Yanlin Wang

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2.

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I–IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2‐dependent yet is gp91phox‐independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue‐derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum‐starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis

Bone marrow-derived fibroblasts may contribute substantially to the pathogenesis of renal fibrosis through the excessive production and deposition of extracellular matrix. However, the mechanisms underlying the accumulation and activation of these fibroblasts are not understood. Here, we used a mouse model of tubulointerstitial fibrosis to determine whether adiponectin, which is elevated in CKD and is associated with disease progression, regulates monocyte-to-fibroblast transition and fibroblast activation in injured kidneys. In wild-type mice, the expression of adiponectin and the number of bone marrow-derived fibroblasts in the kidney increased after renal obstruction. In contrast, the obstructed kidneys of adiponectin-knockout mice had fewer bone marrow-derived fibroblasts. Adiponectin deficiency also led to a reduction in the number of myofibroblasts, the expression of profibrotic chemokines and cytokines, and the number of procollagen-expressing M2 macrophages in injured kidneys. Consistent with these findings, adiponectin-deficiency reduced the expression of collagen I and fibronectin. Similar results were observed in wild-type and adiponectin-knockout mice after ischemia-reperfusion injury. In cultured bone marrow-derived monocytes, adiponectin stimulated the expression of α-smooth muscle actin (SMA) and extracellular matrix proteins and activated AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. Furthermore, specific activation of AMPK increased the expression of α-SMA and extracellular matrix proteins, while inhibition of AMPK attenuated these responses. Taken together, these findings identify adiponectin as a critical regulator of monocyte-to-fibroblast transition and renal fibrosis, suggesting that inhibition of adiponectin/AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

Critical Role of CXCL16 in Hypertensive Kidney Injury and Fibrosis

Recent evidence indicates that inflammation plays a critical role in the initiation and progression of hypertensive kidney disease. However, the signaling mechanisms underlying the induction of inflammation are poorly understood. We found that chemokine (C-X-C motif) ligand 16 (CXCL16) was induced in renal tubular epithelial cells in response to angiotensin II in a nuclear factor-&kgr;B–dependent manner. To determine whether CXCL16 plays a role in angiotensin II–induced renal inflammation and fibrosis, wild-type and CXCL16 knockout mice were infused with angiotensin II at 1500 ng/kg per minute for up to 4 weeks. Wild-type and CXCL16 knockout mice had comparable blood pressure at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between wild-type and CXCL16 knockout mice. CXCL16 knockout mice were protected from angiotensin II–induced renal dysfunction, proteinuria, and fibrosis. CXCL16 deficiency suppressed bone marrow–derived fibroblast accumulation and myofibroblast formation in the kidneys of angiotensin II–treated mice, which was associated with less expression of extracellular matrix proteins. Furthermore, CXCL16 deficiency inhibited infiltration of F4/80+ macrophages and CD3+ T cells in the kidneys of angiotensin II–treated mice compared with wild-type mice. Finally, CXCL16 deficiency reduced angiotensin II–induced proinflammatory cytokine expressions in the kidneys. Taken together, our results indicate that CXCL16 plays a pivotal role in the pathogenesis of angiotensin II–induced renal injury and fibrosis through regulation of macrophage and T cell infiltration and bone marrow–derived fibroblast accumulation.

Anti-Inflammatory and Renal Protective Actions of Stanniocalcin-1 in a Model of Anti-Glomerular Basement Membrane Glomerulonephritis

We have previously shown that stanniocalcin-1 (STC1) inhibits the transendothelial migration of macrophages and T cells, suppresses superoxide generation in macrophages, and attenuates macrophage responses to chemoattractants. To study the effects of STC1 on inflammation, in this study we induced a macrophage- and T-cell-mediated model of anti-glomerular basement membrane disease in STC1 transgenic mice, which display elevated serum STC1 levels and preferentially express STC1 in both endothelial cells and macrophages. We examined the following parameters both at baseline and after anti-glomerular basement membrane antibody treatment: blood pressure; C(3a) levels; urine output; proteinuria; blood urea nitrogen; and kidney C(3) deposition, fibrosis, histological changes, cytokine expression, and number of T cells and macrophages. Compared with wild-type mice, after anti-glomerular basement membrane treatment STC1 transgenic mice exhibited: i) diminished infiltration of inflammatory macrophages in the glomeruli; ii) marked reduction in crescent formation and sclerotic glomeruli; iii) decreased interstitial fibrosis; iv) preservation of kidney function and lower blood pressure; v) diminished C(3) deposition in the glomeruli; and vi) reduced expression of macrophage inhibitory protein-2 and transforming growth factor-beta2 in the kidney. Compared with baseline, wild-type mice, but not STC1 transgenic mice, had higher proteinuria and a marked reduction in urine output. STC1 had minimal effects, however, on both T-cell number in the glomeruli and interstitium and on cytokine expression characteristic of either TH1 or TH2 activation. These data suggest that STC1 is a potent anti-inflammatory and renal protective protein.

Genetic deficiency of adiponectin protects against acute kidney injury

Adiponectin is a multifunctional cytokine that has a role in regulating inflammation. Here we determined if adiponectin modulates ischemic acute kidney injury. Compared with wild-type mice, adiponectin knockout mice were found to have lower serum creatinine and less tubular damage or apoptosis following ischemia/reperfusion injury. This latter process was associated with decreased Bax and reduced activation of p53 and caspase-3. Targeted disruption of adiponectin was also found to inhibit the infiltration of neutrophils, macrophages, and T cells into the injured kidneys. This was associated with an inhibition of NF-κB activation and reduced expression of the proinflammatory molecules IL-6, TNF-α, MCP-1, and MIP-2 in the kidney after ischemia/reperfusion injury. Wild-type mice engrafted with adiponectin null bone marrow had less kidney dysfunction and tubular damage than adiponectin null mice engrafted with wild-type bone marrow. Conversely, adiponectin null mice engrafted with wild-type bone marrow had similar renal dysfunction and tubular damage compared to wild-type mice engrafted with wild-type bone marrow. In cultured macrophages, adiponectin directly promoted macrophage migration; a process blocked by the PI3 kinase inhibitor, LY294002. Thus, our results show that adiponectin plays a pivotal role in the pathogenesis of acute renal ischemia/reperfusion injury and may be a potential therapeutic target.

CXCR6 Plays a Critical Role in Angiotensin II–Induced Renal Injury and Fibrosis

Objective—Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II–induced renal injury and fibrosis. Approach and Results—Wild-type and CXCR6-green fluorescent protein (GFP) knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg per minute after unilateral nephrectomy for ⩽4 weeks. Wild-type and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between wild-type and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II–induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow–derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys after Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80+ macrophages and CD3+ T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow–derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment when compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Conclusions—Our results indicate that CXCR6 plays a pivotal role in the development of Ang II–induced renal injury and fibrosis through regulation of macrophage and T-cell infiltration and bone marrow–derived fibroblast accumulation.

The chemokine receptor CXCR6 contributes to recruitment of bone marrow–derived fibroblast precursors in renal fibrosis

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

CCR2 Regulates the Uptake of Bone Marrow-Derived Fibroblasts in Renal Fibrosis

Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment of bone marrow-derived fibroblasts into the kidney are incompletely understood. Bone marrow-derived fibroblasts express the chemokine receptor - CCR2. In this study, we tested the hypothesis that CCR2 participates in the recruitment of fibroblasts into the kidney during the development of renal fibrosis. Bone marrow-derived collagen-expressing GFP+ fibroblasts were detected in the obstructed kidneys of chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP reporter mice. These bone marrow-derived fibroblasts expressed PDGFR-β and CCR2. CCR2 knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors expressing the hematopoietic marker-CD45 and the mesenchymal markers-PDGFR-β or procollagen I in the obstructed kidneys compared with wild-type mice. Furthermore, CCR2 knockout mice displayed fewer bone marrow-derived myofibroblasts and expressed less α-SMA or FSP-1 in the obstructed kidneys compared with wild-type mice. Consistent with these findings, genetic deletion of CCR2 inhibited total collagen deposition and suppressed expression of collagen I and fibronectin. Moreover, genetic deletion of CCR2 inhibits MCP-1 and CXCL16 gene expression associated with a reduction of inflammatory cytokine expression and macrophage infiltration, suggesting a linear interaction between two chemokines/ligand receptors in tubular epithelial cells. Taken together, our results demonstrate that CCR2 signaling plays an important role in the pathogenesis of renal fibrosis through regulation of bone marrow-derived fibroblasts. These data suggest that inhibition of CCR2 signaling could constitute a novel therapeutic approach for fibrotic kidney disease.

JAK3/STAT6 Stimulates Bone Marrow–Derived Fibroblast Activation in Renal Fibrosis

Renal fibrosis is a final common manifestation of CKD resulting in progressive loss of kidney function. Bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. However, the signaling mechanisms underlying the activation of bone marrow-derived fibroblast precursors in the kidney are not fully understood. In this study, we investigated the role of the Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT6) signaling pathway in the activation of bone marrow-derived fibroblasts. In cultured mouse monocytes, IL-4 or IL-13 activated STAT6 and induced expression of α-smooth muscle actin and extracellular matrix proteins (fibronectin and collagen I), which was abolished by a JAK3 inhibitor (CP690,550) in a dose-dependent manner or blocked in the absence of STAT6. In vivo, STAT6 was activated in interstitial cells of the obstructed kidney, an effect that was abolished by CP690,550. Mice treated with CP690,550 accumulated fewer bone marrow-derived fibroblasts in the obstructed kidneys compared with vehicle-treated mice. Treatment with CP690,550 also significantly reduced myofibroblast transformation, matrix protein expression, fibrosis development, and apoptosis in obstructed kidneys. Furthermore, STAT6-deficient mice accumulated fewer bone marrow-derived fibroblasts in the obstructed kidneys, produced less extracellular matrix protein, and developed much less fibrosis. Finally, wild-type mice engrafted with STAT6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the obstructed kidneys and showed less severe renal fibrosis compared with wild-type mice engrafted with STAT6(+/+) bone marrow cells. Our results demonstrate that JAK3/STAT6 has an important role in bone marrow-derived fibroblast activation, extracellular matrix production, and interstitial fibrosis development.

Human Stanniocalcin-1 Suppresses Angiotensin II-Induced Superoxide Generation in Cardiomyocytes through UCP3-Mediated Anti-Oxidant Pathway

Rationale We have previously shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial membrane and transgenic over-expression of STC1 is associated with increased energy utilization. Objective We examined the hypothesis that STC1 uncouples mitochondrial oxidative phosphorylation - to suppress superoxide generation and modulate neurohormonal effects on cardiomyocytes. Methods and Results Compared to WT mouse heart, STC1 Tg heart displays: 2-fold higher uncoupling protein 3 (UCP3) levels, but no effect on UCP2 protein; 40% lower ATP levels; but similar activities of respiratory chain complexes I-IV. In cultured adult rat and freshly-isolated mouse cardiomyocytes, rSTC1 induces UCP3, but not UCP2. Treatment of cardiomyocytes with STC1 decreases mitochondrial membrane potential and suppresses baseline and angiotensin II (Ang II)-induced superoxide generation. Furthermore, baseline superoxide generation is higher in freshly-isolated adult UCP3−/− mouse cardiomyocytes compared to WT, suggesting an important role for UCP3 in regulating cardiomyocyte ROS under physiologic conditions. Treatment of UCP3−/− cardiomyocytes with rSTC1 failed to suppress superoxide generation, suggesting that the effects of STC1 on superoxide generation in cardiomyocytes are UCP3-dependent. Conclusion STC1 activates a novel anti-oxidant pathway in cardiac myocytes through induction of UCP3, and may play an important role in suppressing ROS in the heart under normal physiologic conditions and ameliorate the deleterious effects of Ang II-mediated cardiac injury. Importantly, our data point to a critical role for the mitochondria in regulating ROS generation in response to Ang II.