Hindbrain Administration of Oxytocin Reduces Food Intake, Weight Gain and Activates Catecholamine Neurons in the Hindbrain Nucleus of the Solitary Tract in Rats.

Existing studies show that CNS oxytocin (OT) signaling is important in the control of energy balance, but it is unclear which neurons may contribute to these effects. Our goals were to examine (1) the dose-response effects of acute OT administration into the third (3V; forebrain) and fourth (4V; hindbrain) ventricles to assess sensitivity to OT in forebrain and hindbrain sites, (2) the extent to which chronic 4V administration of OT reduces weight gain associated with the progression of diet-induced obesity, and (3) whether nucleus tractus solitarius (NTS) catecholamine neurons are downstream targets of 4V OT. Initially, we examined the dose-response effects of 3V and 4V OT (0.04, 0.2, 1, or 5 µg). 3V and 4V OT (5 µg) suppressed 0.5-h food intake by 71.7 ± 6.0% and 60 ± 12.9%, respectively. 4V OT (0.04, 0.2, 1 µg) reduced food intake by 30.9 ± 12.9, 42.1 ± 9.4, and 56.4 ± 9.0%, respectively, whereas 3V administration of OT (1 µg) was only effective at reducing 0.5-h food intake by 38.3 ± 10.9%. We subsequently found that chronic 4V OT infusion, as with chronic 3V infusion, reduced body weight gain (specific to fat mass) and tended to reduce plasma leptin in high-fat diet (HFD)-fed rats, in part, through a reduction in energy intake. Lastly, we determined that 4V OT increased the number of hindbrain caudal NTS Fos (+) neurons (156 ± 25) relative to vehicle (12 ± 3). The 4V OT also induced Fos in tyrosine hydroxylase (TH; marker of catecholamine neurons) (+) neurons (25 ± 7%) relative to vehicle (0.8 ± 0.3%). Collectively, these findings support the hypothesis that OT within the hindbrain is effective at reducing food intake, weight gain, and adiposity and that NTS catecholamine neurons in addition to non-catecholaminergic neurons are downstream targets of CNS OT.

Effects of Combined Oxytocin and Beta-3 Receptor Agonist (CL 316243) Treatment on Body Weight and Adiposity in Male Diet-Induced Obese Rats.

Previous studies have indicated that oxytocin (OT) reduces body weight in diet-induced obese (DIO) rodents through reductions in energy intake and increases in energy expenditure. We recently demonstrated that hindbrain [fourth ventricular (4V)] administration of OT evokes weight loss and elevates interscapular brown adipose tissue temperature (T IBAT ) in DIO rats. What remains unclear is whether OT can be used as an adjunct with other drugs that directly target beta-3 receptors in IBAT to promote BAT thermogenesis and reduce body weight in DIO rats. We hypothesized that the combined treatment of OT and the beta-3 agonist, CL 316243, would produce an additive effect to decrease body weight and adiposity in DIO rats by reducing energy intake and increasing BAT thermogenesis. We assessed the effects of 4V infusions of OT (16 nmol/day) or vehicle (VEH) in combination with daily intraperitoneal injections of CL 316243 (0.5 mg/kg) or VEH on food intake, T IBAT , body weight and body composition. OT and CL 316243 alone reduced body weight by 7.8 ± 1.3% (P < 0.05) and 9.1 ± 2.1% (P < 0.05), respectively, but the combined treatment produced more pronounced weight loss (15.5 ± 1.2%; P < 0.05) than either treatment alone. These effects were associated with decreased adiposity, adipocyte size, energy intake and increased uncoupling protein 1 (UCP-1) content in epididymal white adipose tissue (EWAT) (P < 0.05). In addition, CL 316243 alone (P < 0.05) and in combination with OT (P < 0.05) elevated T IBAT and IBAT UCP-1 content and IBAT thermogenic gene expression. These findings are consistent with the hypothesis that the combined treatment of OT and the beta-3 agonist, CL 316243, produces an additive effect to decrease body weight. The findings from the current study suggest that the effects of the combined treatment on energy intake, fat mass, adipocyte size and browning of EWAT were not additive and appear to be driven, in part, by transient changes in energy intake in response to OT or CL 316243 alone as well as CL 316243-elicited reduction of fat mass and adipocyte size and induction of browning of EWAT.

Beneficial effect on podocyte number in experimental diabetic nephropathy resulting from combined atrasentan and RAAS inhibition therapy.

Podocyte loss and proteinuria are both key features of human diabetic nephropathy (DN). The leptin-deficient BTBR mouse strain with the ob/ob mutation develops progressive weight gain, type 2 diabetes, and diabetic nephropathy that has many features of advanced human DN, including increased mesangial matrix, mesangiolysis, podocyte loss, and proteinuria. Selective antagonism of the endothelin-1 type A receptor (ETAR) by atrasentan treatment in combination with renin-angiotensin-aldosterone system inhibition with losartan has been shown to have the therapeutic benefit of lowering proteinuria in patients with DN, but the underlying mechanism for this benefit is not well understood. Using a similar therapeutic approach in diabetic BTBR ob/ob mice, this treatment regimen significantly increased glomerular podocyte number compared with diabetic BTBR ob/ob controls and suggested that parietal epithelial cells were a source for podocyte restoration. Atrasentan treatment alone also increased podocyte number but to a lesser degree. Mice treated with atrasentan demonstrated a reduction in proteinuria, matching the functional improvement reported in humans. This is a first demonstration that treatment with the highly selective ETAR antagonist atrasentan can lead to restoration of the diminished podocyte number characteristic of DN in humans and thereby underlies the reduction in proteinuria in patients with diabetes undergoing similar treatment. The benefit of ETAR antagonism in DN extended to a decrease in mesangial matrix as measured by a reduction in accumulations of collagen type IV in both the atrasentan and atrasentan + losartan-treated groups compared with untreated controls.

Histopathology of Veins Obtained at Hemodialysis Arteriovenous Fistula Creation Surgery.

Stenosis from venous neointimal hyperplasia is common in native arteriovenous fistulas (AVFs). However, the preexisting histologic characteristics of veins at fistula creation, and associations thereof with baseline patient factors, have not been well characterized. In this study, we conducted histologic analysis of a segment of the vein used for anastomosis creation, obtained during AVF creation from 554 of the 602 participants in the multicenter Hemodialysis Fistula Maturation Cohort Study. We quantified intimal and medial areas and lengths of the internal and external elastic lamina by morphometry and assessed venous wall cells by immunohistochemistry, extracellular matrix with Movat stain, and calcium deposition by alizarin red stain. We also studied a representative subset of veins for markers of monocyte/macrophage content, cell proliferation, apoptosis, and neoangiogenesis. Neointima occupied >20% of the lumen in 57% of fully circumferential vein samples, and neointimal hyperplasia associated positively with age and inversely with black race. The neointima was usually irregularly thickened, sometimes concentric, and contained α-smooth muscle actin-expressing cells of smooth muscle or myofibroblast origin. Proteoglycans admixed with lesser amounts of collagen constituted the predominant matrix in the neointima. In 82% of vein samples, the media of vessel walls contained large aggregates of collagen. A minority of veins expressed markers of inflammation, cell proliferation, cell death, calcification, or neoangiogenesis. In conclusion, we observed preexisting abnormalities, including neointimal hyperplasia and prominent accumulation of extracellular matrix, in veins used for AVF creation from a substantial proportion of this cohort.

Macrophages are essential contributors to kidney injury in murine cryoglobulinemic membranoproliferative glomerulonephritis.

Mice transgenic for thymic stromal lymphopoietin (TSLP), under regulation of the lymphocyte-specific promoter Lck, develop cryoglobulinemia and membranoproliferative glomerulonephritis (MPGN) similar to the disease in patients. To determine whether infiltrating macrophages, a hallmark of this disease, are deleterious or beneficial in the injury process, we developed Lck-TSLP transgenic mice expressing the human diphtheria toxin receptor (DTR) under control of the monocyte/macrophage-restricted CD11b promoter (Lck-TSLP;CD11b-DTR). Treatment with DT resulted in a marked reduction of monocytes/macrophages in the peritoneal cavity of both CD11b-DTR and Lck-TSLP;CD11b-DTR mice and marked reduction of macrophage infiltration in glomeruli of Lck-TSLP;CD11b-DTR mice. Lck-TSLP;CD11b-DTR mice, with or without toxin treatment, had similar levels of cryoglobulinemia and glomerular immunoglobulin deposition as Lck-TSLP mice. Lck-TSLP;CD11b-DTR mice, treated with toxin, had reduced mesangial matrix expansion, glomerular collagen IV accumulation, expression of the activation marker α-smooth muscle actin and transforming growth factor-ß1 in mesangial cells, and proteinuria compared with control mice. Thus, macrophage ablation confers protection in this model and indicates a predominately deleterious role for macrophages in the progression of kidney injury in cryoglobulinemic MPGN.

Deletion of activating Fcgamma receptors does not confer protection in murine cryoglobulinemia-associated membranoproliferative glomerulonephritis.

Many types of glomerulonephritis are initiated by the deposition of immune complexes, which induce tissue injury via either engagement of Fc receptors on effector cells or via complement activation. Four murine Fcgamma receptors (FcgammaRs) have been identified at present. Ligand binding to FcgammaRI, III, and IV induces cell activation via the immunoreceptor tyrosine-based activation motif on the common gamma chain (FcRgamma). In this study, FcRgamma chain knockout (FcRgamma(-/-)) mice were crossed with thymic stromal lymphopoietin transgenic (TSLPtg) mice, which develop cryoglobulinemic membranoproliferative glomerulonephritis (MPGN). Female mice were studied at 30 and 50 days of age, when MPGN is in early and fully developed stages, respectively. Both TSLPtg and TSLPtg/FcRgamma(-/-) mice developed MPGN with massive glomerular immune deposits, mesangial cell proliferation, extensive mesangial matrix accumulation, and macrophage influx. TSLPtg/FcRgamma(-/-) mice had more glomerular immune complex deposits and higher levels of circulating cryoglobulins, IgG2a, IgG2b, and IgM, compared with TSLPtg mice. TSLPtg and TSLPtg/FcRgamma(-/-) mice developed similar levels of proteinuria. These results demonstrated that deletion of activating FcgammaRs does not confer protection in this model of immune complex-mediated MPGN. The findings contradict accepted paradigms on the role of activating FcgammaRs in promoting features of glomerulonephritis as seen in other model systems. We speculate engagement of FcgammaRs on cells such as monocytes/macrophages may be important for the clearance of deposited immune complexes and extracellular matrix proteins.

Imatinib suppresses cryoglobulinemia and secondary membranoproliferative glomerulonephritis.

Imatinib is a receptor tyrosine kinase inhibitor that blocks the activity of c-Abl, c-Kit, and PDGF receptors. We tested the protective effects of imatinib in thymic stromal lymphopoietin transgenic mice, a model of cryoglobulinemia and associated membranoproliferative glomerulonephritis (MPGN), in which some glomerular manifestations likely result from PDGF receptor activation. Surprising, administration of imatinib beginning at weaning suppressed production of cryoglobulin, attenuating both the renal injury and systemic features of cryoglobulinemia. Flow cytometry suggested that inhibition of B cell development in the bone marrow likely caused the reduction in cryoglobulin production. In addition, administration of imatinib to thymic stromal lymphopoietin transgenic mice with established MPGN also diminished cryoglobulin production and reversed the renal and systemic lesions. These data suggest that treatment with imatinib may be a novel therapeutic approach for cryoglobulinemia and MPGN in humans.

Renin-angiotensin system blockade is renoprotective in immune complex-mediated glomerulonephritis.

Blockade of the renin-angiotensin system is renoprotective in a variety of chronic nephropathies, but the direct effect of such treatment in active, immune complex-mediated glomerulonephritis is unknown. This study investigated the short- and long-term effects of an angiotensin-converting enzyme inhibitor (enalapril) and an angiotensin II type 1 receptor blocker (losartan) in thymic stromal lymphopoietin transgenic (TSLPtg) mice, which develop mixed cryoglobulinemia and severe cryoglobulinemia-associated membranoproliferative glomerulonephritis. Enalapril and losartan each reduced hypertension, proteinuria, glomerular extracellular matrix deposition, and mesangial cell activation in TSLPtg mice. These renoprotective effects were not observed with hydralazine treatment, despite a similar antihypertensive effect. Treatment with enalapril or losartan also decreased renal plasminogen activator inhibitor-1 in TSLPtg mice, assessed by immunohistochemistry and quantitative real-time reverse transcriptase-PCR. None of the treatments affected immune complex deposition or macrophage infiltration. Overall, enalapril- and losartan-treated TSLPtg mice survived significantly longer than untreated TSLPtg mice. These studies demonstrate that angiotensin blockade may provide renoprotective benefits, independent of its BP-lowering effect, in the treatment of active immune complex-mediated glomerulonephritis.

TLR4 links podocytes with the innate immune system to mediate glomerular injury.

Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN; mice transgenic for thymic stromal lymphopoietin [TSLP], with or without deletion of the Fcgamma receptor IIb). Expression of TLR1 through 9 and TLR11 mRNA was detectable in whole kidneys and in isolated glomeruli of wild-type mice, with TLR3 and TLR4 having the highest absolute levels of expression. TLR1, 2, and 4 were increased in TSLP transgenic mice and even higher in TSLP transgenic FcgammaRIIb-deficient mice. TLR5 through 9 and 11 were upregulated to similar degrees in TSLP transgenic and TSLP transgenic FcgammaRIIb-deficient mice. Immunohistochemical studies of nephritic glomeruli localized TLR4 protein to podocytes. Cultured podocytes also expressed TLR4, and stimulation with TLR4-specific ligands resulted in a marked induction of chemokines; this was reduced by specific knockdown of TLR4 with siRNA. Fibrinogen, a potential endogenous TLR4 ligand, was shown to induce a similar profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered by the deposition of immune complexes.

Identification of platelet-derived growth factor D in human chronic allograft nephropathy.

Chronic allograft nephropathy (CAN), a descriptive term denoting chronic scarring injury of the renal parenchyma and vasculature in allograft kidneys arising from various etiologies including chronic rejection, is the most common cause of late allograft failure, but mediators of this progressive injury largely remain unknown. We hypothesized that platelet-derived growth factor D (PDGF-D) and its specific receptor PDGF-Rbeta may be an important mediator in the pathogenesis of CAN and, hence, sought to identify its expression in this setting. Allograft nephrectomies demonstrating CAN, obtained from patients with irreversible transplant kidney failure (n = 15), were compared with renal tissues without prominent histopathological abnormalities (n = 18) and a series of renal allograft biopsies demonstrating acute vascular rejection (AVR) (n = 12). Antibodies to PDGF-D and PDGF-Rbeta were used for immunohistochemistry. Double and triple immunohistochemistry was used to identify cell types expressing PDGF-D. PDGF-D was widely expressed in most neointimas in arteries exhibiting the chronic arteriopathy of CAN and only weakly expressed in a small proportion of sclerotic arteries in the other 2 groups. Double and triple immunolabeling demonstrated that the neointimal cells expressing PDGF-D were alpha-smooth muscle actin-expressing cells, but not infiltrating macrophages or endothelial cells. PDGF-Rbeta expression evaluated in serial sections was localized to the same sites where neointimal PDGF-D was expressed. PDGF-Rbeta was expressed in interstitial cells more abundantly in the CAN group compared with the normal and AVR groups, without demonstrable colocalization of PDGF-D. PDGF-D is present in the neointima of the arteriopathy of CAN, where it can engage PDGF-Rbeta to promote mesenchymal cell migration, proliferation, and neointima formation. PDGF-D may engage the PDGF-Rbeta to promote interstitial injury in chronic allograft injury, but its sources within the interstitium were unidentified.

All-trans-retinoic acid aggravates cryoglobulin-associated membranoproliferative glomerulonephritis in mice.

BACKGROUND: Transgenic (tg) mice overexpressing thymic stromal lymphopoietin (TSLP) develop mixed cryoglobulinaemia with renal disease closely resembling human cryoglobulinaemic membranoproliferative glomerulonephritis (MPGN), as well as systemic inflammation involving lung, liver and skin as a result of cryoglobulin deposits. We assessed the effect of all-trans-retinoic acid (ATRA), a powerful anti-inflammatory agent, on this model of cryoglobulinaemic MPGN. METHODS: Groups of male TSLP tg mice and wild-type controls were treated with either ATRA (20 mg/kg) or vehicle 3 times weekly by intraperitoneal injection for 4 or 8 weeks, when mice were then sacrificed. Routine histology and immunohistochemistry for collagen IV, alpha-smooth muscle actin, Mac-2 and Ki67 were performed. Immunoglobulin levels were measured by enzyme-linked immunosorbent assay. RESULTS: ATRA unexpectedly exacerbated renal injury in TSLP tg mice with increased glomerular extracellular matrix, mesangial cell activation, glomerular cell proliferation, glomerular macrophage influx and immune complex deposition. Systemic injuries involving liver and lung, and the amount of circulating cryoglobulins were all worsened by ATRA treatment. Furthermore, ATRA resulted in increased IgG1 and IgM levels, the main components of the cryoglobulins in TSLP tg mice, and a manifestation of an enhanced Th2 immune response. CONCLUSIONS: ATRA is not protective but instead aggravates cryoglobulinaemic MPGN and its systemic manifestations in TSLP tg mice. We speculate these findings may be due to augmented production of pathogenic immunoglobulins and/or an enhanced systemic Th2 response. Although disappointing, our results also suggest caution in the application of retinoid therapy to human disease based on the largely positive animal data reported to date.

Localization of TGF-beta signaling intermediates Smad2, 3, 4, and 7 in developing and mature human and mouse kidney.

Smad proteins are signaling intermediates of the TGF-beta superfamily and are involved in a range of biological activities including development and immune responses. We studied the expression of TGF-beta-receptor activated Smads (Smad2 and Smad3), the common partner Smad (Smad4), an inhibitory Smad (Smad7), and the activated (phosphorylated) Smad2 (pSmad2) in developing and adult kidneys of humans and mice. These studies demonstrate associated expression of these Smads in multiple renal cell types in all developmental stages and in mature non-diseased kidneys. Smad expression is in general most widespread at the earliest stages of nephron development and diminishes as components of the nephrons become more differentiated. Paucity of Smad expression in mesangial cells in contrast to widespread expression of these Smads in glomerular visceral epithelial cells in both developing and mature kidneys was remarkable. Divergent and less extensive expression of Smad4, compared with other Smad proteins, was also demonstrated in tubules of human kidneys. Based on the observed expression patterns, these findings demonstrate, for the first time, expression of the TGF-beta-receptor-activated Smad2 and Smad3, the common mediator Smad4, and the inhibitory Smad7 in the developing human fetal kidney, extending observations previously made in rodent systems to humans.