Janet L. Hobbs

Endothelin A Receptor Blockade Reduces Diabetic Renal Injury via an Anti-Inflammatory Mechanism

Endothelin (ET) receptor blockade delays the progression of diabetic nephropathy; however, the mechanism of this protection is unknown. Therefore, the aim of this study was to test the hypothesis that ET(A) receptor blockade attenuates superoxide production and inflammation in the kidney of diabetic rats. Diabetes was induced by streptozotocin (diabetic rats with partial insulin replacement to maintain modest hyperglycemia [HG]), and sham rats received vehicle treatments. Some rats also received the ETA antagonist ABT-627 (sham+ABT and HG+ABT; 5 mg/kg per d; n = 8 to 10/group). During the 10-wk study, urinary microalbumin was increased in HG rats, and this effect was prevented by ET(A) receptor blockade. Indices of oxidative stress, urinary excretion of thiobarbituric acid reactive substances, 8-hydroxy--deoxyguanosine, and H2O2 and plasma thiobarbituric acid reactive substances were significantly greater in HG rats than in sham rats. These effects were not prevented by ABT-627. In addition, renal cortical expression of 8-hydroxy--deoxyguanosine and NADPH oxidase subunits was not different between HG and HG+ABT rats. ETA receptor blockade attenuated increases in macrophage infiltration and urinary excretion of TGF-beta and prostaglandin E2 metabolites in HG rats. Although ABT-627 did not alleviate oxidative stress in HG rats, inflammation and production of inflammatory mediators were reduced in association with prevention of microalbuminuria. These observations indicate that ETA receptor activation mediates renal inflammation and TGF-beta production in diabetes and are consistent with the postulate that ETA blockade slows progression of diabetic nephropathy via an anti-inflammatory mechanism.

Effect of Epithelial Sodium Channel Blockade on the Myogenic Response of Rat Juxtamedullary Afferent Arterioles

The mechanotransduction mechanism underlying the myogenic response is poorly understood, but evidence implicates participation of epithelial sodium channel (ENaC)-like proteins. Therefore, the role of ENaC on the afferent arteriolar myogenic response was investigated in vitro using the blood-perfused juxtamedullary nephron technique. Papillectomy was used to isolate myogenic influences by eliminating tubuloglomerular feedback signals. Autoregulatory responses were assessed by manipulating perfusion pressure in 30-mm Hg steps. Under control conditions, arteriolar diameter increased by 15% from 13.0±1.3 to 14.7±1.2 &mgr;m (P<0.05) after reducing perfusion pressure from 100 to 70 mm Hg. Diameter decreased to 11.3±1.1 and 10.6±1.0 &mgr;m after increasing pressure to 130 and 160 mm Hg (88±1 and 81±2% of control diameter, P<0.05), respectively. Pressure-mediated autoregulatory responses were significantly inhibited by superfusion of 10 &mgr;mol/L amiloride (102±2, 97±4, and 94±3% of control diameter), or 10 &mgr;mol/L benzamil (106±5, 100±3, and 103±3% of control diameter), and when perfusing with blood containing 5 &mgr;mol/L amiloride (106±2, 97±4, and 97±4% of control diameter). Vasoconstrictor responses to 55 mmol/L KCl were preserved as diameters decreased by 67±4, 55±8, and 60±4% in afferent arterioles superfused with amiloride or benzamil, and perfused with amiloride, respectively. These responses were similar to responses obtained from control afferent arterioles (64±6%, P>0.05). Immunofluorescence revealed expression of the &agr;, &bgr;, and &ggr; subunits of ENaC in freshly isolated preglomerular microvascular smooth muscle cells. These results demonstrate that selective ENaC inhibitors attenuate afferent arteriolar myogenic responses and suggest that ENaC may function as mechanosensitive ion channels initiating pressure-dependent myogenic responses in rat juxtamedullary afferent arterioles.

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.

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.

Mycophenolate mofetil prevents high‐fat diet–induced hypertension and renal glomerular injury in Dahl SS rats

We designed experiments to test the hypothesis that Dahl salt‐sensitive (SS) rats are sensitive to high‐fat diet (HFD)–induced hypertension and renal injury via an inflammatory mechanism. Twelve‐week‐old Dahl SS rats were maintained on a normal diet (ND; 14% fat), HFD (59% fat), or HFD supplemented with the lymphocyte immunosuppressive agent, mycophenolate mofetil (HFD + MMF; 30 mg/kg/day orally in diet), for a period of 4 weeks. Mean arterial pressure (MAP), metabolic parameters, T lymphocyte (CD3+) localization, and renal structural damage were assessed during the studies. Four weeks of HFD significantly elevated MAP and visceral adiposity without changing circulating levels of lipids or adipokines. Immunohistochemical analysis demonstrated that SS rats on HFD had significantly greater numbers of CD3+ cells in renal glomerular and medullary areas compared to ND SS rats. Additionally, HFD led to increased glomerular injury, but did not alter renal medullary injury. Chronic MMF treatment in HFD‐fed Dahl SS rats reduced MAP, visceral adiposity, infiltration of CD3+ cells in the glomerulus, as well as glomerular injury. However, MMF treatment did not alter HFD‐induced infiltration of CD3+ cells in the renal medulla. In conclusion, Dahl SS rats are sensitized to HFD‐induced hypertension and renal glomerular injury via infiltration of T lymphocytes.

Effects of Hydrogen Ion Dissociation and Concentration on The Reactivity of Dichromate-Fixed Tissue Components to the Pas Procedure: Recommendations for Reducing Undesirable Background Staining

The undesirable PAS reactivity of cytoplasmic aldehydes after dichromate fixation can be suppressed without affecting selective staining by lowering the pH of Lillies Cold Schiff reagent to 1.5. Alternatively, dilution of pH 2.2 Cold Schiff reagent with distilled water (1:2) is recommended. Hydrogen ion concentration and dissociation effect the rate of color formation in various PAS positive sites differentially with respect to the time of incubation in Schiff reagent. Based on the experiments, aldehydes exposed in different tissue components appear to be chemically distinct and separable depending on the rate of color formation and duration of incubation in Schiff reagent.

Response to Role of Epithelial Sodium Channels in the Renal Myogenic Response

We appreciate the comments from Loutzenhiser and Aaronson1 regarding our report2 that afferent arteriolar autoregulation was inhibited by amiloride or benzamil (bath; 10 μmol/L) and by perfusion with 5 μmol/L of amiloride. In both conditions, KCl-induced vasoconstriction was preserved, indicating intact voltage-dependent Ca2+ channel function. With immunofluorescent detection of the epithelial sodium channel (ENaC) subunits (α, β, and γ), the data support the hypothesis that ENaC-like proteins may function as mechanosensitive ion channel complexes transducing myogenic responses. As noted, amiloride and benzamil can inhibit other channels and transporters, but they generally require concentrations greater than those used here. The IC50 for amiloride inhibiting the Na+/H+ exchanger is 84 μmol/L and 1100 μmol/L for the Na+/Ca2+ exchanger.3 Benzamil is 10 …