Albuminuria and Estimated Glomerular Filtration Rate Independently Associate with Acute Kidney Injury

Acute kidney injury (AKI) is increasingly common and a significant contributor to excess death in hospitalized patients. CKD is an established risk factor for AKI; however, the independent graded association of urine albumin excretion with AKI is unknown. We analyzed a prospective cohort of 11,200 participants in the Atherosclerosis Risk in Communities (ARIC) study for the association between baseline urine albumin-to-creatinine ratio and estimated GFR (eGFR) with hospitalizations or death with AKI. The incidence of AKI events was 4.0 per 1000 person-years of follow-up. Using participants with urine albumin-to-creatinine ratios <10 mg/g as a reference, the relative hazards of AKI, adjusted for age, gender, race, cardiovascular risk factors, and categories of eGFR were 1.9 (95% CI, 1.4 to 2.6), 2.2 (95% CI, 1.6 to 3.0), and 4.8 (95% CI, 3.2 to 7.2) for urine albumin-to-creatinine ratio groups of 11 to 29 mg/g, 30 to 299 mg/g, and ≥300 mg/g, respectively. Similarly, the overall adjusted relative hazard of AKI increased with decreasing eGFR. Patterns persisted within subgroups of age, race, and gender. In summary, albuminuria and eGFR have strong, independent associations with incident AKI.It has long been recognized that an episode of acute kidney injury (AKI) can have serious health consequences.^1–4 Even a relatively small degree of renal injury increases a patient''s risk of a prolonged hospital stay, chronic kidney disease (CKD), ESRD, and death.^2,5–10 Over the last 2 decades, the incidence of hospitalized AKI has increased dramatically.^11–14 Precise estimations vary depending on population and method of case identification, but a recent community-based study of AKI estimated the incidence of nondialysis requiring AKI at 522 per 100,000 population per year and dialysis-requiring AKI at 30 per 100,000,¹³ which is well over that of ESRD.¹⁴ This increase in the burden of disease, taken with the associated poor long-term outcomes, has established AKI as a major public health issue.¹⁴Beyond routine supportive care, there exists little established medical therapy for AKI.¹⁵ Many current lines of research are focused on the prevention of AKI. However, few prospective, population-based studies have evaluated the development of AKI.^3,13,16 Hsu et al.,^13,17 along with multiple observational series in various clinical settings, have clearly established older age and CKD as risk factors for AKI.^18–24 Other observed associations with AKI include black race and male gender.^11,18,25 Proteinuria, an established risk factor in the development of cardiovascular disease,^26,27 ESRD,²⁸ and death,²⁹ is less studied in its role in the development of AKI. Hsu and colleagues demonstrated the prospective association of proteinuria with dialysis-requiring AKI; however, the proteinuria classification was binary and based on dipstick measurement.¹⁷ To our knowledge, no study has quantified the independent dose response of albuminuria with AKI hospitalization, including less severe AKI. Our study''s objective was thus to characterize prospectively the association between baseline urine albumin-to-creatinine ratio (UACR) and hospitalizations for AKI, controlling for established and potential risk factors such as CKD, age, and cardiovascular comorbidities.     

Acute Kidney Injury Associates with Increased Long-Term Mortality

Acute kidney injury (AKI) associates with higher in-hospital mortality, but whether it also associates with increased long-term mortality is unknown, particularly after accounting for residual kidney function after hospital discharge. We retrospectively analyzed data from US veteran patients who survived at least 90 d after discharge from a hospitalization. We identified AKI events not requiring dialysis from laboratory data and classified them according to the ratio of the highest creatinine during the hospitalization to the lowest creatinine measured between 90 d before hospitalization and the date of discharge. We estimated mortality risks using multivariable Cox regression models adjusting for demographics, comorbidities, medication use, primary diagnosis of admission, length of stay, mechanical ventilation, and postdischarge estimated GFR (residual kidney function). Among the 864,933 hospitalized patients in the study cohort, we identified 82,711 hospitalizations of patients with AKI. In the study population of patients who survived at least 90 d after discharge, 17.4% died during follow-up (AKI 29.8%, without AKI 16.1%). The adjusted mortality risk associated with AKI was 1.41 (95% confidence interval [CI] 1.39 to 1.43) and increased with increasing AKI stage: 1.36 (95% CI 1.34 to 1.38), 1.46 (95% CI 1.42 to 1.50), and 1.59 (95% CI 1.54 to 1.65; P < 0.001 for trend). In conclusion, AKI that does not require dialysis associates with increased long-term mortality risk, independent of residual kidney function, for patients who survive 90 d after discharge. Long-term mortality risk is highest among the most severe cases of AKI.Acute kidney injury (AKI) affects up to 15.3% of all hospitalized patients.^1,2 Regardless of the underlying cause, AKI is associated with significantly increased in-hospital morbidity, mortality, and costs.^2–16 The majority of previous studies linking AKI to mortality examined in-hospital mortality only and did not address postdischarge morbidity and mortality.^{2,4,7,8,10,11,13–16} Studies examining postdischarge mortality have focused primarily on critically ill patients with AKI that requires dialysis.⁹ Consequently, it remains unclear whether AKI that does not require dialysis is associated with a higher long-term risk for all-cause mortality.One of the challenges of long-term mortality studies is to estimate the mortality risk independently associated with AKI from risk associated with chronic kidney disease (CKD). Some patients have incomplete recovery of their kidney function after AKI, and CKD is associated with a higher risk for mortality.^17,18 To evaluate the independent long-term mortality risk of AKI, it is essential to adjust for postdischarge kidney function. The objective of this study was to estimate the postdischarge, long-term mortality risk associated with AKI while adjusting for residual kidney function in a large cohort of US veterans.     

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.     

Macrophage Phenotype Controls Long-Term AKI Outcomes—Kidney Regeneration versus Atrophy

The mechanisms that determine full recovery versus subsequent progressive CKD after AKI are largely unknown. Because macrophages regulate inflammation as well as epithelial recovery, we investigated whether macrophage activation influences AKI outcomes. IL-1 receptor–associated kinase-M (IRAK-M) is a macrophage-specific inhibitor of Toll-like receptor (TLR) and IL-1 receptor signaling that prevents polarization toward a proinflammatory phenotype. In postischemic kidneys of wild-type mice, IRAK-M expression increased for 3 weeks after AKI and declined thereafter. However, genetic depletion of IRAK-M did not affect immunopathology and renal dysfunction during early postischemic AKI. Regarding long-term outcomes, wild-type kidneys regenerated completely within 5 weeks after AKI. In contrast, IRAK-M^−/− kidneys progressively lost up to two-thirds of their original mass due to tubule loss, leaving atubular glomeruli and interstitial scarring. Moreover, M1 macrophages accumulated in the renal interstitial compartment, coincident with increased expression of proinflammatory cytokines and chemokines. Injection of bacterial CpG DNA induced the same effects in wild-type mice, and TNF-α blockade with etanercept partially prevented renal atrophy in IRAK-M^−/− mice. These results suggest that IRAK-M induction during the healing phase of AKI supports the resolution of M1 macrophage– and TNF-α–dependent renal inflammation, allowing structural regeneration and functional recovery of the injured kidney. Conversely, IRAK-M loss-of-function mutations or transient exposure to bacterial DNA may drive persistent inflammatory mononuclear phagocyte infiltrates, which impair kidney regeneration and promote CKD. Overall, these results support a novel role for IRAK-M in the regulation of wound healing and tissue regeneration.As first described in 1967¹, AKI is now considered a predictor of subsequent CKD; however, the pathophysiologic mechanisms underlying this association remain largely unknown.^2–5 Ideally, 100% of nephrons regain their structural integrity and the functional capacity they had before AKI. However, assessing structural recovery is hardly feasible in clinical practice and assessing functional recovery by serum creatinine levels or GFR estimations is impossible, because any loss up to 40%–50% can be missed due to the lack of parameter sensitivity.^6–8 Thus, the clinical observation that AKI is often followed by CKD could simply result from incomplete AKI recovery, implying that AKI episodes involve some irreversible loss of nephrons due to insufficient repair.What mechanisms regulate renal repair upon AKI? Whereas the limited capacity for podocyte regeneration (in adults) often limits glomerular repair, AKI usually involves tubular injury, which has a higher regenerative capacity.^9,10 There is accumulating experimental evidence that the associated immune response is an important determinant of AKI outcomes.¹¹ During the injury phase, necrotic tubules release molecules (e.g., histones or high-mobility group protein B1) that activate Toll-like receptors (TLRs) and inflammasomes on interstitial mononuclear phagocytes to trigger the secretion of proinflammatory cytokines and chemokines.^12–17 Chemokines guide neutrophils and proinflammatory (M1 or classically activated) macrophages to enter the site of injury,¹⁸ which largely account for the extent of tubular necrosis and thereby determine the extent of AKI.¹⁹ The rapid apoptosis of the neutrophils changes the local microenvironment and phagocytic uptake of apoptotic neutrophils induces a functional switch of the macrophages toward an anti-inflammatory (M2 or alternatively activated) phenotype.^19,20 This macrophage phenotype not only supports the resolution of postischemic renal inflammation but also actively promotes healing (i.e., tubular repair).^21–23 In fact, regenerative tubular epithelial cell proliferation starts as early as 3 hours after tubular injury; however, the resolution of renal inflammation seems to be mandatory to shift the balance of tubular repair and ongoing injury toward structural and functional tubular recovery,^24–26 similar to wound healing in general.²⁷ Therefore, factors that regulate macrophage phenotypes might determine AKI recovery and long-term outcomes.^21,28The IL-1 receptor–associated kinases (IRAKs) are important regulators of macrophage phenotype polarization because they are involved in the IL-1R/TLR/Myd88–dependent activation of NF-κB.²⁹ IRAK-4–mediated TNF receptor–associated factor 6 phosphorylation is an essential step of this signaling pathway,³⁰ which is inhibited selectively in monocytes and macrophages by IRAK-M.³¹ This way, the delayed induction of IRAK-M deactivates classically activated macrophages, which contributes to endotoxin tolerance in vitro, resolution of inflammation in vivo, compensatory anti-inflammatory response syndrome during advanced sepsis, and immunosuppressive tumor environments, as well as limits autoimmune tissue injury.^31–39 IRAK-M–mediated deactivation of proinflammatory macrophages also limits immunopathology during infections as well as osteoclast-driven osteoporosis.^36,40We speculated that IRAK-M–mediated deactivation of proinflammatory mononuclear phagocytes is required for the resolution of renal inflammation to allow structural and functional tubular reconstitution as a determinant of long-term outcomes upon AKI. We used IRAK-M–deficient mice and long-term follow-up upon postischemic AKI to address this concept.     

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.     

The RIP1-Kinase Inhibitor Necrostatin-1 Prevents Osmotic Nephrosis and Contrast-Induced AKI in Mice

The pathophysiology of contrast-induced AKI (CIAKI) is incompletely understood due to the lack of an appropriate in vivo model that demonstrates reduced kidney function before administration of radiocontrast media (RCM). Here, we examine the effects of CIAKI in vitro and introduce a murine ischemia/reperfusion injury (IRI)–based approach that allows induction of CIAKI by a single intravenous application of standard RCM after injury for in vivo studies. Whereas murine renal tubular cells and freshly isolated renal tubules rapidly absorbed RCM, plasma membrane integrity and cell viability remained preserved in vitro and ex vivo, indicating that RCM do not induce apoptosis or regulated necrosis of renal tubular cells. In vivo, the IRI-based CIAKI model exhibited typical features of clinical CIAKI, including RCM-induced osmotic nephrosis and increased serum levels of urea and creatinine that were not altered by inhibition of apoptosis. Direct evaluation of renal morphology by intravital microscopy revealed dilation of renal tubules and peritubular capillaries within 20 minutes of RCM application in uninjured mice and similar, but less dramatic, responses after IRI pretreatment. Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 (RIP1) kinase domain, prevented osmotic nephrosis and CIAKI, whereas an inactive Nec-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. In addition, Nec-1 prevented RCM-induced dilation of peritubular capillaries, suggesting a novel role unrelated to cell death for the RIP1 kinase domain in the regulation of microvascular hemodynamics and pathophysiology of CIAKI.Contrast-induced AKI (CIAKI) is the consensus name for what was formally called contrast-induced nephropathy or radiocontrast-induced AKI.^1–3 CIAKI is a common and potentially serious complication⁴ after the administration of contrast media,^5–7 especially in patients who are at risk for AKI, and is the most common cause of iatrogenic, inpatient, drug-induced AKI,^3,8,9 with outstanding implications for patients with diabetes.¹ CIAKI was recognized as the third commonest cause of hospital-acquired renal failure accounting for 11% of the cases¹⁰ even before magnetic resonance imaging contrast media were found to be associated with nephrogenic systemic fibrosis. Preclinical research thus far has failed to unravel the underlying pathophysiology of CIAKI.Programmed cell death (PCD) was used synonymously with apoptosis until regulated necrosis (RN) was discovered.¹¹ Apoptosis has been proposed to contribute to CIAKI^12–14 and asialoerythropoietin was recently demonstrated in this context to prevent CIAKI.¹⁵ Apoptosis is a process that is characterized by the activity of caspases that cleave hundreds of intracellular proteins to ultimately cause membrane blebbing, nuclear fragmentation, and regulated cellular shrinkage as a consequence of their proteolytic activity.^16,17 Within this process, caspases are capable of cleaving NFs like poly(ADP-ribose)-polymerase (PARP)-family proteins.¹⁸ PARP-1 has also been demonstrated to elicit a necrotic phenotype in kidney cells and therefore exhibits a subroutine of the RN.^19,20 It was suggested that tubular cell death by caspase-3–mediated apoptosis substantially contributes to the overall pathogenesis of CIAKI,^14,15 and one report investigated the activation of the cell death molecules PARP, Bad, and BIM.¹⁴ On the basis of these findings, the currently proposed model ascribes apoptosis a major pathophysiologic function in CIAKI.^12,13Apart from PARP-mediated RN, necroptosis, another RN pathway, is mediated by activation of the “necrosome” consisting of receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3).^11,21–23 Necroptosis involves all necrotic cellular hallmarks such as early loss of membrane integrity as well as rupture of the plasma membrane after cellular swelling. We recently described the functional relevance of both apoptosis and necroptosis in AKI.^24,25Here, we demonstrate that necrostatin-1 (Nec-1), a highly specific inhibitor of the RIP1 kinase domain, prevents CIAKI in a new and easy-to-use preclinical model for the in vivo analysis of CIAKI. Our model reliably mimics “osmotic nephrosis,” a pathologic feature that is typical of CIAKI in humans. In vitro and in vivo, we found that apoptosis is of minor pathophysiologic importance. Mechanistically, the data implicate RIP1 in the functional renal failure in vivo and provide evidence for the prevention of CIAKI by the RIP1 kinase inhibitor Nec-1 that also prevented the functional changes in the peritubular vasculature after RCM injection as demonstrated by intravital microscopy. Because of the outstanding specificity of Nec-1 that has been subject to extensive investigation,^26–29 we consider it justified to conclude that a novel non-cell death role of RIP1 might account for the functional kidney failure in CIAKI. In addition, we introduce Nec-1 as a potential inhibitor of CIAKI.     

Renalase Protects against Ischemic AKI

Elevated levels of plasma catecholamines accompany ischemic AKI, possibly contributing the inflammatory response. Renalase, an amine oxidase secreted by the proximal tubule, degrades circulating catecholamines and reduces myocardial necrosis, suggesting that it may protect against renal ischemia reperfusion injury. Here, mice subjected to renal ischemia reperfusion injury had significantly lower levels of renalase in the plasma and kidney compared with sham-operated mice. Consistent with this, plasma NE levels increased significantly after renal ischemia reperfusion injury. Furthermore, renal tubular inflammation, necrosis, and apoptosis were more severe and plasma catecholamine levels were higher in renalase-deficient mice subjected to renal ischemia reperfusion compared with wild-type mice. Administration of recombinant human renalase reduced plasma catecholamine levels and ameliorated ischemic AKI in wild-type mice. Taken together, these data suggest that renalase protects against ischemic AKI by reducing renal tubular necrosis, apoptosis, and inflammation, and that plasma renalase might be a biomarker for AKI. Recombinant renalase therapy may have potential for the prevention and treatment of AKI.Ischemic AKI is a major problem for patients subjected to major surgical procedures involving the kidney, liver, heart, or aorta.¹ Renal ischemia reperfusion (IR) injury is a frequent cause of clinical AKI, with the incidence of AKI exceeding 50% after major cardiac, hepatobiliary, or aortic surgery.^2,3 Furthermore, ischemic AKI is frequently complicated by multi-organ dysfunction, systemic inflammation, sepsis, and death.⁴ Unfortunately, there are no proven therapies to prevent or treat AKI in the perioperative setting.⁵Renalase is a 38-kD, flavin adenine dinucleotide–dependent amine oxidase synthesized and secreted by the renal proximal tubules.⁶ Renalase degrades circulating catecholamines and regulates systemic BP in rodents and humans.⁷ Plasma catecholamines and systemic BP are elevated in patients with chronic kidney dysfunction or end stage renal insufficiency.⁸ Recent studies suggest that renalase deficiency in patients with chronic renal insufficiency leads to increased plasma catecholamine levels and systemic BP.^7,9–11 However, the effect of ischemic AKI on kidney renalase and plasma catecholamine levels remains to be determined.In addition to regulating BP, renalase may protect against inflammatory tissue injury by metabolizing catecholamines. Catecholamines via activation of leukocyte α-adrenergic receptors directly cause inflammation in sepsis and multi-organ dysfunction.^12,13 Indeed, patients with chronic renal insufficiency show increased markers of inflammation that contribute directly to increased morbidity and mortality.¹⁴ In mice, renalase deficiency resulted in exacerbated cardiac IR injury and exogenous renalase administration reduced myocardial necrosis.¹⁵In this study, we hypothesized that ischemic AKI in mice leads to renalase deficiency and this renalase deficiency directly exacerbates ischemic AKI. We performed experiments to test the following: (1) whether ischemic AKI leads to reduced kidney and plasma renalase levels, (2) whether ischemic AKI-induced renalase deficiency leads to elevated plasma catecholamine (NE) levels, (3) whether renalase-deficient mice exhibit increased renal IR injury, and (4) whether exogenous administration of recombinant human renalase directly protects against ischemic AKI in mice.     

Histone Deacetylase Inhibitor Enhances Recovery after AKI

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24–48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.AKI accounts for 2%–7% of inpatient hospital admissions.^1,2 Severe AKI requiring renal replacement therapy occurs in 5%–6% of critically ill patients and has 60% mortality.³ AKI is also a risk factor for progression to CKD,⁴ particularly in patients with severe, dialysis-dependent AKI.^5,6 Currently, there are no established therapies that have been proven to prevent renal injury, accelerate the rate of recovery, or reduce postinjury fibrosis after induction of AKI.⁷ Therapeutic strategies targeting postinjury regenerative responses are attractive, because these therapies may still be efficacious if given days after the initial insult has occurred. After AKI, there is early loss of renal tubular epithelial cells (RTECs). Surviving RTECs undergo transient dedifferentiation, and over a period of days, they proliferate, redifferentiate, and repopulate damaged tubules with functional epithelium.^8–11 Lineage tracing studies indicate that most of regenerating cells are derived from surviving RTECs and not extratubular populations of stem cells.^12,13 Recent studies have also shown that a significant proportion of surviving, proliferating RTECs undergo G2/M arrest after injury, which delays recovery and promotes postinjury fibrosis.^14–16 This finding suggests that G2/M arrest plays an important role in limiting regenerative capacity of surviving RTECs and that therapies that drive cell cycle progression might accelerate recovery and reduce postinjury fibrosis after AKI.To address these questions, we developed a small molecule screen using zebrafish embryos to identify compounds that expand the embryonic renal progenitor cell field.¹⁷ Our rationale was that compounds causing expansion of this proliferative pool of renal progenitor cells might also enhance recovery and reduce postinjury fibrosis after AKI by driving cell cycle progression in surviving RTECs. Using this approach, we identified a new histone deacetylase inhibitor (HDACi), 4-(phenylthio)butanoic acid (PTBA), that expands the pool of renal progenitor cells in zebrafish embryos in a proliferation-dependent manner.¹⁷ Two other HDACis, Trichostatin A (TSA) and phenytbutanoic acid, were also shown to expand the pool. However, PTBA was significantly less toxic to the embryos than TSA and phenytbutanoic acid at their effective doses, and its esterified analog, methyl-4-(phenylthio)butanoate (m4PTB), had even greater activity.¹⁷ We now show that m4PTB accelerates recovery, increases proliferation, and reduces G2/M arrest in surviving RTECs after toxin-induced AKI in zebrafish larvae and ischemia reperfusion-induced AKI (IR-AKI) in mice. In addition, m4PTB reduces postinjury tubular atrophy and interstitial fibrosis after severe IR-AKI. We also show that beneficial effects on functional and structural recovery after AKI occur when m4PTB is administered 24 (mice) to 48 (zebrafish) hours after the initial injury. These studies validate the use of zebrafish embryos as an innovative tool to identify novel therapies for AKI and provide the first evidence of a regenerative agent that is effective when administered after the initial injury.     

Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Predicts Acute Kidney Injury in Critically Ill Adults

Urine neutrophil gelatinase-associated lipocalin (uNGAL) has shown promise as a biomarker for the early detection of acute kidney injury (AKI) in fixed models of injury, but its ability to predict AKI and provide prognostic information in critically ill adults is unknown. We prospectively studied a heterogeneous population of 451 critically ill adults, 64 (14%) and 86 (19%) of whom developed AKI within 24 and 48 h of enrollment, respectively. Median uNGAL at enrollment was higher among patients who developed AKI within 48 h compared with those who did not (190 versus 57 ng/mg creatinine, P < 0.001). The areas under the receiver operating characteristic curves describing the relationship between uNGAL level and the occurrence of AKI within 24 and 48 h were 0.71 (95% Confidence Intervals [CI]: 0.63 to 0.78) and 0.64 (95% CI: 0.57 to 0.71), respectively. Urine neutrophil gelatinase-associated lipocalin remained independently associated with the development of AKI after adjustment for age, serum creatinine closest to enrollment, illness severity, sepsis, and intensive care unit (ICU) location, although it only marginally improved the predictive performance of the clinical model alone. A Cox proportional hazards model using time to first dialysis, adjusted for APACHE II score, suggested that uNGAL independently predicts severe AKI during hospitalization [HR 2.60, 95% CI:1.55 to 4.35]. In summary, although a single measurement of uNGAL exhibited moderate predictive utility for the development and severity of AKI in a heterogeneous ICU population, its additional contribution to conventional clinical risk predictors appears limited.Despite advances in the provision of hospitalized care, the incidence of acute kidney injury (AKI) is increasing and remains an independent predictor of morbidity and mortality.^1–3 An impediment toward improving outcomes has been continued reliance on belated and unreliable markers of injury.⁴ Recent efforts directed toward discovery of biomarkers with early predictive and prognostic potential have yielded several candidates including neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1),⁵ cystatin C,^6,7 Na⁺/H⁺ exchanger isoform 3 (NHE3),⁸ and IL-18 (IL-18).^9–12NGAL is a 25-kD protein of the lipocalin family, whose structure is defined by a calyx that modulates local iron channeling and serves as a growth and differentiation factor for renal tubular epithelia.^13–15 Increased expression in the proximal renal tubular epithelia during ischemic injury has provided a rationale for its use as an early biomarker of AKI.¹⁶ Performance testing of urine NGAL (uNGAL) has yielded particular promise in patient settings with temporally defined mechanisms of injury including both pediatric and adult patients undergoing cardiopulmonary bypass,^17,18 postrenal transplantation,¹⁹ diarrhea-associated hemolytic-uremic syndrome,²⁰ and in a cohort of pediatric patients requiring mechanical ventilation.²¹We assessed the ability of uNGAL to predict both the development and severity of AKI in a mixed adult ICU cohort subject to heterogeneous patterns, timing, and causes of injury. The added and conjoint predictive ability of uNGAL beyond a panel of a priori selected clinical predictors for AKI was also quantified. Data were obtained from subjects enrolled in the ongoing NIH-sponsored Validation of biomarkers in Acute Lung Injury Diagnosis (VALID) study, a single-center, multi-ICU prospective cohort whose primary purpose is to investigate panels of new and existing plasma, serum, or urine protein biomarkers to both diagnose Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) in at-risk patients and identify patients with ALI/ARDS early who are at highest risk for adverse clinical outcomes (Figure 1).Open in a separate windowFigure 1.VALID study scheme.     

B Cells Limit Repair after Ischemic Acute Kidney Injury

There is no established modality to repair kidney damage resulting from ischemia-reperfusion injury (IRI). Early responses to IRI involve lymphocytes, but the role of B cells in tissue repair after IRI is unknown. Here, we examined B cell trafficking into postischemic mouse kidneys and compared the repair response between control (wild-type) and μMT (B cell-deficient) mice with and without adoptive transfer of B cells. B cells infiltrated postischemic kidneys and subsequently activated and differentiated to plasma cells during the repair phase. Plasma cells expressing CD126 increased and B-1 B cells trafficked into postischemic kidneys with distinct kinetics. An increase in B lymphocyte chemoattractant in the kidney preceded B cell trafficking. Postischemic kidneys of μMT mice expressed higher IL-10 and vascular endothelial growth factor and exhibited more tubular proliferation and less tubular atrophy. Adoptive transfer of B cells into μMT mice reduced tubular proliferation and increased tubular atrophy. Treatment with anti-CD126 antibody increased tubular proliferation and reduced tubular atrophy in the late repair phase. These results demonstrate that B cells may limit the repair process after kidney IRI. Targeting B cells could have therapeutic potential to improve repair after IRI.Ischemia is a leading cause of acute kidney injury (AKI) in both native kidneys and allografts. In allografts, ischemic AKI frequently results in delayed graft function.¹ Many studies have demonstrated that both innate and adaptive immune responses are involved in the pathogenesis of renal injury after renal ischemia-reperfusion injury (IRI).^2,3 On the basis of traditional concepts of adaptive immunity, lymphocytes were not expected to play an important role in the early renal injury after IRI; however, T cells were found to mediate the early phase of IRI in kidney and in other organs, both directly and indirectly.^4–6 B cells also seem to participate in the early injury response of renal IRI,⁷ and B cell products are also important in early IRI response in skeletal muscle.⁸B cells have been identified as important mediators of various autoimmune diseases, such as experimental allergic encephalomyelitis (EAE), collagen-induced arthritis, and inflammatory bowel disease.^9–11 In EAE, B cells seem to function as antigen-presenting cells during the initiation phase.^12,13 In a recent report, B cells were involved in both initiation and progression of EAE.¹⁴ Clinical trials using mAb to CD20 expressed on B cells have suggested beneficial effects in autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and multiple sclerosis.^15–18 Although ischemic AKI and autoimmune disease are traditionally viewed as different disease categories, they share a crucial feature: A prominent immune/inflammatory response.It was previously shown that B cells traffic into chronically inflamed organs, activate and form ectopic germinal centers, and locally differentiate to plasma cells.^19,20 A number of studies have demonstrated that B cells infiltrate into renal allografts and contribute to rejection^21,22; however, the exact role of B cells that have infiltrated into renal allografts is still unclear. Some studies reported that B cells could cause transplant acute cellular rejection as well as humoral rejection and increase the risk for graft failure independent of C4d peritubular deposition,^23,24 whereas other studies have not shown this clinical correlation.^25,26 One recent article characterized intragraft B cells during renal allograft rejection: Both mature B cells and interstitial plasmablasts correlated with circulating donor-specific antibody concentration and poor response to steroid therapy during rejection.²⁷ The presence of mature B cells was associated with reduced graft survival.On the basis of recent advances in studies of B cells in auto- and alloimmune diseases, the increasingly recognized pathogenic role for lymphocytes in IRI, and lack of treatment to augment repair, we tested the hypothesis that B cells modulate the repair process after kidney IRI. We analyzed the numbers and phenotypes of kidney-infiltrating B cells and the expression of B lymphocyte chemoattractant (BLC) during the repair phase. We found marked trafficking of B cells into the postischemic kidney during repair, with a distinct phenotype at different time points, along with increased BLC expression. We then evaluated the renal repair status of control (wild-type) mice, mature B cell–deficient (μMT) mice, μMT mice with adoptive B cell transfer, and μMT mice with serum transfer. We found that B cells modify tubular repair and proliferation. Finally, we targeted CD126-expressing plasma cells with an anti-CD126 antibody and found a significant improvement in tissue repair after IRI.     

Ultrasound Prevents Renal Ischemia-Reperfusion Injury by Stimulating the Splenic Cholinergic Anti-Inflammatory Pathway

AKI affects both quality of life and health care costs and is an independent risk factor for mortality. At present, there are few effective treatment options for AKI. Here, we describe a nonpharmacologic, noninvasive, ultrasound-based method to prevent renal ischemia-reperfusion injury in mice, which is a model for human AKI. We exposed anesthetized mice to an ultrasound protocol 24 hours before renal ischemia. After 24 hours of reperfusion, ultrasound-treated mice exhibited preserved kidney morphology and function compared with sham-treated mice. Ultrasound exposure before renal ischemia reduced the accumulation of CD11b⁺Ly6G^high neutrophils and CD11b⁺F4/80^high myeloid cells in kidney tissue. Furthermore, splenectomy and adoptive transfer studies revealed that the spleen and CD4⁺ T cells mediated the protective effects of ultrasound. Last, blockade or genetic deficiency of the α7 nicotinic acetylcholine receptor abrogated the protective effect of ultrasound, suggesting the involvement of the cholinergic anti-inflammatory pathway. Taken together, these results suggest that an ultrasound-based treatment could have therapeutic potential for the prevention of AKI, possibly by stimulating a splenic anti-inflammatory pathway.The immune response after ischemia-reperfusion injury (IRI) contributes to tissue damage and reduced GFR. CD45⁺ leukocyte infiltration begins as early as 30 minutes after reperfusion, with the appearance of CD4⁺ and CD8⁺ T cells, B220⁺ B cells, and the myeloid/monocyte populations (including Ly6G⁺ neutrophils, Ly6C⁺CCR2⁺ monocytes, and F4/80⁺ macrophages).^1,2 Attenuating this ensuing inflammatory response markedly reduces the development of IRI^3–9 by preventing tubular epithelial cell apoptosis, rarefaction, and scarring.¹⁰ The severity of tissue injury depends on the duration of ischemia and results in acute loss of kidney function, progressive kidney fibrosis,¹¹ and, in some cases, CKD or ESRD.^11,12Given the role of the innate immune system in the development of AKI,^1,13,14 treatments targeting inflammation could be valuable therapeutic tools. However, current immunosuppressive agents elicit adverse effects and increase the onset of various comorbid conditions.^15,16 An inherent splenic anti-inflammatory pathway has recently been described, and this pathway can be stimulated pharmacologically with nicotinic agonists or by electrical stimulation of the vagus nerve. Referred to as the cholinergic anti-inflammatory pathway, this cascade depends on the spleen, CD4⁺ T cells, and the α-7 nicotinic acetylcholine receptor (α7nAChR).¹⁷ This pathway modulates inflammation and benefits animals in models of myocardial ischemia,¹⁸ hepatic injury,¹⁹ sepsis and endotoxemia,^17,20,21 IRI,^22–24 and the response of humans injected with lipopolysaccharide.²⁵ Because of its efficacy in humans and the preclinical data from human tissues, the cholinergic anti-inflammatory pathway is a promising therapeutic target. However, improved methods to stimulate this anti-inflammatory pathway are needed.Using a modification of contrast-enhanced ultrasound (CEU), our original intent was to develop a method to precondition the renal vasculature before IRI.²⁶ This concept stems from observations that a modified CEU protocol improves blood flow in ischemic skeletal muscle.^27–29 Serendipitously, results from our initial studies revealed that prior ultrasound (US) exposure alone, in the absence of a contrast agent, prevented kidney IRI. Further studies indicated that the cholinergic anti-inflammatory pathway may be involved because of the dependence of the US treatment on an intact spleen and the α7nAChR. These studies provide evidence for a simple, portable, noninvasive, and nonpharmacologic approach to prevent AKI.     

Recurrence of Lupus Nephritis after Kidney Transplantation

The frequency and outcome of recurrent lupus nephritis (RLN) among recipients of a kidney allograft vary among single-center reports. From the United Network for Organ Sharing files, we estimated the period prevalence and predictors of RLN in recipients who received a transplant between 1987 and 2006 and assessed the effects of RLN on allograft failure and recipients'' survival. Among 6850 recipients of a kidney allograft with systemic lupus erythematosus, 167 recipients had RLN, 1770 experienced rejection, and 4913 control subjects did not experience rejection. The period prevalence of RLN was 2.44%. Non-Hispanic black race, female gender, and age <33 years each independently increased the odds of RLN. Graft failure occurred in 156 (93%) of those with RLN, 1517 (86%) of those with rejection, and 923 (19%) of control subjects without rejection. Although recipients with RLN had a fourfold greater risk for graft failure compared with control subjects without rejection, only 7% of graft failure episodes were attributable to RLN compared and 43% to rejection. During follow-up, 867 (13%) recipients died: 27 (16%) in the RLN group, 313 (18%) in the rejection group, and 527 (11%) in the control group. In summary, severe RLN is uncommon in recipients of a kidney allograft, but black recipients, female recipient, and younger recipients are at increased risk. Although RLN significantly increases the risk for graft failure, it contributes far less than rejection to its overall incidence; therefore, these findings should not keep patients with lupus from seeking a kidney transplant.The frequency and clinical impact of recurrent lupus nephritis (RLN) in the kidney allograft of recipients with systemic lupus erythematosus (SLE) varies considerably in both prospective and retrospective studies.^1–25 In 1996, Mojcik and Klippel²⁶ pooled data from a total of 366 allografts transplanted in 338 recipients. In that review, histologic RLN was present in 3.8% of the grafts. Contrasting, in the studies by Goral et al.²⁷ and Nyberg et al.,¹⁰ RLN was reported in a much higher proportion: 30 and 44% of recipients, respectively.The clinical consequences of RLN on patient and allograft survival have ranged from no effect to a significant increase in the risk for graft loss and patient mortality.^24,27–31 In this case-control study, we estimated the period prevalence of RLN in kidney transplant recipients who had ESRD secondary to lupus nephritis and received a transplant between October 1987 and October 2006. We assessed the effects of RLN on graft failure and recipient survival and the risk factors leading to the development of RLN.     

Pregnancy and Maternal Outcomes Among Kidney Transplant Recipients

Fertility rates, pregnancy, and maternal outcomes are not well described among women with a functioning kidney transplant. Using data from the Australian and New Zealand Dialysis and Transplant Registry, we analyzed 40 yr of pregnancy-related outcomes for transplant recipients. This analysis included 444 live births reported from 577 pregnancies; the absolute but not relative fertility rate fell during these four decades. Of pregnancies achieved, 97% were beyond the first year after transplantation. The mean age at the time of pregnancy was 29 ± 5 yr. Compared with previous decades, the mean age during the last decade increased significantly to 32 yr (P < 0.001). The proportion of live births doubled during the last decade, whereas surgical terminations declined (P < 0.001). The fertility rate (or live-birth rate) for this cohort of women was 0.19 (95% confidence interval 0.17 to 0.21) relative to the Australian background population. We also matched 120 parous with 120 nulliparous women by year of transplantation, duration of transplant, age at transplantation ±5 yr, and predelivery creatinine for parous women or serum creatinine for nulliparous women; a first live birth was not associated with a poorer 20-yr graft or patient survival. Maternal complications included preeclampsia in 27% and gestational diabetes in 1%. Taken together, these data confirm that a live birth in women with a functioning graft does not have an adverse impact on graft and patient survival.One of the many perceived benefits of kidney transplantation has been restoration of pituitary-ovarian function and fertility in women of reproductive age. Prenatal advice for women with a functioning kidney transplant has been primarily based on data derived from observational research,^1–13 and the reported live-birth rates achieved in such women range from 43.2¹⁴ to 82%.¹⁵Although an increased pregnancy event number has been reported for women with a functioning kidney transplant,¹⁶ little is actually known about “pregnancy rate changes” during the past 40 yr. More importantly, long-term graft and maternal survival analyses, referred to when advising women who have undergone transplantation and are considering a pregnancy, have been mostly performed without adequate matching,¹² or, alternatively, matching has been used but outcomes followed up for only brief intervals^14,17,18 and in small cohorts.^19–22 Published graft matching studies to date suggest no adverse impact 10 yr after a live birth.¹⁴In most instances, pregnancies in women with a kidney graft have been encouraged. Historically, renal function,^8,15,17,18 baseline proteinuria,²³ intercurrent hypertension,^1,24 and time from transplantation^{1,3,5,8,14,15,18,24,25} have been used to predict adverse event risks to the mother, kidney, and offspring. To this are added the often unquantifiable inherent risks for genetically transmitted diseases or the problems associated with prematurity.^26,27 More recently, epidemiologic evidence suggests low birth weight may be associated with the development of hypertension,²⁸ cardiovascular disease,²⁹ insulin resistance,³⁰ and end-stage renal failure.³¹ Moreover, low birth weight is associated with an increased risk for hypertension, independent of genetic and shared environmental factors.³²Series published to date have not captured all pregnancy events or their outcomes. Limitations of some of the published studies include short duration of follow-up and studies with no adequate or long-term matching for decade and renal function.We examined fertility rates, pregnancy rates, and pregnancy outcomes over 40 yr in an at-risk population, defined as women who were aged between 15 and 49 and had a functioning kidney transplant, using ANZDATA registry data. In addition, maternal and graft outcomes were analyzed, and, uniquely, a matched cohort analysis of 120 nulliparous and 120 parous women who had undergone transplantation enabled analysis of outcomes at 20 yr.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Racial Composition of Residential Areas Associates with Access to Pre-ESRD Nephrology Care

Referral to a nephrologist before initiation of chronic dialysis occurs less frequently for blacks than whites, but the reasons for this disparity are incompletely understood. Here, we examined the contribution of racial composition by zip code on access and quality of nephrology care before initiation of renal replacement therapy (RRT). We retrospectively studied a cohort study of 92,000 white and black adults who initiated RRT in the United States between June 1, 2005, and October 5, 2006. The percentage of patients without pre-ESRD nephrology care ranged from 30% among those who lived in zip codes with <5% black residents to 41% among those who lived in areas with >50% black residents. In adjusted analyses, as the percentage of blacks in residential areas increased, the likelihood of not receiving pre-ESRD nephrology care increased. Among patients who received nephrology care, the quality of care (timing of care and proportion of patients who received a pre-emptive renal transplant, who initiated therapy with peritoneal dialysis, or who had a permanent hemodialysis access) did not differ by the racial composition of their residential area. In conclusion, racial composition of residential areas associates with access to nephrology care but not with quality of the nephrology care received.Clinical practice guidelines for chronic kidney disease emphasize the importance of timely referral to a nephrologist for patients expected to require renal replacement therapy.^1–3 Nevertheless, approximately 33% of end-stage renal disease (ESRD) patients in the United States do not see a nephrologist before initiation of chronic dialysis.^4,5 Lack of timely access to nephrology care is associated with several adverse outcomes after initiation of dialysis including higher mortality rates,^6–9 higher rates of hospitalization,¹⁰ lower rates of renal transplantation,^11,12 delayed creation of arteriovenous fistulae,¹³ lower rates of achievement of dialysis treatment targets,^14,15 and a lower likelihood of receiving home-based dialysis therapies such as peritoneal dialysis and home hemodialysis.^3,16–18In the United States, black dialysis patients are less likely than white patients to have received nephrology care before onset of ESRD.^9,19–21 They are also less likely to receive a pre-emptive kidney transplant,^22,23 select peritoneal dialysis over hemodialysis,²⁴ and have a vascular access in place at onset of hemodialysis.^25,26 Several factors may contribute to these disparities including differences in insurance status, level of education, physician knowledge or biases, and patient preferences.^9,27–29 In addition, geographic factors such as proximity to dialysis facilities and degree of urbanization also affect access to nephrology care.^30–33A substantial proportion of black patients are also more likely to live in areas where most other residents are black. A recent study demonstrated that both black and white dialysis patients living in predominantly black zip codes were less likely to receive a kidney transplant than patients living in other areas.³⁴ Although levels of income, wealth and education tended to be lower among residents of predominantly black zip codes, lower transplant rates among dialysis patients living in these areas were not completely explained by these measures. Patients who live in predominantly black zip codes may face unique barriers to care that are not explained by measured socioeconomic characteristics of those zip codes. We therefore hypothesized that dialysis patients living in zip codes with a greater proportion of black residents would be less likely to have received pre-ESRD care and less likely to have received high-quality nephrology care than patients living in other zip codes. We also hypothesized that these relationships would not be completely explained by differences in zip code socioeconomic status or patient race.     

Severe Renal Mass Reduction Impairs Recovery and Promotes Fibrosis after AKI

Preexisting CKD may affect the severity of and/or recovery from AKI. We assessed the impact of prior graded normotensive renal mass reduction on ischemia-reperfusion–induced AKI. Rats underwent 40 minutes of ischemia 2 weeks after right uninephrectomy and surgical excision of both poles of the left kidney (75% reduction of renal mass), right uninephrectomy (50% reduction of renal mass), or sham reduction of renal mass. The severity of AKI was comparable among groups, which was reflected by similarly increased serum creatinine (S_Cr; approximately 4.5 mg/dl) at 2 days, tubule necrosis at 3 days, and vimentin-expressing regenerating tubules at 7 days postischemia-reperfusion. However, S_Cr remained elevated compared with preischemia-reperfusion values, and more tubules failed to differentiate during late recovery 4 weeks after ischemia-reperfusion in rats with 75% renal mass reduction relative to other groups. Tubules that failed to differentiate continued to produce vimentin, exhibited vicarious proliferative signaling, and expressed less vascular endothelial growth factor but more profibrotic peptides. The disproportionate failure of regenerating tubules to redifferentiate in rats with 75% renal mass reduction associated with more severe capillary rarefaction and greater tubulointerstitial fibrosis. Furthermore, initially normotensive rats with 75% renal mass reduction developed hypertension and proteinuria, 2–4 weeks postischemia-reperfusion. In summary, severe (>50%) renal mass reduction disproportionately compromised tubule repair, diminished capillary density, and promoted fibrosis with hypertension after ischemia-reperfusion–induced AKI in rats, suggesting that accelerated declines of renal function may occur after AKI in patients with preexisting CKD.Clinical studies suggest that AKI worsens preexisting CKD and accelerates progression to end stage because of residual structural and functional deficits.^1–7 CKD, per se, may increase the risk and severity of AKI and the likelihood of incomplete recovery from AKI.⁸ Thus, AKI and CKD reinforce each other to increase nephron loss and tubulointerstitial fibrosis (TIF).⁹ Nevertheless, causal relationships for both aspects of the AKI–CKD nexus¹⁰ (AKI resulting in CKD/ESRD and CKD, per se, predisposing to AKI) have been questioned.^11,12 These concerns were recently reviewed.¹³ AKI–CKD relationships have also been questioned on the grounds that mechanisms for AKI–CKD interactions are ill-defined and controversial.^14–16We addressed these uncertainties by investigating the impact of normotensive renal mass reduction (RMR; 0%, 50%, and 75%) of 2-weeks duration on AKI induced by ischemia-reperfusion (I/R) in rats. We addressed three questions. (1) Does prior RMR increase AKI severity? (2) Does prior RMR impair recovery from AKI? (3) Does prior RMR predispose to the development of more severe TIF during recovery from AKI? To achieve 75% RMR, we used the normotensive rat remnant kidney model produced by uninephrectomy and excision of approximately one half of the contralateral kidney.¹⁷ Normotensive 75% RMR, per se, causes minimal TIF even after 4 months,^17–19 despite adaptations (e.g., glomerular hyperfiltration, hypertrophy, oxidative stress, etc.) that are postulated to cause fibrosis in hypertensive RMR models.^20–22