzed for ATP by luciferin-luciferase assay and AngII by EIA. At the end of the experiment, the hearts and kidneys were collected and analyzed for oxidative stress by EPR (Electron Paramagnetic Resonance) spectroscopy using CMH and CPH spin probes. Results: ATP and AngII levels were elevated from 31.65±4.4ng/μl (day 0) to 130.96±2.9 ng/μl (day 14) and 0.1±0.01 ng/ml (day 0) to 0.247±0.02 ng/ml(day 14), respectively. Elevation of peroxynitrite and superoxide anions were observed in the hearts and kidneys of insulin treated animals when compared to the control group. Conclusion: Thus the present study utilizes real-time chronic collections of renal interstitial samples to identify a potential mechanism where iatrogenic hypoglycemia promotes hypertension via a synergistic relationship between interstitial ATP, AngII and developed oxidative stress.

Sildenafil citrate for prophylaxis of nephropathy in an animal model of contrast-induced acute kidney injury.

Contrast-induced acute kidney injury (CIAKI) is one of the commonest complications associated with contrast media (CM). Although the exact etiology of CIAKI remains unclear, one hypothesis involves vasoconstriction of afferent arterioles resulting in renal ischemia. Increased renal blood flow, therefore, might represent an attractive target for the treatment of CIAKI. In this study we evaluated the protective effects of the phosphodiesterase type 5 (PDE5) inhibitor, sildenafil citrate, in a rabbit model of CIAKI. New Zealand white rabbits were used due to their susceptibility to CIAKI. To evaluate the effects of sildenafil, the drug was administered before CM infusion and repeatedly throughout the remainder of the experiment (6 mg/kg, p.o.). Animals were sacrificed after 48 hours and kidneys were prepared for histological evaluation. Intravenous administration of CM produced marked kidney injury. Serum creatinine concentrations were elevated within two hours of the infusion and remained elevated for the duration of the experiment. Histological evaluation of the kidneys revealed significant tubular necrosis. The effects of the CM were dose dependent. Treatment with sildenafil was associated with lesser degree of histological injury, attenuation in markers of acute kidney injury (48 hour creatinine 1.54±0.21 versus 4.42±1.31 mg/dl, p<0.05) and reduction in electrolyte derangement (percent change in serum K+ at 48 hours 2.55±3.80% versus 15.53±4.47%, p<0.05; serum Na+ at 48 hours −0.14±0.26% versus −1.97±1.29%, p = 0.20). The results suggest a possible role for PDE5 inhibitors in the treatment of CIAKI and warrant further evaluation to determine the exact mechanism of protection.

Sildenafil citrate attenuates acute renal injury in an animal model of contrast‐induced nephropathy (LB573)

Contrast‐induced nephropathy (CIN) is defined as acute renal failure occurring within 48 hr of exposure to intravascular contrast agents. Although the exact etiology of CIN remains unclear, one hypothesis involves vasoconstriction of afferent arterioles resulting in renal ischemia. Increased renal blood flow, therefore, might represent an attractive target for the treatment of CIN. In this study we evaluated the effect of inhibition of cGMP‐specific phosphodiesterase type 5 (PDE5), a process resulting in renal vasodilation, in an animal model of CIN.New Zealand white rabbits were used due to their susceptibility to CIN. Contrast agent (ioxilan, 2.5‐10.0 g I/kg) was infused intravenously over 30 m. Another group of animals received an equiosmolar NaCl solution and served as the control. To evaluate the effects of PDE5 inhibition, a final group was treated with sildenafil citrate before contrast infusion and repeatedly throughout the remainder of the experiment (6 mg/kg, p.o., t.i.d.). The animals were sacrificed after 48 hr and the kidneys were prepared for histological evaluation.Intravenous administration of ioxilan produced marked kidney injury. Serum creatinine concentrations were elevated within two hr of the infusion and remained elevated for the duration of the experiment. Histological evaluation of the kidneys revealed significant tubular necrosis. The effects of the contrast agent were dose dependent, but absent in the animals that were administered NaCl solution. Notably, the effects of contrast infusion were attenuated by simultaneous treatment with sildenafil citrate.The conclusions of this study are two fold. First, the absence of deleterious effects in animals treated with equiosmolar NaCl solution suggests that the mechanism by which the contrast agent induced kidney injury was independent of osmolality. Second, selective PDE5 inhibition protected the kidney from CIN. The results suggest a possible role for PDE5 inhibitors in the treatment of CIN and warrant further evaluation to determine the exact mechanism of protection.

Adaptation of chronic renal microdialysis to evaluate the role of interstitial ATP and angiotensin II in accelerating nephropathy and oxidative stress during insulin induced hypertension (1088.3)

Various studies demonstrated the role of ATP in regulating hemodynamic functions and play a key role in maintaining tubuloglomerularfeedback (TGF) and renal vascular resistance. Previous studies also demonstrated the role of Ang II and HO‐1 in regulating blood pressure. We thus hypothesize that insulin induced hypoglycemia augments interstitial ATP levels which in turn increases AngII. This stimulates afferent arteriolar vasoconstriction interrupting normal TGF mechanism resulting in hypertension leading to oxidative stress and ultimately renal damage. Male Sprague Dawley rats (200‐225g) were anesthetized and microdialysis probe was inserted into their renal cortex. Post surgery rats were treated with insulin (7U/kg body weight) or vehicle for 2 weeks. Dialysate was collected daily after insulin dosing and analyzed for ATP by luciferin‐luciferase assay and Ang II by ELISA. ATP levels were elevated from 4.75 ng (day 0) to 36.47 ng (day 14). Heart and kidneys were collected at the end and analyzed for oxidative stress using EPR (Electron Paramagnetic Resonance). Oxidative stress was observed from the spectra obtained by using spin probes CMH and CPH. Thus the present study demonstrates a correlation between interstitial ATP and oxidative stress by real‐time chronic collection of renal interstitial samples, demonstrating the ability of hypoglycemia to promote the development of hypertension in turn accelerating oxidative stress.Grant Funding Source: Board of Regents Research Competitiveness 2010

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.

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Introduction: Chronic allograft nephropathy is the primary cause of late graft dysfunction in the renal transplant population. The causes are multi-factorial including calcineurin inhibitor nephrotoxicity. Calcineurin inhibitors (CNI), such as tacrolimus and cyclosporine are commonly used in solid organ transplant patients to prevent allograft rejection. They contribute to chronic allograft nephropathy and cause nephrotoxicity in all solid organ transplant populations via afferent arteriolar vasoconstriction. Fenoldopam is a dopamine receptor-1 agonist, which causes vasodilatation in the proximal tubule, and theoretically will directly antagonize the deleterious effects of CNI induced renal failure; with in-vitro data to suggest its efficacy in preventing CNI induced renal failure. Methods: A retrospective chart review was performed on transplant patients, in acute renal failure, admitted to the University of Kentucky medical center from 2004- 2011. Our primary objective was to compare changes in renal function values: serum creatinine (Scr) and urine output (UOP) of patients admitted with acute renal failure due to CNI who received either fenoldopam or no drug. Patient data was collected at baseline (time 0) and then for 4 continuous days upon admission (non-fenoldopam group) or at the point of initiation of fenoldopam (time 1) until day 5, or the end of the infusion. Demographic data were analyzed using either a two sample T-test or Mann-Whitney U test, when appropriate. Repeated measures ANOVA with and without adjustment for covariates for the outcomes of Scr and UOP were performed using SAS version 9.3 by SAS Institute (Cary, NC). Results: A total of 87 patients were analyzed (36 non-fenoldopam; 51 fenoldopam). Differences in demographics were significant for a higher SOFA score in the fenoldopam group, (4.8 ± 2.77 vs 2.97 ± 1.68 p=0.0005) After controlling for confounders, the effect of fenoldopam on urine output (UOP) was not statistically significant (p=0.08771) at any time point throughout the infusion time (days 1-4, or end of infusion, day 5) compared to the non-drug group. The non-drug group saw a statistically significant decrease in Scr over the 4 days (-1.0173 ± 0.2143 p= <0.001). The fenoldopam group saw a decrease in Scr from time 1 (start of infusion) over the 4 time points, however this effect did not reach statistical significance (-0.13 ± 0.2 p=0.5194). Conclusions: Although in-vitro data exist to suggest fenoldopam's efficacy in preventing CNI induced renal failure, fenoldopam was not associated with improvement in renal function parameters in this transplant population. As this is the first reported use of fenoldopam for this indication in humans, there is an opportunity for a randomized trial to determine the utility in CNI induced renal failure.

Afferent arteriolar responses to β,γ-methylene ATP and 20-HETE are not blocked by ENaC inhibition

Afferent arteriolar myogenic and tubuloglomerular feedback responses are critical for the proper maintenance of renal hemodynamics and water and electrolyte homeostasis. Adenosine triphosphate (ATP) P2X receptor activation and 20-hydroxyeicosatetraenoic acids (20-HETE) have been implicated in afferent arteriolar autoregulatory responses. Besides these two participants, members of the degenerin/epithelial Na+ channel (DEG/ENaC) family have been demonstrated to play a pivotal role in the afferent arteriolar myogenic response. The aim of this study was to determine if ENaC contributes to P2X receptor- or 20-HETE-mediated afferent arteriolar vasoconstriction. As previously demonstrated, afferent arteriolar diameter responses to increasing perfusion pressure from 100 to 160 mmHg were abolished by ENaC inhibitors amiloride or benzamil. Afferent arteriolar diameter decreased by 29% under control conditions and by 1% and 5% in the presence of amiloride or benzamil, respectively. The P2X receptor agonist β,γ-methylene ATP decreased afferent arteriolar diameter by 3 ± 1%, 7 ± 1%, 12 ± 2%, and 17 ± 3% in response to 0.1, 1, 10, and 100 μmol/L, respectively. ENaC inhibition did not alter the afferent arteriolar vasoconstrictor response to the P2X receptor agonist β,γ-methylene ATP. Like P2X receptor activation, 20-HETE dose-dependently decreased afferent arteriolar diameter and this vasoconstrictor response was not altered by the presence of ENaC inhibitors amiloride or benzamil. These results suggest that DEG/ENaC channels are required for afferent arteriolar autoregulatory responses; however, DEG/ENaC channels do not contribute to P2X receptor- or 20-HETE-mediated afferent arteriolar vasoconstriction.

Abstract 551: Inhibition of Sphingosine-1-phosphate Receptor 2 Activation Protects against Renal Ischemia-Reperfusion Injury via Reduction of Reactive Oxygen Species

Sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of the preglomerular microvasculature. Our recent studies indicate that S1P-mediated afferent arteriolar vasoconstriction is enhanced in ischemia-reperfusion (IR) induced renal injury and S1P2 receptor protein expression is increased in renal microvessels. Reactive oxygen species (ROS) play a critical role in the development of IR renal injury. We hypothesized that inhibition of S1P2 receptor activation protects against renal IR injury via ROS reduction. Renal IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. In some rats, IR was induced during treatment with the selective S1P2 receptor antagonist, JTE-013 (0.1mg/kg BW, IP) prior to ischemia and repeated at the time of reperfusion. After reperfusion, plasma was collected and kidneys were harvested for histological analysis and ROS detection. Plasma creatinine (Cr) concentration was markedly increased in IR rats (4.2±0.1 mg/dl, n=6) compared to sham controls (1.1±0.1 mg/dl, n=5, p&lt;0.05). JTE-013 treatment (n=4) significantly reduced plasma Cr in IR rats (2.0±0.3 mg/dl, p&lt;0.05). Histological analysis revealed no significant cell damage in control kidney sections stained with hematoxylin and eosin whereas severe cell damage was detected in IR kidneys, including cell necrosis, disruption of the plasma membrane, nuclear pyknosis, cellular vacuolization, and the presence of luminal casts and sloughed cells in proximal tubules, particularly in the outer stripe of the outer medulla. Additionally, there was significant trapping of erythrocytes in the medulla. In contrast, JTE-013 treated IR rats showed less necrosis and cell damage in the proximal tubules. Renal ROS levels were assessed in frozen kidney sections using the fluorogenic probe, H 2 DCFDA. Fluorescence intensity was markedly increased in IR rats (18.7±2.4 in cortex and 19.0±2.5 in medulla) compared to control (10.9±0.7 and 9.6±0.7, p&lt;0.05, respectively). JTE-013 treatment reduced ROS levels to values not different from control (13.5±1.1 and 15.4±1.5, p&gt;0.05 vs. control, respectively). These data suggest that blockade of S1P2 receptors protects against renal IR injury by reducing tissue ROS accumulation.

Synthetic oleanane triterpenoids: magic bullets or not?

A recent clinical trial in patients with chronic kidney disease (CKD) and diabetic nephropathy demonstrated that bardoxolone methyl (CDDO-ME) increases estimated glomerular filtration rate (eGFR) by an unknown mechanism. The paper by Ding et al. suggests that short-term administration of a CDDO-ME analog increases GFR by increasing glomerular surface area. However, changes in other renal hemodynamic parameters cannot be excluded. Vigorous testing of CDDO-ME and highly purified analogs is warranted to determine their physiology, pharmacology, and efficacy and to exclude serious side effects.

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.