Fan Chi Chang

Targeting Endothelium-Pericyte Cross Talk by Inhibiting VEGF Receptor Signaling Attenuates Kidney Microvascular Rarefaction and Fibrosis

Microvascular pericytes and perivascular fibroblasts have recently been identified as the source of scar-producing myofibroblasts that appear after injury of the kidney. We show that cross talk between pericytes and endothelial cells concomitantly dictates development of fibrosis and loss of microvasculature after injury. When either platelet-derived growth factor receptor (R)-β signaling in pericytes or vascular endothelial growth factor (VEGF)R2 signaling in endothelial cells was blocked by circulating soluble receptor ectodomains, both fibrosis and capillary rarefaction were markedly attenuated during progressive kidney injury. Blockade of either receptor-mediated signaling pathway prevented pericyte differentiation and proliferation, but VEGFR2 blockade also attenuated recruitment of inflammatory macrophages throughout disease progression. Whereas injury down-regulated angiogenic VEGF164, the dys-angiogenic isomers VEGF120 and VEGF188 were up-regulated, suggesting that pericyte-myofibroblast differentiation triggers endothelial loss by a switch in secretion of VEGF isomers. These findings link fibrogenesis inextricably with microvascular rarefaction for the first time, add new significance to fibrogenesis, and identify novel therapeutic targets.

Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis

Pericytes are the major source of scar-producing myofibroblasts following kidney injury; however, the mechanisms of this transition are unclear. To clarify this, we examined Collagen 1 (α1)-green fluorescent protein (GFP) reporter mice (pericytes and myofibroblasts express GFP) following ureteral obstruction or ischemia-reperfusion injury and focused on the role of platelet-derived growth factor (PDGF)-receptor (PDGFR) signaling in these two different injury models. Pericyte proliferation was noted after injury with reactivation of α-smooth muscle actin expression, a marker of the myofibroblast phenotype. PDGF expression increased in injured tubules, endothelium, and macrophages after injury, whereas PDGFR subunits α and β were expressed exclusively in interstitial GFP-labeled pericytes and myofibroblasts. When PDGFRα or PDGFRβ activation was inhibited by receptor-specific antibody following injury, proliferation and differentiation of pericytes decreased. The antibodies also blunted the injury-induced transcription of PDGF, transforming growth factor β1, and chemokine CCL2. They also reduced macrophage infiltration and fibrosis. Imatinib, a PDGFR tyrosine kinase inhibitor, attenuated pericyte proliferation and kidney fibrosis in both fibrogenic models. Thus, PDGFR signaling is involved in pericyte activation, proliferation, and differentiation into myofibroblasts during progressive kidney injury. Hence, pericytes may be a novel target to prevent kidney fibrosis by means of PDGFR signaling blockade.

Preoperative Proteinuria Predicts Adverse Renal Outcomes after Coronary Artery Bypass Grafting

Whether preoperative proteinuria associates with adverse renal outcomes after cardiac surgery is unknown. Here, we performed a secondary analysis of a prospectively enrolled cohort of adult patients undergoing coronary artery bypass grafting (CABG) at a medical center and its two affiliate hospitals between 2003 and 2007. We excluded patients with stage 5 CKD or those who received dialysis previously. We defined proteinuria, measured with a dipstick, as mild (trace to 1+) or heavy (2+ to 4+). Among a total of 1052 patients, cardiac surgery-associated acute kidney injury (CSA-AKI) developed in 183 (17.4%) patients and required renal replacement therapy (RRT) in 50 (4.8%) patients. In a multiple logistic regression model, mild and heavy proteinuria each associated with an increased odds of CSA-AKI, independent of CKD stage and the presence of diabetes mellitus (mild: OR 1.66, 95% CI 1.09 to 2.52; heavy: OR 2.30, 95% CI 1.35 to 3.90). Heavy proteinuria also associated with increased odds of postoperative RRT (OR 7.29, 95% CI 3.00 to 17.73). In summary, these data suggest that preoperative proteinuria is a predictor of CSA-AKI among patients undergoing CABG.

Novel insights into pericyte–myofibroblast transition and therapeutic targets in renal fibrosis

Renal fibrosis is a disease affecting millions worldwide and is a harbinger of progressive renal failure. Understanding the mechanisms of renal fibrosis is important for discovering new therapies that are required to prevent loss of renal function. Recently, we identified pericytes that line the kidney microvasculature as the precursor cells of the scar-producing myofibroblasts during kidney injury. Kidney pericytes are extensively branched cells embedded within the capillary basement membrane and stabilize the capillary network through tissue inhibitor of metalloproteinase 3 and angiogenic growth factors. Pericytes detach from endothelial cells and migrate into the interstitial space where they undergo a transition into myofibroblasts after injury. Activation of endothelium, pericyte-myofibroblast transition, and recruitment of inflammatory macrophages lead to capillary rarefaction and fibrosis. Targeting endothelium-pericyte crosstalk by inhibiting vascular endothelial cell growth factor receptors and platelet-derived growth factor receptors in response to injury have been identified as new therapeutic interventions. Furthermore, targeting macrophage activation has also been proven as a novel and safe therapeutic approach for pericyte-myofibroblast transition. However, we are still far from understanding the interaction between pericytes and other cellular elements in normal physiology and during kidney fibrosis. Further studies will be required to translate into more specific therapeutic approaches.

Angiopoietin-2 Is Associated with Albuminuria and Microinflammation in Chronic Kidney Disease

Although cardiovascular disease (CVD) is the leading cause of mortality in patients with chronic kidney disease (CKD), the pathophysiology is not thoroughly understood. Given that elevated albuminuria or circulating angiopoietin-2 associates with CVD and mortality in CKD patients, we were intrigued by the relationship between albuminuria and angiopoietin-2. A total of 416 patients with CKD stages 3 to 5 were stratified by urine albumin-creatinine ratio as normoalbuminuria (<30 mg/g), microalbuminuria (30–300 mg/g), or macroalbuminuria (>300 mg/g). The levels of plasma angiopoietin-2 and vascular endothelial growth factor (VEGF) increased, and soluble Tie-2 decreased in the subgroups of albuminuria; whereas angiopoietin-1 did not change. Linear regression showed a positive correlation between urine albumin-creatinine ratio (ACR) and plasma angiopoietin-2 (correlation coefficient r = 0.301, 95% confidence interval 0.211–0.386, P<0.0001), but not between ACR and VEGF or soluble Tie-2. Multivariate linear regression analysis showed that plasma angiopoietin-2 was independently associated with ACR (P = 0.025). Furthermore, plasma angiopoietin-2 was positively correlated with high sensitive C-reactive protein (r = 0.114, 95% confidence interval 0.018–0.208, P = 0.020). In conclusion, plasma angiopoietin-2 was associated with albuminuria and markers of systemic microinflammation in CKD patients. Although previous evidence has shown that angiopoietin-2 destabilizes vasculature and induces inflammation in different scenarios, further study will be required to delineate the role of angiopoietin-2 in albuminuria and microinflammation in CKD patients.

The role played by perivascular cells in kidney interstitial injury.

Fibrosis of the kidney is a disease affecting millions worldwide and is a harbinger of progressive loss of organ function resulting in organ failure. Recent findings suggest that understanding mechanisms of development and progression of fibrosis will lead to new therapies urgently required to counteract loss of organ function. Recently, little-known cells that line the kidney microvasculature, known as pericytes, were identified as the precursor cells which become the scar-forming myofibroblasts. Kidney pericytes are extensively branched cells located in the wall of capillaries, embedded within the microvascular basement membrane, and incompletely envelope endothelial cells with which they establish focal contacts. In response to kidney injuries, pericytes detach from endothelial cells and migrate into the interstitial space where they undergo a transition into myofibroblasts. Detachment leads to fibrosis but also leaves an unstable endothelium, prone to rarefaction. Endothelial-pericyte crosstalk at the vascular endothelial growth factor receptors and platelet derived growth factor receptors in response to injury have been identified as major new targets for therapeutic intervention.

Angiopoietin-2–Induced Arterial Stiffness in CKD

The mechanism of vascular calcification in CKD is not understood fully, but may involve collagen deposition in the arterial wall upon osteo/chondrocytic transformation of vascular smooth muscle cells (VSMCs). Increased levels of circulating angiopoietin-2 correlate with markers of CKD progression and angiopoietin-2 regulate inflammatory responses, including intercellular and vascular adhesion and recruitment of VSMCs. Here, we investigate the potential role of angiopoietin-2 in the pathogenesis of arterial stiffness associated with CKD. In a cohort of 416 patients with CKD, the plasma level of angiopoietin-2 correlated independently with the severity of arterial stiffness assessed by pulse wave velocity. In mice subjected to 5/6 subtotal nephrectomy or unilateral ureteral obstruction, plasma levels of angiopoietin-2 also increased. Angiopoietin-2 expression markedly increased in tubular epithelial cells of fibrotic kidneys but decreased in other tissues, including aorta and lung, after 5/6 subtotal nephrectomy. Expression of collagen and profibrotic genes in aortic VSMCs increased in mice after 5/6 subtotal nephrectomy and in mice producing human angiopoietin-2. Angiopoietin-2 stimulated endothelial expression of chemokines and adhesion molecules for monocytes, increased Ly6C(low) macrophages in aorta, and increased the expression of the profibrotic cytokine TGF-β1 in aortic endothelial cells and Ly6C(low) macrophages. Angiopoietin-2 blockade attenuated expression of monocyte chemokines, profibrotic cytokines, and collagen in aorta of mice after 5/6 subtotal nephrectomy. This study identifies angiopoietin-2 as a link between kidney fibrosis and arterial stiffness. Targeting angiopoietin-2 to attenuate inflammation and collagen expression may provide a novel therapy for cardiovascular disease in CKD.

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1+ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRβ, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-β1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.

The role of angiopoietin-2 in progressive renal fibrosis

With increasing prevalence worldwide, chronic kidney disease (CKD) has drawn great attention. In the case of progressive renal disease, decline in renal function correlates with the extent of interstitial fibrosis, irrespective of the initiating pathology. From a histological perspective, the disease is characterized by myofibroblast accumulation, extracellular matrix (ECM) deposition, inflammation, tubular atrophy, and microvascular rarefaction. The complex intertexture between cellular elements and molecular mediators in tissue damage may be initially adaptive, leading to functional and architectural regeneration; with chronic stimuli turning out to be maladaptive, resulting in permanent scar formation and irreversible organ failure. Scar-producing myofibroblasts or activated fibroblasts, characterized by the expression of cytoskeletal protein a-smooth muscle actin and contractile function, deposit collagenous ECM and play a crucial role in fibrotic diseases. In animal models of progressive renal disease, loss of the microvasculature integrity also correlates with the degree of glomerulosclerosis and tubulointerstitial fibrosis. Persistent or repeated injuries lead to peritubular capillary rarefaction, chronic hypoxia, tubular cell loss, cytokine release, inflammatory cell recruitment, pericyte

Angiopoietins modulate endothelial adaptation, glomerular and podocyte hypertrophy after uninephrectomy.

Glomerular capillary remodeling is an essential process in the development of glomerular hypertrophy. Angiopoietins, which are important regulators in angiogenesis, plays a role in the development of glomerulus during embryogenesis. Here, we evaluated the influence of angiopoietin on glomerular components and hypertrophy after uninephrectomy in adult male BALB/c mice. The actions of angiopoietin 1 or 2 were systemically antagonized by the subcutaneous administration of antagonists. We observed that the angiopoietin system was activated after uninephrectomy, and that the blockade of angiopoietin 1 or 2 decreased the activation of the angiopoietin receptor—tyrosine kinase with Ig and EGF homology domains-2—and attenuated the development of glomerular and podocyte hypertrophy. The increase in endothelial density staining (anti-CD31) following uninephrectomy was also reversed by angiopoietin 1 or 2 blockades. Glomerular basement thickness and foot process width were observed to decrease in the angiopoietin blockade groups. These changes were associated with the down regulation of the expression of genes for the glomerular matrix and basement membrane, including collagen type IV α1, collagen type IV α2, collagen type IV α5, and laminin α5. Thus, angiopoietin 1 or 2 may play an important role in the development of glomerular hypertrophy after uninephrectomy. A blockade of the angiopoietin system not only influenced the endothelium but also the podocyte, leading to diminished gene expression and morphological changes after uninephrectomy.