Huixia Jin

Tumor Necrosis Factor-α Produced in the Kidney Contributes to Angiotensin II–dependent Hypertension

Immune system activation contributes to the pathogenesis of hypertension and the resulting progression of chronic kidney disease. In this regard, we recently identified a role for proinflammatory Th1 T-lymphocyte responses in hypertensive kidney injury. Because Th1 cells generate interferon-&ggr; and tumor necrosis factor-&agr; (TNF-&agr;), we hypothesized that interferon-&ggr; and TNF-&agr; propagate renal damage during hypertension induced by activation of the renin–angiotensin system. Therefore, after confirming that mice genetically deficient of Th1 immunity were protected from kidney glomerular injury despite a preserved hypertensive response, we subjected mice lacking interferon-&ggr; or TNF-&agr; to our model of hypertensive chronic kidney disease. Interferon deficiency had no impact on blood pressure elevation or urinary albumin excretion during chronic angiotensin II infusion. By contrast, TNF-deficient (knockout) mice had blunted hypertensive responses and reduced end-organ damage in our model. As angiotensin II–infused TNF knockout mice had exaggerated endothelial nitric oxide synthase expression in the kidney and enhanced nitric oxide bioavailability, we examined the actions of TNF-&agr; generated from renal parenchymal cells in hypertension by transplanting wild-type or TNF knockout kidneys into wild-type recipients before the induction of hypertension. Transplant recipients lacking TNF solely in the kidney had blunted hypertensive responses to angiotensin II and augmented renal endothelial nitric oxide synthase expression, confirming a role for kidney-derived TNF-&agr; to promote angiotensin II–induced blood pressure elevation by limiting renal nitric oxide generation.

Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor–mediated kidney fibrosis

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 microg) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 microg. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats.

Bladder fibrosis during outlet obstruction is triggered through the NLRP3 inflammasome and the production of IL-1β.

Bladder outlet obstruction (BOO) triggers inflammation in the bladder through the NLRP3 inflammasome. BOO also activates fibrosis, which is largely responsible for the decompensation of the bladder in the chronic state. Because fibrosis can be driven by inflammation, we have explored a role for NLRP3 (and IL-1β produced by NLRP3) in the activation and progression of BOO-induced fibrosis. Female rats were divided into five groups: 1) control, 2) sham, 3) BOO + vehicle, 4) BOO + the NLRP3 inhibitor glyburide, or 5) BOO + the IL-1β receptor antagonist anakinra. Fibrosis was assessed by Massons trichrome stain, collagen secretion via Sirius Red, and protein localization by immunofluorescence. BOO increased collagen production in the bladder, which was blocked by glyburide and anakinra, clearly implicating the NLRP3/IL-1β pathway in fibrosis. The collagen was primarily found in the lamina propria and the smooth muscle, while IL-1 receptor 1 and prolyl 4-hydroylase (an enzyme involved in the intracellular modification of collagen) both localized to the urothelium and the smooth muscle. Lysyl oxidase, the enzyme involved in the final extracellular assembly of mature collagen fibrils, was found to some extent in the lamina propria where its expression was greatly enhanced during BOO. In vitro studies demonstrated isolated urothelial cells from BOO rats secreted substantially more collagen than controls, and collagen expression in control cultures could be directly stimulated by IL-1β. In summary, NLRP3-derived-IL-1β triggers fibrosis during BOO, most likely through an autocrine loop in which IL-1β acts on urothelia to drive collagen production.

The effects of Ins2(Akita) diabetes and chronic angiotensin II infusion on cystometric properties in mice

Diabetes is associated with both dysfunction of the lower urinary tract (LUT) and overactivity of the renin‐angiotensin system (RAS). Although it is well known that the RAS affects normal LUT function, very little is known about RAS effects on the diabetic LUT. Accordingly, we investigated the effects of chronic angiotensin II (AngII) treatment on the LUT in a model of type 1 diabetes.

NLRP3/IL-1β mediates denervation during bladder outlet obstruction in rats

Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3‐mediated inflammation on bladder denervation during BOO.

MP38-05 NLRP3 INFLAMMASOME MEDIATED INFLAMMATION AND DENERVATION ARE CRITICAL PROCESSES IN THE DEVELOPMENT OF DIABETIC BLADDER DYSFUNCTION

METHODS: Primary rat bladder cell culture was used to evaluate the effect of bAR agonist and mAchR antagonist on extracellular matrix (ECM) production, oxidative stress and EMT markers in hypoxic environment. In the animal study, we used the well-established partial pelvic ischemia rat model by ligation of the common iliac artery. Rats were divided into five groups, including control group, ischemic bladder (IB) group, bAR agonist treated IB group, mAchR non-selective antagonist treated IB group, and mAchR selective antagonist treated IB group. Bladders were later be retrieved for evaluation of oxidative stress, along with ECM deposition and EMT markers RESULTS: In cell line study, response to hypoxia of bladder mucosa cells would be modified by both bAR agonist and mAchR antagonist. Decreased HIF-1a was found in treated cells, even after hypoxia. However, cells would demonstrate prominent EMT with collagen deposition, especially when treated with bAR agonist. In animal study, total glutathione can be a good indicator of ischemic severity. Both bAR agonist and mAchR antagonist can help improve urodynamic parameters from IB. This correlates with EMT after ischemic change but not with collagen and TGF-b expression. Bladder remodeling seemed to be associated more with selective mAchR antagonist treatment. Pathological evidence of ECM deposition can also be found in immunohistochemical stain of bladder. CONCLUSIONS: Ischemia does affect EMT status of bladder, which can be modified by both bAR agonist and mAchR antagonist. But it does not correlate well with collagen expression. This is more related with TGF-b, which is predominantly affected by types of treatment. The selective mAchR antagonist, Tolterodine, induced the strongest TGF-b expression in our study, which is followed by collagen I/ III expression. Besides, both types of OAB treatment may affect TGF-b and collagen expression by themselves.

Bladder decompensation and reduction in nerve density in a rat model of chronic bladder outlet obstruction is attenuated with the NLRP3 inhibitor glyburide

Bladder outlet obstruction (BOO) leads to progressive voiding dysfunction. Acutely, obstruction triggers inflammation that drives bladder dysfunction. Over time, inflammation leads to decreased bladder nerve density and increased fibrosis, responsible for eventual decompensation and irreversibility. We have previously shown that BOO triggers inflammation, reduced bladder nerve density and increased fibrosis via activation of the NLRP3 inflammasome in an acutely obstructed (12-day) rat model. However, as BOO progresses, the bladder may become decompensated with an increase in postvoid residual volume and decreased voiding efficiency. Currently, we have examined rat bladder function and nerve densities after chronic BOO to determine whether NLRP3 plays a role in the decompensation at this stage. Four groups were examined: control, sham-operated, BOO, or BOO+gly (glyburide; an NLRP3 inhibitor). After 42 days, bladder weight, inflammation (Evans blue), urodynamics, and nerve density were measured. BOO greatly enhanced bladder weights and inflammation, while inflammation was prevented by glyburide. Voiding pressures were increased, and flow rates decreased in BOO and BOO+gly groups, demonstrating physical obstruction. No difference in frequency or voided volume was detected. However, postvoid residual volumes were greatly increased in BOO rats while BOO+gly rats were not different than controls. Moreover, there was a dramatic decrease in voiding efficiency in the chronic BOO rats, which was prevented with glyburide treatment. Finally, a reduction in nerve density was apparent with BOO and attenuated with glyburide. Together the results suggest a critical role for NLRP3 in mediating bladder decompensation and nerve density during chronic BOO.

TNF-alpha Produced in the Kidney Contributes to Angiotensin II-dependent Hypertension

Immune system activation contributes to the pathogenesis of hypertension and the resulting progression of chronic kidney disease. In this regard, we recently identified a role for proinflammatory Th1 T-lymphocyte responses in hypertensive kidney injury. Because Th1 cells generate interferon-&ggr; and tumor necrosis factor-&agr; (TNF-&agr;), we hypothesized that interferon-&ggr; and TNF-&agr; propagate renal damage during hypertension induced by activation of the renin–angiotensin system. Therefore, after confirming that mice genetically deficient of Th1 immunity were protected from kidney glomerular injury despite a preserved hypertensive response, we subjected mice lacking interferon-&ggr; or TNF-&agr; to our model of hypertensive chronic kidney disease. Interferon deficiency had no impact on blood pressure elevation or urinary albumin excretion during chronic angiotensin II infusion. By contrast, TNF-deficient (knockout) mice had blunted hypertensive responses and reduced end-organ damage in our model. As angiotensin II–infused TNF knockout mice had exaggerated endothelial nitric oxide synthase expression in the kidney and enhanced nitric oxide bioavailability, we examined the actions of TNF-&agr; generated from renal parenchymal cells in hypertension by transplanting wild-type or TNF knockout kidneys into wild-type recipients before the induction of hypertension. Transplant recipients lacking TNF solely in the kidney had blunted hypertensive responses to angiotensin II and augmented renal endothelial nitric oxide synthase expression, confirming a role for kidney-derived TNF-&agr; to promote angiotensin II–induced blood pressure elevation by limiting renal nitric oxide generation.

Tumor Necrosis Factor-α Produced in the Kidney Contributes to Angiotensin II–dependent HypertensionNovelty and Significance

Immune system activation contributes to the pathogenesis of hypertension and the resulting progression of chronic kidney disease. In this regard, we recently identified a role for proinflammatory Th1 T-lymphocyte responses in hypertensive kidney injury. Because Th1 cells generate interferon-&ggr; and tumor necrosis factor-&agr; (TNF-&agr;), we hypothesized that interferon-&ggr; and TNF-&agr; propagate renal damage during hypertension induced by activation of the renin–angiotensin system. Therefore, after confirming that mice genetically deficient of Th1 immunity were protected from kidney glomerular injury despite a preserved hypertensive response, we subjected mice lacking interferon-&ggr; or TNF-&agr; to our model of hypertensive chronic kidney disease. Interferon deficiency had no impact on blood pressure elevation or urinary albumin excretion during chronic angiotensin II infusion. By contrast, TNF-deficient (knockout) mice had blunted hypertensive responses and reduced end-organ damage in our model. As angiotensin II–infused TNF knockout mice had exaggerated endothelial nitric oxide synthase expression in the kidney and enhanced nitric oxide bioavailability, we examined the actions of TNF-&agr; generated from renal parenchymal cells in hypertension by transplanting wild-type or TNF knockout kidneys into wild-type recipients before the induction of hypertension. Transplant recipients lacking TNF solely in the kidney had blunted hypertensive responses to angiotensin II and augmented renal endothelial nitric oxide synthase expression, confirming a role for kidney-derived TNF-&agr; to promote angiotensin II–induced blood pressure elevation by limiting renal nitric oxide generation.