Tubular von Hippel-Lindau Knockout Protects against Rhabdomyolysis-Induced AKI

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA–based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.No specific therapy is currently available for human AKI, a clinical entity of increasing incidence and high morbidity and mortality.^1–4 Rhabdomyolysis, one of the leading causes of AKI, develops after trauma, drug toxicity, infections, burns, and physical exertion.^5–8 The animal model using an intramuscular glycerol injection with consequent myoglobinuria is closely related to the human syndrome of rhabdomyolysis.⁹ Experimental data demonstrate renal vasoconstriction,^9–15 tubular hypoxia,^15,16 normal or even reduced intratubular pressure,^9–11 as well as large variation in single nephron GFR.^10,11 Intratubular myoglobin casts, a histologic hallmark, seem not to cause tubular obstruction,^9–11 but rather scavenge nitric oxide^17,18 and generate reactive oxygen species¹⁹ followed by vasoconstriction.The traditional discrimination between ischemic and toxic forms of AKI has been challenged because an increasing amount of evidence suggests that renal hypoxia is a common denominator in AKI of different etiologies.²⁰ Pimonidazole adducts, which accumulate in tissues at oxygen tensions <10 mmHg,²¹ have been demonstrated in various AKI forms.^16,22–24 During AKI, hypoxia-inducible factors (HIFs), which are mainly regulated by oxygen-dependent proteolysis, were found to be upregulated in different renal tubular segments.^{16,20,22,24,25} HIFs are heterodimers of a constitutive β subunit, HIF-β (ARNT), and one of three oxygen-dependent α-subunits, HIF-1α, HIF-2α, and HIF-3α. The α-β dimers bind to hypoxia-response elements (HREs) in the promoter-enhancer region of HIF target genes.^26–28 Although the 5′-RCGTG-3′ (R = A or G) core HRE appears >1 million times in the entire genome²⁹ and in >4000 promoter regions of validated genes,³⁰ a recent study demonstrated HIF binding in roughly 350 genes.³¹ Multiple HIF-based biologic effects are known, and it is widely accepted that a broad panel of these promote cellular survival in a hostile and oxygen-deprived environment.^27–29 In all types of AKI tested thus far, HIF activation along the nephron correlates with tubular survival, and the cells most vulnerable to injury exhibit no or only very limited HIF activity.²⁰ This observation led to the concept of insufficient HIF-based hypoxic adaptation in AKI. Consequently, maneuvers of preconditional HIF activation are utilized to ameliorate AKI. Indeed, many of these attempts are successful but the majority are conducted in ischemia-reperfusion injury.²⁰ It is largely unclear whether HIF can rescue kidneys exposed to AKI forms other than ischemia-reperfusion injury, and it is unclear which HIF target genes are involved in AKI protection if so. In many tumors, constitutive HIF activation promotes anaerobic ATP production, a process known as the Warburg effect.³²von Hippel-Lindau protein (VHL) is a ubiquitin ligase engaged in the stepwise HIF-α degradation process, which constantly occurs during normoxia.³³ Inducible Pax8-rtTA–based knockout of VHL (VHL-KO) achieves strong, selective, and persistent upregulation of HIF in all nephron segments.³⁴ In this study, we use this transgenic technique in conjunction with rhabdomyolysis in mice to address two issues: (1) Does HIF activation through VHL-KO protect from rhabdomyolysis-induced AKI? (2) If so, what are the biologic mechanisms and HIF target genes that are responsible for renal protection against acute injury? We demonstrate that indeed VHL-KO mice are largely protected against rhabdomyolysis-induced AKI, and provide evidence for a metabolic shift toward anaerobic ATP generation as the central protective mechanism.     

NAD(P)H Oxidase Mediates TGF-β1–Induced Activation of Kidney Myofibroblasts

TGF-β1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase–derived ROS in TGF-β1–induced activation of rat kidney fibroblasts and expression of α-smooth muscle actin (α-SMA) and Fn-ED-A. In vitro, TGF-β1 stimulated formation of abundant stress fibers and increased expression of both α-SMA and Fn-ED-A. In addition, TGF-β1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-β1–induced stimulation of NADPH oxidase activity and reduced α-SMA and Fn-ED-A expression. Inhibition of TGF-β1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-β1–enhanced NADPH oxidase activity; and decreased expression of Nox4, α-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-β1–induced Smad3 but reduced both α-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked α-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-β1 stimulated phosphorylation of extracellular signal–regulated kinase (ERK1/2), and this was inhibited by blocking TGF-β1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased α-SMA and Fn-ED-A. Taken together, these results suggest that TGF-β1–induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.Progression of renal fibrosis involves expansion of interstitial myofibroblasts and extracellular matrix accumulation, resulting in the loss of function and ultimately renal failure.^1,2 The origin of myofibroblasts is under extensive investigation, and evidence indicates the cells may be derived from several sources, including an expansion of activated resident fibroblasts, perivascular adventitial cells, blood-borne stem cells that migrate into the glomerular mesangial or interstitial compartment, or tubular epithelial-to-mesenchymal transition and migration into the peritubular interstitial space. Regardless of their origin, there is common agreement that the myofibroblast is the cell most responsible for interstitial expansion and matrix accumulation during the course of renal fibrosis. TGF-β1 is the predominant growth factor responsible for matrix synthesis by mesenchymal cells such as fibroblasts in vitro and during renal fibrosis.^3,4 Indeed, there is a close correlation in the cellular expression of TGF-β1, a fibroblast transition to an activated, α-smooth muscle actin (α-SMA)-positive myofibroblast phenotype, and synthesis of an alternatively spliced isoform of fibronectin, Fn-ED-A.⁵ TGF-β1 differentially regulates the expression of Fn-ED-A in fibroblasts^6–8 and induces expression of α-SMA in a variety of mesenchymal cells in culture.^9,10 Indeed, a functional ED-A domain is mandatory for α-SMA induction by TGF-β1.^7,8,10 Moreover, TGF-β1 is frequently associated with a myofibroblast phenotype in liver, lung, and kidney disease,^1,11–13 and all three proteins frequently co-localize in these disease settings. In addition, a co-localization of α-SMA and Fn-ED-A is frequently observed in fibrotic disease as well as in glomerular and interstitial lesions in kidney diseases previously investigated in our laboratory.^14–17Accumulating evidence also indicates that reactive oxygen species (ROS), mainly in the form of superoxide, play a significant role in the initiation and progression of cardiovascular^18,19 and renal^20–25 disease. ROS are involved in distinct cell functions, including hypertrophy, migration, proliferation, apoptosis, and regulation of extracellular matrix.^25–28 More specific, the NAD(P)H oxidases of the Nox family have gained heightened attention as mediators of injury associated with vascular diseases, including hypertension, atherosclerosis, heart disease, and diabetes.^18,19,29,30 NAD(P)H oxidase generation of superoxide is recognized as an important mediator of cell proliferation in glomerulonephritis²² and matrix accumulation in diabetic nephropathy^25,31–33 and fibrosis.^21,24 Adventitial fibroblasts are also a major source of superoxide in the aorta,^19,34–36 therefore being highly relevant to renal disease. This is because the renal perivascular space is noticeably reactive and is the site where myofibroblasts may first appear during the course of renal disease and fibrosis.^17,37–39The observations that both TGF-β1 and ROS induce fibroblasts to α-SMA–positive myofibroblast phenotype^40–42 suggest that these two pathways are interrelated and may share signaling pathways in kidney disease. TGF-β signaling occurs through a well-established process involving two downstream pathways: Smad and extracellular signal–regulated kinase (ERK).^43–45 TGF-β/Smad signaling (Smad2 and Smad3) is tightly controlled by mitogen-activated protein kinase (MAPK; ras/MEK/ERK) signaling cascades.⁴⁶ A regulatory role for ROS in PDGF and angiotensin II–induced signal transduction has gained recognition^47,48; however, a role for ROS in TGF-β signaling is less well understood. It is also unknown whether kidney myofibroblasts express NAD(P)H oxidase homologs or generate ROS in response to TGF-β1. Given TGF-β1–induced myofibroblast activation and matrix synthesis during renal disease may be linked to ROS, we examined a role for NAD(P)H oxidase in TGF-β1–induced Smad3 and ERK signaling as well as kidney myofibroblast activation, as assessed by a switch to an α-SMA–positive phenotype and expression of Fn-ED-A expression in vitro.     

Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury

Fibrosis of the peritoneal cavity remains a serious, life-threatening problem in the treatment of kidney failure with peritoneal dialysis. The mechanism of fibrosis remains unclear partly because the fibrogenic cells have not been identified with certainty. Recent studies have proposed mesothelial cells to be an important source of myofibroblasts through the epithelial–mesenchymal transition; however, confirmatory studies in vivo are lacking. Here, we show by inducible genetic fate mapping that type I collagen–producing submesothelial fibroblasts are specific progenitors of α-smooth muscle actin–positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-β1. Similar genetic mapping of Wilms’ tumor-1–positive mesothelial cells indicated that peritoneal membrane disruption is repaired and replaced by surviving mesothelial cells in peritoneal injury, and not by submesothelial fibroblasts. Although primary cultures of mesothelial cells or submesothelial fibroblasts each expressed α-smooth muscle actin under the influence of TGF-β1, only submesothelial fibroblasts expressed α-smooth muscle actin after induction of peritoneal fibrosis in mice. Furthermore, pharmacologic inhibition of the PDGF receptor, which is expressed by submesothelial fibroblasts but not mesothelial cells, attenuated the peritoneal fibrosis but not the remesothelialization induced by hypochlorite. Thus, our data identify distinctive fates for injured mesothelial cells and submesothelial fibroblasts during peritoneal injury and fibrosis.Many patients with kidney failure rely on the peritoneal membrane to perform life-saving dialysis.^1–3 In addition to changes in permeability of the peritoneal membrane, the dialysis process itself frequently triggers a fibrosing process that progressively reduces membrane function resulting in dialysis failure, sometimes with high patient mortality.^4–7 In a small percentage of patients, severe fibrosis occurs primarily in the visceral peritoneum, resulting in encapsulating peritoneal sclerosis (EPS), a catastrophic complication with obscure pathogenesis and a high mortality rate.^6,7 Such dialysis failure is characterized by progressive peritoneal fibrosis that can be seen as with thickening of basal lamina and accumulation of α-smooth muscle actin (αSMA)⁺ myofibroblasts.^4,5,8–10The peritoneum is composed of mesothelium, basal lamina, and submesothelial (SM) connective tissue.^11–13 The mesothelium consists of a single layer of flattened mesothelial cells (MCs) that lines the peritoneal cavity and internal organs.^12,14–16 In many circumstances such as organ development or tissue injury repair, MCs are the cellular source of growth factors including TGF-β1, PDGF, and vascular endothelial growth factor, which support cell proliferation and differentiation of parenchymal and stromal cells as well as angiogenesis.^8,17–22 By contrast, SM connective tissue containing plexuses of blood vessels, lymphatic channels, and scattered fibroblasts has drawn much less attention.^22–24 A number of studies suggested MCs as the major source of myofibroblasts through the epithelial–mesenchymal transition (EMT) during peritoneal fibrosis.^8,18,25–27 However, these studies relied predominantly on in vitro experiments to show that MCs can be stimulated to express αSMA and produce matrix proteins outside of the body under the influence of profibrotic agents such as TGF-β1.^8,25–28 Nevertheless, confirmatory studies of mesothelial EMT in vivo are lacking even though costaining of cytokeratin and αSMA was previously shown.^8,18 The contribution of SM fibroblasts to myofibroblasts in vivo is not clear despite some studies in vitro have suggested.^22–24A conditional cell lineage analysis using WT1^CreERT2/+ mice demonstrated that Wilms’ tumor-1 (WT1)⁺ septum transversum mesenchyme gives rise to MCs, SM fibroblasts covering the liver, and hepatic stellate cells within the liver during hepatic development.^12,13 Using WT1^CreERT2/+ mice, a recent study reported that WT1⁺ MCs may differentiate into myofibroblasts in liver injury.²⁹ WT1 expression in MCs is observed in both embryonic development and adult peritoneum; however, the expression of WT1 by SM fibroblasts is not clearly defined in the adult peritoneum.^{12,13,21,30–32} Hence, the progenitors of myofibroblasts in the injured liver and the peritoneum remain controversial despite these studies.Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we performed lineage tracing of both MCs and SM fibroblasts in models of peritoneal fibrosis induced by sodium hypochlorite solution, hyperglycemic dialysis solution, or adenovirus-expressing TGF-β1 (AdTGF-β1). Although these models are more akin to EPS than the progressive thickening peritoneum seen in humans on peritoneal dialysis, they represent robust tools to study the pathogenesis of peritoneal fibrosis in the laboratory.^33–36 Contrary to the prevailing model, our findings indicate that peritoneal myofibroblasts derive from SM fibroblasts and peritoneal membrane disruption is repaired by surviving MCs.     

Renal Dendritic Cells Ameliorate Nephrotoxic Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury. Dendritic cells (DCs) are immune sentinels with the ability to induce immunity or tolerance, but whether they mediate acute kidney injury is unknown. Here, we studied the distribution of DCs within the kidney and the role of DCs in cisplatin-induced acute kidney injury using a mouse model in which DCs express both green fluorescence protein and the diphtheria toxin receptor. DCs were present throughout the tubulointerstitium but not in glomeruli. We used diphtheria toxin to deplete DCs to study their functional significance in cisplatin nephrotoxicity. Mice depleted of DCs before or coincident with cisplatin treatment but not at later stages experienced more severe renal dysfunction, tubular injury, neutrophil infiltration and greater mortality than nondepleted mice. We used bone marrow chimeric mice to confirm that the depletion of CD11c-expressing hematopoietic cells was responsible for the enhanced renal injury. Finally, mixed bone marrow chimeras demonstrated that the worsening of cisplatin nephrotoxicity in DC-depleted mice was not a result of the dying or dead DCs themselves. After cisplatin treatment, expression of MHC class II decreased and expression of inducible co-stimulator ligand increased on renal DCs. These data demonstrate that resident DCs reduce cisplatin nephrotoxicity and its associated inflammation.Innate immune responses are pathogenic in both ischemic and toxic acute renal failure. In response to renal injury, inflammatory chemokines and cytokines are produced both by renal parenchymal cells, such as proximal tubule epithelial cells, and resident or infiltrating leukocytes.^1–4 The elaborated chemokines and cytokines, including TNF-α, IL-18, keratinocyte-derived chemokine, and monocyte chemoattractant protein 1, subsequently recruit additional immune cells to the kidney, such as neutrophils, T cells, monocytes, and inflammatory dendritic cells (DCs), which may cause further injury through pathways that are not fully defined.^2,5–12 DCs are sentinels of the immune system and under steady-state conditions induce tolerance by various mechanisms, including production of TGF-β, IL-10, or indoleamine 2,3-dioxygenase^13–16; expression of PDL-1, PDL-2, or FcγR2B^17,18; clonal deletion of autoreactive T cells¹⁹; and induction of T regulatory cells via the inducible co-stimulator (ICOS) pathway.^20–23 In response to pathogens or products of tissue injury, DCs mature and initiate immunity or inflammatory diseases.^24,25 Monocytes recruited to inflamed tissue can also differentiate into inflammatory DCs and mediate defense against pathogens or contribute to inflammatory tissue responses.^12,26–28Although DCs represent a major population of immune cells in the kidney,²⁹ their role in renal disease is poorly defined. Liposomal clodronate has been used to study the pathophysiologic role of phagocytic cells, which include DCs and macrophages.^3,30–32 An alternative DC-specific approach uses expression of the simian diphtheria toxin receptor (DTR) driven by the CD11c promoter to target DCs for DT-mediated cell death.²⁴ This model has been used extensively to study the role of DCs in various physiologic and pathophysiologic contexts^32,33; however, its application in kidney disease has been limited to recent studies of immune complex–mediated glomerulonephritis.^12,23We have reported that inflammation plays an important role in the pathogenesis of cisplatin-induced acute kidney injury (AKI).^1,4,5,34 Given the dearth of information regarding the role of renal DCs in AKI, this study examined the renal DC population and the impact of its depletion on cisplatin nephrotoxicity. We show that DCs are the most abundant population of renal resident leukocytes and form a dense network throughout the tubulointerstitium. Renal DCs displayed surface markers that distinguished them from splenic DCs. Using a conditional DC depletion model, we determined that DC ablation markedly exacerbates cisplatin-induced renal dysfunction, structural injury, and infiltration of neutrophils.     

PKCδ Impaired Vessel Formation and Angiogenic Factor Expression in Diabetic Ischemic Limbs

Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates the role of PKCδ in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and actions. Ischemic adductor muscles of diabetic Prkcd^+/+ mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic Prkcd^+/+ mice. By contrast, diabetic Prkcd^−/− mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various PKC isoforms was unchanged, activation of PKCδ was increased in diabetic Prkcd^+/+ mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic Prkcd^+/+ mice and were normalized in diabetic Prkcd^−/− mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-β (PDGFR-β) were blunted in diabetic Prkcd^+/+ mice but elevated in diabetic Prkcd^−/− mice. The inhibition of VEGFR2 and PDGFR-β activity was associated with increased SHP-1 expression. In conclusion, our data have uncovered the mechanisms by which PKCδ activation induced poor collateral vessel formation, offering potential novel targets to regulate angiogenesis therapeutically in diabetic patients.The main long-term complications from diabetes are vascular diseases, which are in turn the main causes of morbidity and mortality in diabetic patients (1). Diabetic vascular complications affect several important organs, including the retina, kidney, and arteries (2,3). Peripheral vascular diseases are the major risk factor for nontraumatic lower limb amputation in patients with diabetes (4), characterized by collateral vessel development insufficient to support the loss of blood flow through occluded arteries in the ischemic limbs (5). Multiple abnormalities in the angiogenic response to ischemia have been documented in the diabetic state and depend on complex interactions of multiple growth factors and vascular cells.Experiments to improve angiogenesis and vascular cell survival by local infusion of vascular endothelial growth factor (VEGF) or angiopoietin by increasing its expression have also been reported in nondiabetic animal models (6,7). Moreover, animal studies have used platelet-derived growth factor (PDGF) to improve collateral vessel formation and vascular healing in the diabetic state (8). Clinical trials using recombinant growth factors have noted transient improvement of myocardial and distal leg circulation (9–11). However, these favorable vascular effects appeared to produce limited clinical benefits (12). Local administration of growth factors, such as VEGF by gene therapy in the setting of diabetes, does not appear to have the beneficial long-term effects seen in the absence of diabetes or to improve quality of life (13,14). One potential problem with normalizing VEGF or PDGF action alone is that a variety of growth factors may be needed to establish and maintain the capillary bed.Various studies have clearly identified that the expression of growth factors, such as VEGF, PDGF, and stromal-derived factor-1 (SDF-1), are critically important in the formation of collateral vessels in response to ischemia (15–17). Previous studies suggested that hyperglycemia attenuates VEGF production and levels in myocardial tissue and in animal models of wound repair (5,18). Furthermore, decreased VEGF and PDGF expression in the peripheral limbs and nerves of diabetic animals and rodents has been reported (19–21). Although the underlying mechanism of reduction of VEGF and PDGF expression in diabetes is not clear, it is well-known that the major inducers of VEGF and PDGF (i.e., hypoxia and oxidants) can both play a role in diabetes. We and other researchers have reported that variation in PDGF signaling, rather than expression, is linked to morphological abnormalities in the retina and in collateral capillary formation in an ischemic limb model of diabetic animals (22,23). Clearly, poor collateral vessel formation during diabetes-induced ischemia is attributable to the lack of production and/or action of critical growth factors such as VEGF and PDGF. Therefore, further studies of the basic mechanisms of hyperglycemia-induced activation of toxic metabolites, such as activation of protein kinase C (PKC), are needed to identify how these proteins contribute to growth factor deregulation.PKC, a member of a large family of serine/threonine kinases, is involved in the pathophysiology of vascular complications. When activated, PKC phosphorylates specific serine or threonine residues on target proteins that vary, depending on cell type. PKC has multiple isoforms that function in a wide variety of biological systems (24). PKC activation increases endothelial permeability and decreases blood flow and the production and response of angiogenic growth factors that contribute to the loss of capillary pericytes, retinal permeability, ischemia, and neovascularization (25–29).Previous data have demonstrated that high glucose levels in smooth muscle cells activate PKCα, -β, -δ, and -ε but not the atypical PKCζ (30,31). In general, high levels of glucose-induced PKC activation cause vascular dysfunction by altering the expressions of growth factors such as VEGF, PDGF, transforming growth factor-β, and others (32–34). PKCδ has been proposed to participate in smooth muscle cell apoptosis, and deletion of this PKC isoform led to increased arteriosclerosis (35). Moreover, we previously demonstrated that diabetes-induced PKCδ activation generates PDGF unresponsiveness, causing pericyte apoptosis, acellular capillaries, and diabetic retinopathy (23). We therefore hypothesized that PKCδ activation could be involved in proangiogenic factor inhibition that triggers poor collateral vessel formation in diabetes.     

Advanced Glycation End-Products Induce Tubular CTGF via TGF-β–Independent Smad3 Signaling

Advanced glycation end-products (AGEs) can induce expression of connective tissue growth factor (CTGF), which seems to promote the development of diabetic nephropathy, but the exact signaling mechanisms that mediate this induction are unknown. Here, AGEs induced CTGF expression in tubular epithelial cells (TECs) that either lacked the TGF-β1 gene or expressed dominant TGF-β receptor II, demonstrating independence of TGF-β. Furthermore, conditional knockout of the gene encoding TGF-β receptor II from the kidney did not prevent AGE-induced renal expression of CTGF and collagen I. More specific, AGEs induced CTGF expression via the receptor for AGEs-extracellular signal–regulated kinase (RAGE-ERK)/p38 mitogen-activated protein kinase–Smad cross-talk pathway because inhibition of this pathway by several methods (anti-RAGE antibody, specific inhibitors, or dominant negative adenovirus to ERK1/2 and p38) blocked this induction. Overexpressing Smad7 abolished AGE-induced Smad3 phosphorylation and CTGF expression, demonstrating the necessity for activation of Smad signaling in this process. More important, knockdown of either Smad3 or Smad2 demonstrated that Smad3 but not Smad2 is essential for CTGF induction in response to AGEs. In conclusion, AGEs induce tubular CTGF expression via the TGF-β–independent RAGE-ERK/p38-Smad3 cross-talk pathway. These data suggest that overexpression of Smad7 or targeting Smad3 may have therapeutic potential for diabetic nephropathy.Connective tissue growth factor (CTGF; CCN2), a member of CCN family of growth factors, plays an important role in connective tissue homeostasis and fibroblast proliferation, migration, adhesion, and extracellular matrix expression.¹ Clinically, renal expression of CTGF is increased in patients with diabetic nephropathy (DN), and its expression correlates closely with the degree of albuminuria.^2,3 In addition, studies in human renal biopsy show that CTGF expression significantly augments glomerular and tubulointerstitial injury with α-smooth muscle actin cell accumulation.⁴ Several pieces of evidence from recent rodent studies further support the notion that CTGF is important in the pathogenesis of DN. For example, the thickening of glomerular basement membrane is attenuated in CTGF^+/− mice.⁵ In type 1 diabetic mouse model, cell-specific overexpression of CTGF in podocytes of CTGF transgenic mice is able to intensify proteinuria and mesangial expansion.⁶ The co-localization of increased renal CTGF expression and AGE accumulation in diabetic rats indicates a causal link between AGE deposition and CTGF expression.⁷ This is supported by the ability of the AGE inhibitor to suppress CTGF expression and reduce renal fibrosis.⁷ Although the mechanisms that regulate renal CTGF function are not clearly understood, CTGF should play an essential role in DN.Engagement of AGEs to the receptor (RAGE) has been shown to play a critical role in diabetic complications, including DN.⁸ Indeed, AGE-induced tubular epithelial-to-mesenchymal transition (EMT) and renal fibrosis are RAGE dependent.^8,9 Under diabetic conditions, although treatments with high glucose and angiotensin II are also able to upregulate CTGF expression in glomerular mesangial cells (MCs) and TECs,^2,10–12 it is clear that AGEs mediate CTGF expression by stimulating TGF-β expression.^13,14 It is generally believed that TGF-β/Smad signaling should be responsible for inducing CTGF expression because CTGF is a downstream mediator of TGF-β signaling^{11,12,15–17}; however, the exact mode of signaling mechanisms by which AGEs induce CTGF expression remains largely unclear.Our previous study of MCs, TECs, and vascular smooth muscle cells (VSMCs) showed that AGEs are able to induce Smad2/3 phosphorylation markedly in TGF-β receptor I (TβRI) and TβRII mutant cell lines via the extracellular signal–regulated kinase (ERK)/p38 mitogen-activate protein kinase (MAPK)-dependent mechanism.¹⁸ This demonstrates a critical role for the TGF-β–independent Smad pathway in AGE-mediated fibrotic response. This is further supported by the finding that blockade of TGF-β1 with specific small hairpin RNA (shRNA) and a neutralizing antibody is unable to inhibit significantly AGE-induced CTGF mRNA expression.¹⁹ All of these studies suggest a TGF-β–independent mechanism in regulating CTGF expression in response to AGEs. Because AGEs are capable of activating the TGF-β/Smad signaling pathway via the ERK/p38 MAPK-dependent mechanism and because CTGF is a target gene of TGF-β/Smad signaling,^7,15,19–22 we thus hypothesized that AGEs might induce CTGF expression via the TGF-β–independent Smad3 signaling pathway. This was tested in mouse TECs lacking TGF-β1 gene²³ and rat TEC lines overexpressing the dominant negative TβRII or Smad7 or having a knockdown of Smad2 or Smad3. Finally, the functional importance of the TGF-β–independent signaling pathway in AGE-mediated CTGF expression and renal fibrosis was tested in mice that had conditional knockout (KO) for TβRII from the kidney.     

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.C-peptide and insulin are cosecreted in equimolar amounts into the circulation from the pancreatic β-cells of Langerhans (1). C-peptide deficiency is a prominent attribute of type 1 diabetes (1). Deficiencies of C-peptide and insulin may also occur in the late stages of type 2 diabetes as a result of progressive loss of β-cells (2–4). Recent evidence demonstrates a beneficial role for C-peptide in diabetic neuropathy (1,5,6), nephropathy (1,6,7), and vascular dysfunction (1,5) and inflammation (1). C-peptide protects against diabetic vascular damage by promoting nitric oxide (NO) release (8) and suppressing nuclear factor-κB (9), which suppresses leukocyte-endothelium interactions (8,9). C-peptide may prevent atherosclerosis by inhibiting vascular smooth muscle proliferation and migration (10) and reducing venous neointima formation (11). However, the molecular mechanism by which C-peptide prevents diabetes complications is not understood well enough to permit its clinical implementation.Generation of reactive oxygen species (ROS) in response to high glucose is the leading cause of endothelial damage and diabetic vasculopathy (12). Protein kinase C (PKC)-dependent NADPH oxidase is considered a major cytosolic mediator of ROS generation in endothelial cells (13,14) that play a central role in hyperglycemia-induced endothelial cell apoptosis and vascular complications (15–17). Overproduction of intracellular ROS by mitochondria also occurs during the development of hyperglycemia-induced vascular complications (12,18,19). Altered mitochondrial dynamics due to mitochondrial fission were recently linked with endothelial dysfunction in diabetes (20,21). However, the mechanisms regulating production of cytosolic and mitochondrial ROS and their individual functions in regulating mitochondrial dynamics and apoptosis remain to be elucidated.AMP-activated protein kinase (AMPK) is an intracellular energy and stress sensor (22) and is an emerging target for preventing diabetes complications (23), as exhibited by the most common antihyperglycemic drugs, rosiglitazone (24) and metformin (25). AMPK prevents apoptosis of endothelial cells (26–28) by inhibiting ROS generation by NADPH oxidase (24,29) and mitochondria (30). Additionally, AMPK dephosphorylation is associated with diabetes (22,31,32). It has been reported that C-peptide inhibits high glucose–induced mitochondrial superoxide generation in renal microvascular endothelial cells (7). We recently demonstrated a key role for C-peptide in preventing high glucose–induced ROS generation and apoptosis of endothelial cells through inhibition of transglutaminase (17). However, the mechanism underlying C-peptide–mediated inhibition of intracellular ROS production and subsequent apoptosis remains unclear. Thus, we hypothesized that the potential protective role of C-peptide could be attributed to activation of AMPK, which results in reduced hyperglycemia-induced production of intracellular ROS and altered mitochondrial dynamics that suppress apoptosis of endothelial cells.In this study, we sought to elucidate the mechanism by which C-peptide protects against hyperglycemia-induced ROS production and subsequent endothelial cell damage. We examined the beneficial effect of C-peptide through AMPKα activation and subsequent protection against hyperglycemia-induced production of intracellular ROS, dysregulation of mitochondrial dynamics, mitochondrial membrane potential (∆Ψ_m) collapse, and apoptosis of endothelial cells. These studies were confirmed in vivo in mice with streptozotocin-induced diabetes using C-peptide supplement therapy delivered through osmotic pumps. Thus, our study implicates C-peptide replacement therapy as a potentially significant approach for preventing diabetes complications.     

Direct Effects of TNF-α on Local Fuel Metabolism and Cytokine Levels in the Placebo-Controlled,Bilaterally Infused Human Leg: Increased Insulin Sensitivity,Increased Net Protein Breakdown,and Increased IL-6 Release

Tumor necrosis factor-α (TNF-α) has widespread metabolic actions. Systemic TNF-α administration, however, generates a complex hormonal and metabolic response. Our study was designed to test whether regional, placebo-controlled TNF-α infusion directly affects insulin resistance and protein breakdown. We studied eight healthy volunteers once with bilateral femoral vein and artery catheters during a 3-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. One artery was perfused with saline and one with TNF-α. During the clamp, TNF-α perfusion increased glucose arteriovenous differences (0.91 ± 0.17 vs. 0.74 ± 0.15 mmol/L, P = 0.012) and leg glucose uptake rates. Net phenylalanine release was increased by TNF-α perfusion with concomitant increases in appearance and disappearance rates. Free fatty acid kinetics was not affected by TNF-α, whereas interleukin-6 (IL-6) release increased. Insulin and protein signaling in muscle biopsies was not affected by TNF-α. TNF-α directly increased net muscle protein loss, which may contribute to cachexia and general protein loss during severe illness. The finding of increased insulin sensitivity, which could relate to IL-6, is of major clinical interest and may concurrently act to provide adequate tissue fuel supply and contribute to the occurrence of systemic hypoglycemia. This distinct metabolic feature places TNF-α among the rare insulin mimetics of human origin.Originally, tumor necrosis factor-α (TNF-α) was identified as an endogenous pyrogen or “cachectin” (1) because of its biological properties of inducing fever, cachexia, and muscle protein loss in various states of disease (2–4). TNF-α is a key component of an inflammatory response and one of the most potent proinflammatory cytokines released by innate immune cells that induces release of other cytokines, including interleukin-6 (IL-6) (5,6). TNF-α plays an important role in the pathophysiology of sepsis, and there seems to be a relation between the TNF-α level and the severity of disease (7–9). Finally, TNF-α has been associated with states of constant low-grade inflammation, eventually leading to insulin resistance and overt diabetes (10,11). In line with this, it has been shown that plasma levels of TNF-α are correlated with BMI; weight loss leads to a decrease in plasma levels of TNF-α (12,13).Systemic infusion of TNF-α induces insulin resistance and increased lipolysis in humans (6,14,15), whereas the effects on protein metabolism are less clear (16). A number of studies have shown that anti–TNF-α treatment increases insulin sensitivity in patients with inflammatory chronic diseases (17–19), whereas other reports have failed to confirm this relationship (20–23). Furthermore, studies investigating TNF-α neutralization in type 2 diabetic patients and in patients with metabolic syndrome show no effect of anti–TNF-α treatment on insulin sensitivity (24,25). TNF-α activates the hypothalamopituitary axis and stimulates the release of stress hormones, such as epinephrine, glucagon, cortisol, and growth hormone into the blood (26,27); all of these counter-regulatory stress hormones generate insulin resistance (27–29), and glucocorticoids generate muscle loss (30). Thus, TNF-α invariably generates release of both other cytokines and stress hormones, and it is uncertain to which extent the metabolic actions of TNF-α are intrinsic or caused by other cytokines or stress hormones in humans.The current study was therefore designed to define the direct metabolic effects of TNF-α in human muscle. Since all previous human studies assessing the metabolic actions of TNF-α have used systemic administration, making discrimination between direct and indirect effects impossible, we infused TNF-α directly into the femoral artery and compared the effects to the saline-infused contralateral leg.     

The NLRP3 Inflammasome Promotes Renal Inflammation and Contributes to CKD

Inflammation significantly contributes to the progression of chronic kidney disease (CKD). Inflammasome-dependent cytokines, such as IL-1β and IL-18, play a role in CKD, but their regulation during renal injury is unknown. Here, we analyzed the processing of caspase-1, IL-1β, and IL-18 after unilateral ureteral obstruction (UUO) in mice, which suggested activation of the Nlrp3 inflammasome during renal injury. Compared with wild-type mice, Nlrp3^−/− mice had less tubular injury, inflammation, and fibrosis after UUO, associated with a reduction in caspase-1 activation and maturation of IL-1β and IL-18; these data confirm that the Nlrp3 inflammasome upregulates these cytokines in the kidney during injury. Bone marrow chimeras revealed that Nlrp3 mediates the injurious/inflammatory processes in both hematopoietic and nonhematopoietic cellular compartments. In tissue from human renal biopsies, a wide variety of nondiabetic kidney diseases exhibited increased expression of NLRP3 mRNA, which correlated with renal function. Taken together, these results strongly support a role for NLRP3 in renal injury and identify the inflammasome as a possible therapeutic target in the treatment of patients with progressive CKD.Chronic kidney disease (CKD) is a significant cause of morbidity and mortality in the general population.^1,2 In nondiabetic CKD, the progression from mild/moderate kidney disease to ESRD is a complex process that involves many factors, including tubulointerstitial inflammation and fibrosis. The involvement of mononuclear inflammatory cells in the damaged renal interstitium is a universal finding in failing kidneys and correlates inversely with renal function.^3–9 The molecular mechanisms that regulate inflammation in CKD, however, remain unclear.An inflammatory response is induced during cellular injury such as necrosis.¹⁰ Cellular contents that are inappropriately released after loss of plasma membrane, integrity are endogenous adjuvants or danger-associated molecular patterns (DAMPs).^11–13 These DAMPs alert the innate immune system to cellular injury and produce a proinflammatory response to aid the repair of damaged tissues. Although beneficial in the case of pathogens, the reaction to endogenous (nonmicrobial) injury can contribute to tissue damage and disease progression.The NOD-like receptors (NLRs) compose a group of pattern recognition receptors involved in a wide variety of host innate immune responses to microbial and nonmicrobial stimuli.¹⁴ The best understood members include NOD2 (NLRC2, implicated in Crohn''s disease)¹⁵ and NLRP3 (also known as NALP3 or cryopyrin). Upon activation, the NLRP3 proteins oligomerize and recruit via homotypic molecular interactions, the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and the protease caspase-1 to form a protein complex termed “the inflammasome.”¹⁶ The formation of the inflammasome induces caspase-1 autoprocessing and activation that results in the processing of cellular substrates including the cytokines pro-IL-1β and pro-IL-18.^17,18 In the case of IL-1β, caspase-1 cleaves the 35-kD pro-IL-1β to generate the mature and secreted 17-kD cytokine.Recent reports have implicated the NLRP3 inflammasome in the recognition of endogenous danger signals released from damaged and dying cells. DAMPs capable of activating the NLRP3 inflammasome include reactive oxygen species, extracellular ATP, monosodium urate crystals, nucleic acids, and extracellular matrix components including hyaluronan and biglycan.^19–24 Consistent with these observations, the NLRP3 inflammasome has been implicated in the pathogenesis of various nonmicrobial diseases, including diabetes, gout, silicosis, and acetaminophen liver toxicity.^19,20,25,26 The coexistence of cellular injury and inflammation suggests that the NLRP3 inflammasome may also play a role in regulating inflammation in CKD. Furthermore, the NLRP3 agonist biglycan and cytokines such as IL-1β, IL-18, and the IL-1 receptor all contribute to renal inflammation and fibrosis.^24,27–30 In this study, we demonstrated that the Nlrp3 inflammasome regulates renal inflammation and fibrosis during unilateral ureteral obstruction (UUO) in mice. In addition, studies of humans demonstrated increased NLRP3 in a variety of nondiabetic kidney diseases and CKD. These data provide valuable insight into the processes driving renal inflammation and CKD progression and identify NLRP3 as a novel target for therapeutic intervention.