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Blood pressure impacts the renal T cell profile of male and female spontaneously hypertensive ratsOf the 68 million Americans with hypertension, fewer than 46% have their blood pressure (BP) adequately controlled and women are more likely than men to have uncontrolled hypertension. This underscores the critical need for new treatment options; however, this is a challenge due to our lack of knowledge regarding the mechanism(s) driving essential hypertension. T cells have been implicated in hypertension in males. Prior to our work, the role of T cells in hypertensive females had been unexplored. We demonstrate that female spontaneously hypertensive rats (SHR) have a decrease in BP in response to an immunosuppressant, supporting an immune component to their hypertension. We further defined a sex difference in the renal T cell and cytokine profile in SHR. Female SHR have a more anti-inflammatory immune profile in their kidneys than males. To gain insight into the mechanisms mediating sex differences in the immune profile, male and female SHR were gonadectomized. Gonadectomy increased pro-inflammatory markers in both sexes and attenuated anti-inflammatory markers particularly in females. Therefore, while both male and female sex hormones promote an anti-inflammatory immune profile, female ii sex hormones contribute greater to their more anti-inflammatory profile, but do not explain the sex difference. To determine the impact of hypertension on the renal immune profile, experiments measured renal T cells and cytokines in hypertensive male and female SHR, normotensive Wistar Kyoto rats (WKY), and SHR treated with antihypertensive therapy. All T cells and cytokines measured were higher in SHR compared to the same sex WKY. Moreover, antihypertensive therapy decreased renal Tregs only in female SHR. These data suggest that increased BP in both sexes is associated with an increase in renal inflammation; however female SHR have a compensatory increase in renal Tregs in response to increases in BP. TGF-β is a key cytokine regulating Treg and Th17 differentiation and we found that female SHR express more TGF-β than males. Experiments assessed if female SHR possessed a sex hormone or BP-mediated increase in renal TGF- β corresponding with increases in Tregs. We determined that loss of female sex hormones and increased BP in female SHR increase renal TGF-β expression. We conclude that BP status drive sex differences in the renal T cell and cytokine profile of SHR.
Differential effects of taurine treatment and taurine deficiency on the outcome of renal ischemia reperfusion injuryTaurine possesses membrane stabilization, osmoregulatory and antioxidant properties, aspects of relevance to ischemic injury. We tested the hypothesis that body taurine status is a determinant of renal ischemic injury. Accordingly, renal function and structure were examined in control (C), taurine-treated (TT) and taurine deficient (TD) rats that were subjected to bilateral renal ischemia (60 min) followed by reperfusion (IR); sham operated rats served as controls. Baseline urine osmolality was greater in the TD group than in the control and the TT groups, an effect associated with increased renal aquaporin 2 level. The IR insult reduced urine osmolality (i.e., day-1 post insult); the TD/IR group displayed a more marked recovery in urine osmolality by day-6 post insult than the other two groups. Fluid and sodium excretions were lower in the TD/IR group, suggesting propensity to retention. Histopathological examination revealed the presence of tubular necrotic foci in the C/IR group than sham controls. While renal architecture of the TD/IR group showed features resembling sham controls, the TT/IR group showed dilated tubules, which lacked immunostaining for aquaporin 2, but not 1, suggestive of proximal tubule origin. Finally, assessment of cell proliferation and apoptosis revealed lower proliferation but higher apoptotic foci in the TT/IR group than other IR groups. Collectively, the results indicate that body taurine status is a major determinant of renal IR injury.
Role of Endothelin-1 (ET-1) in Glomerular Inflammation and Glomerular Permeability in Normal and Diabetic KidneysEndothelin-1 (ET-1) is a potent vasoactive peptide implicated in the pathogenesis of hypertension and renal disease. The overall specific aim of this dissertation is to investigate the role of ET-1 in mediating glomerular inflammation and permeability, especially in diseases characterized by high activity of the ET-1 system, such as diabetic nephropathy. The first study was designed to test the hypotheses that ET-1 increases albumin permeability of glomeruli isolated from normal rats and that chronic ET-1 infusion will increase glomerular permeability and inflammation independent of blood pressure. Glomerular permeability to albumin (Palb) was determined from the change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients. Incubation of glomeruli taken from normal rats with ET-1 at a concentration that did not produce direct glomerular contraction (1 nM) significantly increased Palb, reaching a maximum after 4 hrs. Chronic ET-1 infusion for 2 weeks in Sprague-Dawley (SD) rats significantly increased Palb and nephrin excretion rate, effects that were attenuated in rats given an ETA receptor antagonist, ABT-627. Urinary protein and albumin excretion and mean arterial pressure (telemetry) were not changed by ET-1 infusion. Acute incubation of glomeruli isolated from ET-1-infused rats with the selective ETA antagonist significantly reduced Palb, an effect not observed with acute treatment with a selective ETB antagonist. Chronic ET-1 infusion increased glomerular and plasma sICAM-1 and MCP-1 and elevated the number of macrophages and lymphocytes in renal cortices (CD68- and CD3-positive staining, respectively). These effects were all attenuated in rats given an ETA selective antagonist. These data support the hypothesis that ET-1 directly increases glomerular permeability to albumin and renal inflammation via ETA receptor activation independent of changes in arterial pressure. The second study was designed to test the hypothesis that ETA receptor activation increases Palb and elevates pro-inflammatory markers in hyperglycemic rats. Male SD rats were given streptozotocin (STZ) or saline (sham). Half of the animals in each group received ABT- 627 beginning immediately after hyperglycemia had been confirmed. Glomeruli were isolated by sieving and Palb determined from the change in glomerular volume induced by exposing glomeruli to oncotic gradients of albumin. Glomerular nephrin expression was assessed by immunofluorescence, whereas urinary nephrin was measured by enzymelinked immunosorbent assay. Three and 6 weeks after STZ injection, proteinuria was significantly increased compared to sham controls and was significantly reduced by ABT-627 treatment. Palb was also increased at 3 and 6 wk post-STZ; ABT-627 had no effect on Palb or protein excretion in sham rats. Glomerular and plasma content of sICAM-1 and MCP-1 were significantly increased 6 wk after STZ. ABT-627 attenuated these increases. After 6 weeks of hyperglycemia, glomerular nephrin expression was decreased with a concurrent increase in urinary nephrin excretion; ABT-627 prevented glomerular nephrin loss in the hyperglycemic rats. These observations support the hypothesis that ET-1, via the ETA receptor, mediates the increase in proteinuria and Palb, possibly via nephrin loss, as well as early inflammation in the hyperglycemic rat. In the third study, we determined the actions of ETA and ETB receptors on measures of glomerular function and renal inflammation in the early stages of diabetic renal injury in rats. Six weeks after STZ-induced hyperglycemia, rats were given ABT-627 (5 mg/kg/d) a selective ETA antagonist; A-182086 (10 mg/kg/d), a combined ETA/B antagonist; or vehicle for 1 week. Sham controls received STZ vehicle (saline). Hyperglycemia led to significant proteinuria, increased Palb, nephrinuria, and an increase in total matrix metalloprotease (MMPs) and transforming growth factor-beta 1 (TGF-β1) activities in glomeruli. Plasma and glomerular sICAM-1 and MCP-1 were elevated after 7 weeks of hyperglycemia. Daily administration of both ABT-627 and A-182086 for 1 week significantly attenuated proteinuria, the increase in Palb, nephrinuria, and total MMPs and TGF-β1 activity. However, glomerular sICAM-1 and MCP-1 expression was attenuated with ABT-627, but not A-182086 treatment. In summary, both selective ETA and combined ETA/B antagonists reduced proteinuria, glomerular permeability and restored glomerular filtration barrier components integrity, but only ETA selective blockade had anti-inflammatory and anti-fibrotic effects. We conclude that selective ETA antagonists are more likely to be preferred for treatment of diabetic kidney disease.
Sex Differences in Renal Inner Medullary Nitric Oxide Synthase Regulation and Nitric Oxide Synthase Contribution to Blood Pressure Control in HypertensionThere are sex differences in the development of hypertension with young males developing a more severe pathology faster than age-matched females; however, with advancing age this “protection” in females is lost. The mechanisms responsible for the sex difference in hypertension are unclear but the vasodilator nitric oxide (NO)/NO synthase (NOS) pathway which is important in blood pressure (BP) regulation has been implicated. Systemic inhibition of NOS using L-NAME (2, 5, and 7 mg/kg/day at 4 days per dose in drinking water) in male and female spontaneously hypertensive rats (SHR) resulted in dose-dependent increases in BP measured via telemetry; however, females exhibited greater increases in BP than males. Treatment of male and female SHR chronically with L-NAME at a dose of 7 mg/kg/day for 2 weeks significantly increased BP in both sexes, however, a previous exposure to L-NAME increased BP sensitivity to chronic NOS inhibition in females exclusively; this confirmed our hypothesis that female SHR are more dependent on NOS for BP control compared to male. Important for BP control, the renal inner medulla (IM) is the only region of the kidney to exhibit sex differences in NOS enzymatic activity. Female SHR have greater total NOS activity than males and we observed that it is not due to differences in phosphorylation or protein expression. Therefore, we examined potential molecular mechanisms to explain the sexual dimorphism in renal IM NOS activity. The endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) has been indicated in hypertension. However, HPLC analysis of ADMA and the essential NOS substrate L-arginine were equal between the sexes in plasma and renal IM of SHR and thus do not contribute to the sex differences in renal IM NOS activity. Tetrahydrobiopterin (BH4) is an essential NOS cofactor and decreased BH4 availability has been indicated to be elevated in patients and animal models with essential hypertension. BH4 levels can be decreased via oxidation and male SHR have higher levels of oxidative stress compared to females. HPLC analysis of biopterin levels in control and tempol (antioxidant) treated SHR showed that female SHR have greater total biopterin, BH4 and BH2 levels than males in the renal IM and that these sex differences were dependent on the presence of oxidative stress. Studies next examined if greater biopterin levels in females translated into greater NOS activity in females. In vitro analysis of NOS enzymatic activity confirmed that greater oxidative stress and deficiency of BH4 of male SHR in the renal IM resulted in lower levels of NOS activity relative to female SHR. In addition, in vitro analysis of renal IM NOS activity revealed that 1) female SHR exhibit a sex hormone-dependent increase in renal IM NOS activity from sexually immature, pre-hypertensive age to sexually mature, hypertensive age that is not evident in male SHR and 2) that the ability of female sex hormones to stimulate NOS activity is time-dependent. In conclusion, the combination of BH4 deficiency in males caused by elevated oxidative stress and the ability of female sex hormones to stimulate NOS activity in female SHR and not ADMA or L-arginine, contribute to the sexual dimorphism in renal IM NOS activity. In addition, differences in sensitivity to NOS levels in SHR aid in creating sex differences in BP control.