Aluminum Citrate Prevents Renal Injury from Calcium Oxalate Crystal Deposition

Calcium oxalate monohydrate crystals are responsible for the kidney injury associated with exposure to ethylene glycol or severe hyperoxaluria. Current treatment strategies target the formation of calcium oxalate but not its interaction with kidney tissue. Because aluminum citrate blocks calcium oxalate binding and toxicity in human kidney cells, it may provide a different therapeutic approach to calcium oxalate-induced injury. Here, we tested the effects of aluminum citrate and sodium citrate in a Wistar rat model of acute high-dose ethylene glycol exposure. Aluminum citrate, but not sodium citrate, attenuated increases in urea nitrogen, creatinine, and the ratio of kidney to body weight in ethylene glycol–treated rats. Compared with ethylene glycol alone, the addition of aluminum citrate significantly increased the urinary excretion of both crystalline calcium and crystalline oxalate and decreased the deposition of crystals in renal tissue. In vitro, aluminum citrate interacted directly with oxalate crystals to inhibit their uptake by proximal tubule cells. These results suggest that treating with aluminum citrate attenuates renal injury in rats with severe ethylene glycol toxicity, apparently by inhibiting calcium oxalate’s interaction with, and retention by, the kidney epithelium.Ethylene glycol (EG) is a common household poison found in antifreeze, automotive engine coolants, and water-based latex paints. Approximately 5000 accidental or intentional EG ingestions occur per year in the United States, resulting in about 20–30 deaths.¹ Acute EG poisoning can result in central nervous system depression, metabolic acidosis, acute renal failure, coma, and death.² Ethylene glycol itself is nontoxic. However, the end metabolite, oxalate, is insoluble in the presence of calcium and forms oxalate crystals (primarily calcium oxalate monohydrate [COM]) that are deposited in the kidney tissue. Pathologic studies have shown that COM accumulation in the tubule correlates strongly with the degree of proximal tubule cell necrosis and with renal failure.^3,4 Experiments using kidney cell cultures have convincingly shown that COM, and not the metabolites glycolate, glyoxylate, or ionic oxalate, is the metabolite responsible for the renal toxicity associated with EG poisoning.^5–9 COM crystals can bind to kidney cell membranes and can be internalized by kidney cells,^7,10–12 where they induce mitochondrial dysfunction leading to cell death.^12–14 The ability to induce cell death is closely linked with the degree of cellular internalization of COM crystals.¹²EG is metabolized fairly rapidly, so there is little time between ingestion and the formation of the toxic metabolites; thus, quick and aggressive treatment is required.^2,15 With early diagnosis, inhibition of the enzyme alcohol dehydrogenase using fomepizole or ethanol can block the metabolism of EG, effectively preventing the formation of COM. If renal failure has already occurred, long-term hemodialysis (2–6 months) must be used to restore kidney function.² Primary hyperoxaluria, a genetic disease caused by deficiencies in the glyoxalate-metabolizing enzymes, alanine-glyoxylate aminotransferase (type 1) or glyoxylate reductase/hydroxypyruvate reductase (type 2), also results in COM crystal deposits and ultimately kidney injury.¹⁶ Potassium citrate and sodium citrate, which raise the urinary excretion of citrate to chelate calcium and retard the formation of oxalate crystals,¹⁷ are used clinically to minimize crystal formation during hyperoxaluria and can be used to treat kidney stone recurrence,¹⁸ but neither citrate blocks the toxicity from COM per se.¹⁹ As such, for patients with renal damage associated with EG poisoning and primary hyperoxaluria, there are no good therapies to block the effects of COM crystals at the primary site of action in the kidney lumen and the subsequent renal damage.Aluminum citrate has been shown to be a possible therapeutic agent by blocking COM toxicity in vitro and to operate by a mechanism of action unique from the citrate salts used clinically.¹⁹ Of the citrate salts (aluminum, calcium, ammonium, sodium, and potassium) tested against COM-induced cytotoxicity in human proximal tubule (HPT) cells, only aluminum citrate significantly reduces cell death.¹⁹ Also, treatment with aluminum chloride does not reduce COM-induced toxicity on kidney cells or erythrocytes, suggesting that efficacy is not due to the aluminum moiety but rather to aluminum complexed with citrate. Aluminum is primarily excreted by the kidneys, and when complexed with citrate, aluminum is freely filtered at the glomerulus and removed from the body.²⁰ For the purposes of treating COM toxicity, the body’s propensity to filter aluminum citrate into the urine is ideal,²¹ so that it is present at the primary site of action in the proximal tubule lumen of the nephron. Aluminum accumulation has been linked to many diseases, including microcytic anemia, bone disease, and neurologic disorders.²² We are aware that aluminum citrate will probably never be a suitable drug candidate for treating COM toxicities because of the controversy surrounding its potential toxicities, but studies of aluminum citrate’s efficacy and mechanism of action are necessary for developing alternative drug therapies for diseases involving COM, including EG poisoning and severe hyperoxaluria. The purpose of this study was to examine the efficacy of aluminum citrate in treating COM toxicity in a rat model of acute EG poisoning and to understand the mechanism of this inhibition of toxicity.     

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.     

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.     

Glucose- and hormone-induced cAMP oscillations in α- and β-cells within intact pancreatic islets

OBJECTIVE

cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.

RESEARCH DESIGN AND METHODS

The subplasma-membrane cAMP concentration ([cAMP]_pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells.

RESULTS

In islets exposed to 3 mmol/L glucose, [cAMP]_pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]_pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]_pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]_pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in the β-cells but not caused by the changes in [Ca²⁺]_i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]_pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively.

CONCLUSIONS

Oscillatory [cAMP]_pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]_pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.Cyclic AMP and Ca²⁺ are key messengers in the regulation of insulin and glucagon secretion from pancreatic β- and α-cells, respectively, by nutrients, hormones, and neural factors. Glucose stimulation of insulin secretion involves uptake and metabolism of the sugar in the β-cells, closure of ATP-sensitive K⁺ channels, and depolarization-induced Ca²⁺ entry generating oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that trigger periodic exocytosis of secretory granules (1,2). This process is amplified by mechanism(s) acting distal to the elevation of Ca²⁺ (3). cAMP promotes exocytosis by facilitating the generation of Ca²⁺ signals (4,5), by sensitizing the secretory machinery to Ca²⁺ (4,6), and by stimulating mobilization and priming of granules via protein kinase A– and Epac-dependent pathways (7,8).Measurements of the cAMP concentration beneath the plasma membrane ([cAMP]_pm) in individual INS-1 β-cells showed that glucagon-like peptide-1(7-36)-amide (GLP-1) induces [cAMP]_pm elevation, often manifested as oscillations (9). Glucose has been regarded to only modestly raise islet cAMP, supposedly by amplifying the effect of glucagon (10), but single-cell cAMP recordings have recently shown that glucose alone induces marked elevation of cAMP in MIN6 β-cells (11,12) and primary mouse β-cells (12,13). Although one study reported that the glucose-induced cAMP response depends on elevation of [Ca²⁺]_i (11), other studies show only partial or no Ca²⁺-dependence of the cAMP signal (12,13). Like hormone stimulation, glucose induces oscillations of [cAMP]_pm, and coordination of the [cAMP]_pm and [Ca²⁺]_i elevations generates pulsatile insulin release (12,14).There are 10 isoforms of cAMP-generating adenylyl cyclases (ACs) with different regulatory properties, nine of which are transmembrane (tm) proteins stimulated by Gα_s and the plant diterpene forskolin. Such tmACs mediate the cAMP-elevating action of glucagon and incretin hormones (15). β-cells express multiple tmACs (16), and the Ca²⁺-stimulated AC8 has been proposed to be particularly important for integrating hormone- and depolarization-evoked signals (17). Soluble AC (sAC) is the only isoform that lacks transmembrane domains. It is insensitive to forskolin and G-proteins but stimulated by bicarbonate (18) and Ca²⁺ (19). Although sAC was first found in the testis, it also seems to be expressed in other tissues and was recently proposed to be involved in glucose-induced cAMP production in insulin-secreting cells (20).Like insulin secretion, exocytosis of glucagon from the α-cells is triggered by an increase of [Ca²⁺]_i (21). Glucagon release is stimulated by absence of glucose and is maximally inhibited when the sugar concentration approaches the threshold for stimulation of insulin secretion (22). Under hypoglycemic conditions, glucagon secretion is also stimulated by adrenaline, which raises [Ca²⁺]_i and [cAMP]_pm via α₁- and β-adrenergic mechanisms (23,24). There are fundamentally different ideas about the mechanisms underlying glucose inhibition of glucagon secretion, including paracrine influences from β- and δ-cells (25–29) and direct actions of glucose on the α-cells, resulting in depolarization- (30) or hyperpolarization-mediated (22) inhibition of exocytosis. Apart from the inhibitory effect of glucose, we observed that very high glucose concentrations unexpectedly stimulate glucagon secretion (31). The stimulatory component may be important under physiological conditions because the hormone is released in pulses from rat (32) and human (33) islets. Glucose thus causes alternating periods of stimulation and inhibition resulting in time-average reduction of glucagon secretion. Ca²⁺ is probably not the only messenger in glucose-regulated glucagon release (29). Like for insulin secretion, cAMP is believed to promote glucagon release by enhancing intracellular Ca²⁺ mobilization, Ca²⁺ influx through the plasma membrane, and mobilization of secretory granules (23,24,34,35). However, it has also been suggested that cAMP-mediated reduction of N-type Ca²⁺ currents can explain the inhibitory effect of GLP-1 on glucagon secretion (36).Until now, nothing was known about cAMP kinetics in α-cells and all information on primary β-cells was based on studies of dispersed islet cells. However, as a result of gap junctional coupling and paracrine influences, the electrophysiological characteristics and [Ca²⁺]_i signaling in intact islets differ considerably from those in dispersed β-cells (2). Therefore, the aim of the current study was to clarify how glucose, glucagon, and GLP-1 affect cAMP signaling in α- and β-cells within intact islets of Langerhans.     

Cyclic GMP kinase I modulates glucagon release from pancreatic α-cells

OBJECTIVE

The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.

RESEARCH DESIGN AND METHODS

Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iβ rescue mice [α/βRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²⁺ ([Ca²⁺]_i) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.

RESULTS

Immunohistochemistry showed that cGKI is present in α- but not in β-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²⁺]_i levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²⁺]_i or hormone release in cGKI-deficient islets.

CONCLUSIONS

We propose that cGKI modulates glucagon release by suppression of [Ca²⁺]_i in α-cells.The complex and tightly controlled process of glucagon secretion from pancreatic α-cells is important for the maintenance of blood glucose homeostasis (1). Glucagon release is physiologically regulated by multiple signaling pathways that include neuronal control of α-cell function, paracrine factors such as insulin (2,3), and/or γ-aminobutyric acid (GABA) (4) released from β-cells, somatostatin (SST) secreted from adjacent δ-cells (5), and the inhibitory role of high blood glucose itself that directly acts on α-cells to suppresses glucagon release (3,6).Controversial data have been reported for the physiologic significance of the nitric oxide (NO) pathway for islet functions. Both types of constitutive NOS (eNOS, nNOS) isozymes have been identified in islets (7–11). It was suggested that NO stimulates glucose-induced insulin release (7,10), was a negative modulator of insulin release (8,12,13) or had no effect (14). These discrepant results are probably caused by the analysis of various β-cell–derived cell lines compared with intact islets, the use of different types and concentrations of NO-donors and NOS-inhibitors. Additional data suggested that iNOS-derived NO is involved in autoimmune reactions that cause β-cell–dysfunction leading to insulin-dependent diabetes (15,16).It has been difficult to discriminate between a direct action of NO on α-cells and an indirect effect of NO via β-cells since β-cell factors are potent inhibitors of α-cell activity (12,17,18). An important target of NO is the soluble guanylyl cyclase (sGC) that generates the second messenger cyclic guanosine-3′-5′-monophosphate (cGMP) (19). Some studies detected increased islet cGMP levels upon treatment with cytokines and l-arginine (12,15,20). cGMP analogues were reported to potentiate insulin release directly (21), suggesting that cGMP-dependent effectors are involved in the control of islet activity. The cGMP-dependent protein kinase type I (cGKI) is an important intracellular mediator of NO/cGMP signaling in many cells (22). The analysis of cGKI-deficient mice revealed that cGKI mediates the inhibitory effects of NO on platelet aggregation, the negative inotropic effect of NO/cGMP in the murine heart, and the NO-induced relaxation of blood vessels (22). However, cGKI knockout mice could not be analyzed reliably for a distorted islet function because they display abnormalities of various organ systems and die within the first 6 weeks (23). Recently, we generated an improved mouse model to study the specific roles of cGKI in vivo (24,25). These mice express either the cGKIα or cGKIβ isozyme selectively in smooth muscle cells (SMCs) on a cGKI-deficient genetic background. Since the animals show a prolonged life expectancy and normal SMC functions they were termed cGKIα and cGKIβ rescue mice (αRM and βRM, respectively).We examined the role of cGKI for the regulation of glucose homeostasis using cGKI-KO mice (23) and rescue mice (RM) (24,25) models. We show that cGKI is expressed in the α-cells of the endocrine pancreas, whereas in different gene-targeted animals the cGKI protein was not detectable. Furthermore, we demonstrate that islet cGKI regulates glucagon release by modulation of the glucose-dependent changes of [Ca²⁺]_i that trigger exocytosis. These ex-vivo findings were supported by elevated serum levels of basal glucose and glucagon in intact RM animals.     

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.     

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.     

Connexin 30 Deficiency Impairs Renal Tubular ATP Release and Pressure Natriuresis

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na⁺ excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel–dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.It is well established that renal tubular epithelial cells release ATP,^1–4 which then binds to purinergic receptors along the nephron and collecting ducts to regulate salt and water reabsorption^5–12; however, the molecular mechanism of ATP release and the identity of ATP channels are less clear. The possible mechanisms include vesicular release,^1,2 pannexin or connexin hemichannels,^13–16 ATP-permeable anion channels such as the cystic fibrosis transmembrane conductance regulator,¹⁷ or the maxi anion channel.¹⁸ Previous work also suggested that the release of ATP in epithelia may be triggered by mechanical stimulation,^19–21 including elevations in tubular fluid flow rates. For example, tubular flow–dependent ATP release resulting in purinergic calcium signaling has been well characterized in the cortical collecting duct (CCD)^21–23; however, the ATP release mechanism has not been identified.Connexin 30 (Cx30) is a member of the connexin (Cx) family of transmembrane proteins (more than 20 isoforms). Various Cx proteins can form large pores in the nonjunctional plasma membrane of cells (gap junction hemichannels) before the assembly of two Cx hemichannels into complete gap junction channels between adjacent cells.¹⁴ Cx hemichannels are large, mechanosensitive ion channels that allow the passage of a variety of small molecules and metabolites, including ATP.^14–16 Our laboratory localized Cx30 in the nonjunctional apical plasma membrane of cells in the distal nephron,²⁴ suggesting that Cx30 may function as an ATP-releasing, luminal membrane hemichannel in this nephron segment. To address this hypothesis, we applied a recently developed ATP biosensor technique¹⁸ combined with a Cx30 knockout mouse model²⁵ to show tubular flow–induced ATP release through luminal Cx30 hemichannels in the renal collecting duct. The physiologic significance of this mechanically induced Cx30-mediated luminal ATP release was further studied by assessing its involvement in pressure natriuresis, a multifactorial, multimechanistic intrarenal phenomenon that causes diuresis and natriuresis in response to elevations in systemic BP.²⁶ Pressure natriuresis involves proximal and distal nephron components; hemodynamic factors such as medullary blood flow and renal interstitial hydrostatic pressure; and renal autocoids such as nitric oxide, prostaglandins, kinins, and angiotensin II.^26–32 Because pressure natriuresis is related to mechanical factors and Cx hemichannels are mechanosensitive, we hypothesized that Cx30-mediated ATP release is involved, at least in part, in pressure natriuresis, a critically important phenomenon in the maintenance of body fluid balance and BP.     

Enhanced Glucose Tolerance by SK4 Channel Inhibition in Pancreatic ��-Cells

OBJECTIVE

Ca²⁺-regulated K⁺ channels are involved in numerous Ca²⁺-dependent signaling pathways. In this study, we investigated whether the Ca²⁺-activated K⁺ channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic β-cell function.

RESEARCH DESIGN AND METHODS

Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

RESULTS

SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO β-cells stimulated with 15 mmol/l glucose had an increased Ca²⁺ action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca²⁺]_c. In addition, glucose responsiveness of membrane potential, [Ca²⁺]_c, and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K⁺ currents and concomitant membrane hyperpolarization could be evoked in WT β-cells by the SK4 channel opener DCEBIO (100 μmol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 μmol/l) partly inhibited K_Ca currents and induced electrical activity at a threshold glucose concentration. In stimulated WT β-cells, TRAM-34 further increased [Ca²⁺]_c and broadened action potentials similar to those seen in SK4-KO β-cells. SK4 channels were found to substantially contribute to K_slow (slowly activating K⁺ current).

CONCLUSIONS

SK4 channels are involved in β-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated β-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.SK4 channels are Ca²⁺-activated K⁺ channels of intermediate conductance (synonymous with IK1 and KCa3.1) encoded by the KCNN4 gene. They are primarily expressed in cells of the hematopoietic system, where they represent the Gardos channel (1). Channel activation requires Ca²⁺ increase and determines the cell volume of T-cells and erythrocytes by elevating K⁺ efflux. In organs regulating salt and fluid transport (e.g., colon, salivary glands, and lung), SK4 current provides the driving force for secondary electrogenic ion transport (2–4). SK4 channels are suggested to be involved in mast cell stimulation (5), and channel upregulation is important for lymphocyte activation and cell proliferation (6,7). For enteric neurons, SK4 channels seem to mediate the late after-hyperpolarization (8). In 1997, SK4 channels were cloned from human pancreatic tissue (9). A detailed investigation of mRNA and protein expression of K_Ca channels of intermediate (SK4) and small conductance (SK1–3) was performed by Tamarina et al. (10) showing mRNA expression of these channels in murine islets.In the past, ATP-sensitive K⁺ (K_ATP) channels were considered to be essential for glucose homeostasis. Consequently, K_ATP channel inhibitors are important drugs to augment insulin secretion in type 2 diabetic subjects. However, with the generation of two K_ATP channel-deficient mouse models (SUR1 and Kir6.2 knockout), it was shown that K_ATP channels are not indispensable for glycemic control (11–14). Neither SUR1 nor Kir6.2 knockout mice show severe hypoglycemia or any symptoms of insulin hypersecretion. Several reports provide evidence that efficient blood glucose regulation and even glucose-dependent insulin secretion (15–17) is possible despite K_ATP channel ablation. In the search for compensatory mechanisms, modulation of insulin release by other K⁺ channels gains particular interest.Besides K_Ca channels, pancreatic β-cells express K⁺ channels exclusively regulated by voltage (K_v channels) (10,18,19). Several studies indicate that K_v channel activation plays a role in action potential (AP) repolarization (20–22). Blocking these channels broadens APs and increases insulin secretion (23–25). Recently, it was shown that K_v2.1 ablation drastically reduces K_v currents of isolated β-cells (26). Interestingly, this coincides with improved glucose tolerance pointing to a specific role for K_v2.1 in the regulation of insulin secretion.For decades, it was discussed whether K_Ca channels participate in the regulation of β-cell activity (27). An early report (28) described K_Ca currents that were periodically activated by inositol-trisphosphate–dependent Ca²⁺ mobilization. The existence of large conductance K_Ca channels (BK channels) in pancreatic β-cells and insulin-secreting cell lines has been verified by several groups (29–31). However, since blockage of BK channels does not alter membrane potential oscillations (31,32), these channels are not considered to play a major role in glucose-stimulated insulin release. In 1999, a K⁺ current activating with increasing Ca²⁺ influx during burst phases of glucose-stimulated β-cells was detected (33). The current, termed K_slow because of its delayed and slow onset, strongly depends on [Ca²⁺]_c. Further analysis suggested that ∼50% could be ascribed to K_ATP current (34). However, the remaining sulfonylurea-insensitive component of K_slow does not resemble the characteristics of any known K_Ca channel (33), and its precise nature remains to be identified. It has been suggested that K_Ca channels of small conductance (SK1–3) play a functional role in β-cells (10,35), but at present, there is only limited information about their contribution to glucose handling of the whole organism.Because up to now nothing is known about the significance of SK4 channels in pancreatic β-cells, this study was performed to elucidate whether SK4 channels are suitable candidates for modulation of β-cell function. We demonstrate that SK4 channels are expressed in murine islets and investigated the influence of constitutive SK4 channel knockout (SK4-KO) and of pharmacological SK4 channel inhibition on glucose homeostasis, insulin sensitivity, and the stimulus-secretion cascade of murine pancreatic β-cells.     

TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca²⁺-permeable TRPV4 channel underlies flow-dependent Ca²⁺ signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca²⁺]_i, and loss of flow-mediated [Ca²⁺]_i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca²⁺ signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca²⁺ signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.Polycystic kidney disease (PKD) is a cohort of monogenic disorders that result in development and subsequent growth of renal cysts filled with fluid.^1–5 Cyst enlargement compromises function of surrounding nephrons and progresses to ESRD.^1,6 In the more common form of PKD, autosomal dominant PKD (ADPKD), which is caused by mutations of polycystin 1 (PC1) and polycystin 2 (PC2), renal cysts are formed along the full length of the nephron with prevalence to the collecting duct (CD).^1,7 In the rarer and more severe autosomal recessive PKD (ARPKD), renal cyst formation is virtually restricted to the CD.^1,2,5,8 Mutations of the PKHD1 gene encoding fibrocystin underlie the genetic basis of the disease.^6,8,9 Although the exact function of the protein is unknown, fibrocystin was shown to be expressed in primary cilia where it can interact and form complexes with PC2, possibly participating in mechanotransduction.^10–12It is accepted that the CD cells elevate [Ca²⁺]_i in response to mechanical stress arising from variations in tubular flow or tubular composition.^13–23 Impaired mechanosensitive [Ca²⁺]_i responses, reported for both cultured ADPKD²⁴ and ARPKD^25,26 cells, point to a possible fundamental role of disrupted [Ca²⁺]_i signaling in cystogenesis. The central cilia and cilia-associated PC1 and PC2 were proposed to mediate flow-induced cellular responses.^19,27 However, homomeric PC2 channels are not mechanosensitive and fail to increase [Ca²⁺]_i in response to flow and hypotonicity.^28,29 Furthermore, intercalated cells, which lack primary cilia, respond to flow changes with comparable increases in [Ca²⁺]_i as observed in principal cells, which have primary cilia.^16,30 Therefore, additional mechanisms conferring mechanosensitivity to the CD cells need to be considered.Transient receptor potential (TRP) channels are known to participate in cellular responses to a variety of environmental stimuli, including thermosensation, chemosensation, and mechanical forces (reviewed in Song and Yuan³¹). Several TRP channels, including TRPC3, TRPC6, and TRPV4, can be detected in the native CD cells and CD-originated cultured lines.^19,32–34 Among these channels, TRPV4 has routinely been shown to be activated by mechanical stimuli.^34–38 Indeed, we documented that endogenous TRPV4 in M-1 CD cells is stimulated by increases in flow, a response that is abolished by TRPV4 small interfering RNA knockdown.^34,38 We further demonstrated a lack of flow-mediated [Ca²⁺]_i elevations in CD from TRPV4−/− mice.³⁰ Consistently, flow-mediated Ca²⁺-dependent K⁺ secretion in the CD is disrupted in TRPV4 knockout animals.³⁹ TRPV4 directly interacts with PC2 to form mechanosensitive heteromeric complexes.^28,29 The fact that PC2 interacts with both PC1⁴⁰ and fibrocystin^10–12 suggests that TRPV4 could be an essential part of this mechanotransducing sensory complex.Current PKD management is directed toward pharmacologic interference with abnormal signaling pathways causing exaggerated cell proliferation, dedifferentiation, apoptosis, and cyst growth.⁴¹ Specifically, PKD is associated with elevated circulating vasopressin levels, increased basal cellular cAMP levels, and strong upregulation of cAMP-dependent fluid secretion and proliferation.^42,43 V2 antagonism greatly diminishes disease progression in rodent models of both ADPKD and ARPKD.^42,44 Elevated cAMP levels might be directly related to the reduced [Ca²⁺]_i, possibly due to impaired ability to sense changes in flow.^42,44,45 This raises the possibility that manipulation with the mechanosensitivity in the CD along the TRPV4 axis modulates [Ca²⁺]_i signalization and, in turn, renal cystogenesis.In this study, we developed a new approach to isolate native CD-derived cyst monolayers and nondilated CDs from a rat model of ARPKD to thoroughly investigate how functional TRPV4 status determines the development and growth of renal cysts. We found that the disease leads to disruption of mechanosensitive [Ca²⁺]_i signaling and impaired TRPV4 activity specifically in CD cysts but not in nondilated CDs. Long-term pharmacologic potentiation of TRPV4 activity gradually restores mechanosensitivity in cyst cells and greatly blunts renal ARPKD progression. From a global prospective, this study establishes a temporal link between disruption of TRPV4-based mechanosensitivity in the CD and cystogenesis. This also suggests pharmacologic potential of targeting TRPV4 activity as a treatment strategy in retarding development of ARPKD.     

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.     

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.     

The Calcineurin Homologous Protein-1 Increases Na+/H+-Exchanger 3 Trafficking via Ezrin Phosphorylation

The Na⁺/H⁺-exchanger 3 (NHE3) is essential for regulation of Na⁺ transport in the renal and intestinal epithelium. Although changes in cell surface abundance control NHE3 function, the molecular signals that regulate NHE3 surface expression are not well defined. We found that overexpression of the calcineurin homologous protein-1 (CHP1) in opossum kidney cells increased NHE3 transport activity, surface protein abundance, and ezrin phosphorylation. CHP1 knockdown by small interfering RNA had the opposite effects. Overexpression of wild-type ezrin increased both NHE3 transport activity and surface protein abundance, confirming that NHE3 is downstream of ezrin. Expression of a pseudophosphorylated ezrin enhanced these effects, whereas expression of an ezrin variant that could not be phosphorylated prevented the downstream effects on NHE3. Furthermore, CHP1 knockdown reversed the activation of NHE3 by wild-type ezrin but not by the pseudophosphorylated ezrin. Taken together, these results demonstrate that CHP1 increases NHE3 abundance and constitutive function in a manner dependent on ezrin phosphorylation.Na⁺/H⁺-exchanger 3 (NHE3), in the brush border membrane of renal proximal tubule cells,^1,2 is of major importance in mediating the absorption of the bulk of filtered sodium and fluid.^3,4 NHE3 is a member of the mammalian NHE superfamily that mediates electroneutral countertransport of H⁺ for Na⁺. At least ten NHE isoforms (NHE1–10), a cluster of distant NHE-related genes (NHA1–2), and one sperm-specific NHE are found in mammals.^5–7 Gene disruption of NHE3 in mice causes hypotension, acidosis, and hypovolemia. NHE3 knockout mice have decreased renal absorption of Na⁺, fluid, and HCO₃⁻, diarrhea, and universal mortality when fed with a low-salt diet.⁸The current structural model of NHE3 predicts two major domains, an amino-terminal transmembrane domain and a carboxyl-terminal cytoplasmic domain.⁹ The latter functions as a regulatory domain involving the dynamic association with accessory proteins that form a protein network jointly modulating NHE3 expression, traffic, and activity. A number of NHE3 binding partners and regulatory proteins have been identified, such as megalin, PDZK1, DPPIV, PP2A, Hsp70, and Shank2.^10–17 The functional roles of most of these associations remain elusive.Moreover, NHE3 associates with the actin cytoskeleton by binding to ezrin, which provides a regulated linkage between the plasma membrane and the actin cytoskeleton.^18–23 Ezrin is a member of the ezrin/radixin/moesin family and is highly enriched in the microvilli on the apical side of epithelial cells. NHE3 binds to ezrin both directly and indirectly. Direct binding of NHE3 to ezrin (amino acids 475 to 589 of the NHE3 C terminus²⁴) likely affects many aspects of basal NHE3 trafficking, including delivery from the synthetic pathway, basal exocytosis, and movement of NHE3 along the brush border, that may contribute to NHE3 endocytosis.²⁴ Indirect binding, via the PDZ-domain-containing proteins Na⁺/H⁺-exchanger regulatory factor (NHERF) 1 and 2 (amino acids 585 to 689 of the NHE3 C terminus^25,26), mediates several aspects of NHE3 regulation, including regulation by intracellular Ca^{2 +},^27,28 cAMP,^25,26,29 cGMP,³⁰ and lysophosphatidic acid.^28,31 Ezrin also has been proposed to mediate the Na⁺/glucose-cotransporter-induced activation and translocation of NHE3.³²Ezrin is responsible for membrane targeting^33,34 by direct association of its N terminus with the cytoplasmic domain of several integral membrane proteins (CD43, CD44, ICAM-1, -2, and -3, and NHE1 and 3^18,19,23); it also binds F-actin via its C terminus.³⁵ In the cytoplasmic dormant form of ezrin, intramolecular association of the N terminus with the C terminus masks binding sites for membrane proteins and F-actin.^33,36 Phosphoinositol-(4,5)-bisphosphate binding to the N terminus of ezrin followed by phosphorylation of threonine 567 at the C terminus of ezrin exposes both membrane protein and actin binding sites³⁶ and thus activates ezrin.³⁷ Ezrin in its closed conformation does not bind NHE3; only the open, active form of ezrin directly binds NHE3 in vitro.²⁴The calcineurin homologous protein (CHP) family belongs to an EF-hand (calcium-binding motifs composed of two helixes E and F joined by loop) subfamily of calcium-binding proteins that has similarity to the B regulatory subunit of the heterodimeric protein phosphatase calcineurin or Ca²⁺-calmodulin.³⁸ The CHP family (CHP1–3) associates with NHE isoforms, including NHE3,^38–41 in close proximity to the direct NHE3–ezrin binding region.^42–44 CHP1 is a widely expressed and highly conserved cytosolic Ca²⁺-binding protein^38,45 that regulates the phosphatase calcineurin⁴⁶ and apoptosis-inducing protein kinase DRAK2 activity.⁴⁷ CHP1 appears to be a crucial partner of several NHE isoforms for basal and regulated activity.^{38–40,48,49} Indeed, CHP1 sets the resting intracellular pH sensitivity of the transporter,^39,50 permits NHE3 regulation by adenosine A₁ receptors,⁴⁸ and stabilizes NHE1 on the plasma membrane.⁴⁹ CHP1 was found also to regulate targeting, docking, and fusion of transcytotic membrane vesicles⁴⁵ and associate with microtubules and affect their organization.^51,52 Similarly, CHP3 promotes NHE1 abundance, cell surface stability, and optimal transport function.⁴¹These collective properties of CHP1 render it a multipotent regulatory protein with several functions and make it a plausible candidate to regulate NHE3 traffic. Here, we propose that CHP1 is an essential signal molecule that controls the level of basal NHE3 expression and function, and we present a model that links CHP1–ezrin–NHE3 in a regulatory complex.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

Kallikrein 5-Mediated Inflammation in Rosacea: Clinically Relevant Correlations with Acute and Chronic Manifestations in Rosacea and How Individual Treatments May Provide Therapeutic Benefit

Rosacea is a chronic inflammatory condition of facial skin estimated to affect more than 16 million Americans. Although the pathogenesis of rosacea is not fully understood, recent evidence in vitro as well as in vivo has supported the role of increased levels of the trypsin-like serine protease, kallikrein 5, in initiating an augmented inflammatory response in rosacea. The increase in the quantity and magnitude of biological activity of kallikrein 5 leads to production of greater quantities of cathelicidin (LL-37), an antimicrobial peptide associated with increases in innate cutaneous inflammation, vasodilation, and vascular proliferation, all of which are characteristic features of rosacea. In this article, the authors review the literature supporting the role of kallikrein 5 in the pathophysiology of rosacea, including how therapeutic interventions modulate the effects of kallikrein 5, thus providing further support for this pathophysiological model that at least partially explains many of the clinical features of cutaneous rosacea.Cutaneous rosacea (rosacea) is a chronic inflammatory facial skin disorder noted most commonly in individuals of northern European descent, although people of any ethnicity or skin color may be affected.1-4 The visible manifestations with central facial predominance are characteristic of rosacea, including erythema, papules, pustules, telangiectasias, and phymatous changes.1-4 However, persistent (nontransient) erythema involving the central face that intensifies during flares and the presence of telangiectasias, which are also accentuated mostly on the central face, are the core clinical features that support a diagnosis of rosacea.1-9 Papules and pustules are not consistently present in rosacea, characterizing only those individuals with rosacea who exhibit the papulopustular subtype of the disease.3-6 In fact, papulopustular lesions never emerge in many individuals affected by rosacesa, and phymatous changes affect only a relatively small number of the rosacea-affected population; however, central facial erythema is present to some extent in essentially all people with rosacea.1-8Why do some people get rosacea and others do not? Although the entire explanation that would fully answer this question remains elusive, current evidence suggests that individuals affected by rosacea exhibit rosacea-prone skin, which inherently displays dysregulation of two main systems present within skin—the neurovascular/neuroimmune system and the immune detection/response system (innate immunity).3,5-8,1–9 Both of these systems normally serve physiological functions related to how skin responds to exogenous changes or insults (i.e., changes in temperature, exposure to microbial pathogens). However, in rosacea, both the cutaneous neurovascular/neuroimmune system and the immune detection/response system are dysregulated, with both demonstrating augmented responses that correlate with clinical manifestations commonly seen in patients with cutaneous rosacea.Neurovascular/neuroimmune dysregulation, which includes both anatomic and physiochemical differences present in rosacea-prone skin as compared to healthy facial skin, appears to be a major contributor that exacerbates the vasodilation of facial skin vasculature with increased facial blood flow that occurs during a rosacea flare.3,5-7,17,18,20 This increased vasodilation in rosacea-affected skin, which can be acute or subacute in onset, is commonly referred to as flushing.1,3,4,6,7,17,20 Neurosensory symptoms (i.e., stinging, burning) are often associated with or exacerbated during a rosacea flare.1,3-8 Exogenous factors that are commonly recognized by patients as triggers, which seem to induce a flare, include increased ambient heat/warmth and certain spices (i.e., capsaicin), all of which can induce signaling of neurogenic inflammation via specific receptor channels (transient receptor potential vanilloid [TRPV] subfamily) shown to be increased in rosacea-prone skin.3,6,17,18 The immune detection/response dysregulation of rosacea is evidenced by the upregulation of the pattern recognition receptor, toll-like receptor 2 (TLR2) and the cathelicidin innate immunity pathway.3,5-17,19,21,22 Ultraviolet light (UV) exposure, another recognized trigger factor associated with flares of rosacea, produces changes that induce ligand-binding of TLR2, which signals innate inflammation.3,5-16,19,21 Lastly, upregulated production of several matrix metalloproteases (MMPs) has been demonstrated in rosacea, further contributing to cascades of inflammation and degradation of the dermal matrix.1,3,5-7,19 Accentuated immune detection/response as a major component of the pathophysiology of rosacea has been discussed extensively in the literature and is addressed in more detail as a major subject of this article.5-7,10-16,19,21,22Although the pathophysiology of rosacea is not completely understood, dysregulation of the innate immune detection/response system plays a significant role in the inflammatory and vascular responses seen in this condition.5-7,10-16,19,21,22 As a known inducer of innate and cellular inflammation, increased vascularity, and angiogenesis, cathelicidin (LL-37), an antimicrobial peptide that physiologically provides near-immediate innate defense against several microbial organisms, has been investigated to determine its potential role in the pathophysiology of rosacea.10,11,23,24 Results have shown that patients with rosacea express elevated levels of LL-37 in facial skin, with this increased expression attributed to abnormally high levels of the trypsin-like serine protease enzyme, kallikrein 5 (KLK5), which selectively cleaves an inactive precursor protein (hCAP18) to form the biologically active antimicrobial peptide (LL-37).10,22 Investigations of the mechanism of action of two agents proven to be effective in reducing papulopustular lesions and perilesional erythema in rosacea, topical azelaic acid (AzA) and oral doxycycline, demonstrated direct and indirect inhibition of KLK5, respectively.25-29 In one study with AzA 15% gel, the reduction in KLK5 activity correlated with clinical improvement of rosacea.29 In this review, the authors further describe the role of KLK5 in the pathophysiology of rosacea, including the inflammatory cascades that result from increased KLK5 expression, as well as a more detailed discussion of different therapies shown to inhibit the progression of this cascade.     

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.     

Cutaneous Collagenous Vasculopathy

Cutaneous collagenous vasculopathy is a rare microangiopathy of dermal blood vessels. Clinically indistinguishable from generalized essential telangiectasia, this condition is diagnosed by its unique histological appearance. In contrast to other primary telangiectatic processes, cutaneous collagenous vasculopathy has dilated vascular structures that contain deposits of eosinophilic hyaline material within the vessel walls. To date, cutaneous collagenous vasculopathy has been described in a total of 19 cases in the medical literature. The first several cases were described exclusively in middle-aged to elderly men. Though it has now been described in both men and women, cutaneous collagenous vasculopathy is still most often described in middle-aged to older adults. No particular disease or medication has been linked to the development of cutaneous collagenous vasculopathy, and the etiology remains unknown. In this case series, the authors present three additional patients diagnosed with cutaneous collagenous vasculopathy and discuss their clinical and histopathologic features.Cutaneous collagenous vasculopathy (CCV) is a rare, idiopathic microangiopathy first reported in 2000 by Salama and Rosenthal.1 CCV has characteristic microscopic findings, including dilated capillaries and post-capillary venules with marked collagen deposition,2 which are features essential to diagnosis. Clinically, CCV presents as blanchable, non-urticating macules that typically begin on the lower extremities and then spread to the trunk and upper extremities. 1,3-6 Due to its clinical similarity to generalized essential telangiectasia (GET), dermatologists may not biopsy these patients, potentially causing CCV to be underdiagnosed and under-reported. 4,5,7,8 To date, CCV has been described in approximately equal numbers in men and women of Caucasian race with patients ranging from 16 to 83 years of age.4,9 The majority of cases have been diagnosed in patients with other concomitant diseases, most commonly hypertension and cardiovascular disease,3,5,6,9,10 but also in patients with autoimmune conditions2,5,8,9 and diabetes mellitus.5,10 Additionally, in most described cases, patients were taking at least one medication on an intermittent or ongoing basis. 1,2,4-7,9,1° Despite this observation, specific medical conditions or medications are yet to be linked to the development of CCV. In this article, the authors present three female patients who have been diagnosed with CCV along with their clinical and histopathological features.     

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.