Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.Acute kidney injury (AKI) leading to renal failure is a devastating disorder,¹ with a prevalence varying from 30 to 50% in the intensive care unit.² AKI during sepsis results in significant morbidity, and is an independent risk factor for mortality.^3,4 In patients with severe sepsis or shock, the reported incidence ranges from 23 to 51%^5–7 with mortality as high as 70% versus 45% among patients with AKI alone.^1,8The pathogenesis of AKI during sepsis involves hemodynamic alterations along with microvascular impairment.⁴ Although many factors change during sepsis, suppression of the plasma serine protease, protein C (PC), has been shown to be predictive of early death in sepsis models,⁹ and clinically has been associated with early death resulting from refractory shock and multiple organ failure in severe sepsis.¹⁰ Moreover, low levels of PC have been highly associated with renal dysfunction and pathology in models of AKI.¹¹ During vascular insult, PC becomes activated by the endothelial thrombin-thrombomodulin complex, and the activated protein C (APC) exhibits both antithrombotic and cytoprotective properties. We have previously demonstrated that APC administration protects from renal dysfunction during cecal ligation and puncture and after endotoxin challenge.^11,12 In addition, recombinant human APC [drotrecogin alfa (activated)] has been shown to reduce mortality in patients with severe sepsis at high risk of death.¹³ Although the ability of APC to protect from organ injury in vivo is well documented,^11,14,15 the precise mechanism mediating the response has not been ascertained.APC exerts anticoagulant properties via feedback inhibition of thrombin by cleavage of factors Va and VIIIa.¹⁶ However, APC bound to the endothelial protein C receptor (EPCR) can also exhibit direct potent cytoprotective properties by cleaving protease-activated receptor-1 (PAR-1).¹⁷ Various cell culture studies have demonstrated that the direct modulation of PAR-1 by APC results in cytoprotection by several mechanisms, including suppression of apoptosis,^18,19 leukocyte adhesion,^19,20 inflammatory activation,²¹ and suppression of endothelial barrier disruption.^22,23 In vivo, the importance of the antithrombotic activity of APC is well established in model systems^24,25 and in humans.²⁶ However, the importance of PAR-1-mediated effects of APC also has been clearly defined in protection from ischemic brain injury²⁷ and in sepsis models.²⁸ Hence, there has been significant debate whether the in vivo efficacy of APC is attributed primarily to its anticoagulant (inhibition of thrombin generation) or cytoprotective (PAR-1-mediated) properties.^17,29The same active site of APC is responsible for inhibition of thrombin generation by the cleavage of factor Va and for PAR-1 agonism. Therefore, we sought to generate point mutations that would not affect catalytic activity, but would alter substrate recognition to distinguish the two functions. Using these variants, we examined the relative role of the two known functions of APC in a model of LPS-induced renal microvascular dysfunction.     

Dietary Intake Relative to Cardiovascular Disease Risk Factors in Individuals With Chronic Spinal Cord Injury: A Pilot Study

Background:

The relationship between cardiovascular disease (CVD) risk factors and dietary intake is unknown among individuals with spinal cord injury (SCI).

Objective:

To investigate the relationship between consumption of selected food groups (dairy, whole grains, fruits, vegetables, and meat) and CVD risk factors in individuals with chronic SCI.

Methods:

A cross-sectional substudy of individuals with SCI to assess CVD risk factors and dietary intake in comparison with age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Dietary history, blood pressure, waist circumference (WC), fasting blood glucose, high-sensitivity C-reactive protein (hs-CRP), lipids, glucose, and insulin data were collected from 100 SCI participants who were 38 to 55 years old with SCI >1 year and compared to 100 matched control participants from the CARDIA study.

Results:

Statistically significant differences between SCI and CARDIA participants were identified in WC (39.2 vs 36.2 in.; P < .001) and high-density lipoprotein cholesterol (HDL-C; 39.2 vs 47.5 mg/dL; P < .001). Blood pressure, total cholesterol, triglycerides, glucose, insulin, and hs-CRP were similar between SCI and CARDIA participants. No significant relation between CVD risk factors and selected food groups was seen in the SCI participants.

Conclusion:

SCI participants had adverse WC and HDL-C compared to controls. This study did not identify a relationship between consumption of selected food groups and CVD risk factors.Key words: cardiovascular disease risk factors, dietary intake, spinal cord injuryCardiovascular disease (CVD) is a leading cause of death in individuals with chronic spinal cord injuries (SCIs).^1–5 This is partly because SCI is associated with several metabolic CVD risk factors, including dyslipidemia,^6–10 glucose intolerance,^6,11–14 and diabetes.^15–17 In addition, persons with SCI exhibit elevated markers of inflammation^18,19 and endothelial activation²⁰ that are correlated with higher CVD prevalence.^21–23 Obesity, and specifically central obesity, another CVD risk factor,^24–26 is also common in this population.^12,27–29Dietary patterns with higher amounts of whole grains and fiber have been shown to improve lipid abnormalities,³⁰ glucose intolerance, diabetes mellitus,^31–34 hypertension,³⁵ and markers of inflammation³⁶ in the general population. These dietary patterns are also associated with lower levels of adiposity.³¹ Ludwig et al reported that the strong inverse associations between dietary fiber and multiple CVD risk factors – excessive weight gain, central adiposity, elevated blood pressure, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), high low-density lipoprotein cholesterol (LDL-C), and high fibrinogen – were mediated, at least in part, by insulin levels.³⁷ Whole-grain food intake is also inversely associated with fasting insulin, insulin resistance, and the development of type 2 diabetes.^32,38,39Studies in the general population have also shown a positive association between the development of metabolic syndrome as well as heart disease and consumption of a Western diet, a diet characterized by high intake of processed and red meat and low intake of fruit, vegetables, whole grains, and dairy.^40,41 Red meat, which is high in saturated fat, has been shown to have an association with adverse levels of cholesterol and blood pressure and the development of obesity, metabolic syndrome, and diabetes.^40,42,43Numerous studies have shown that individuals with chronic SCI have poor diet quality.^44–49 A Canadian study found that only 26.7% of their sample was adherent to the recommendations about the consumption of fruit, vegetables, and grains from the “Eating Well with Canada’s Food Guide.”⁴⁴ Individuals with chronic SCI have also been found to have low fiber and high fat intakes when their diets were compared to dietary recommendations from the National Cholesterol Education Program,⁴⁶ the 2000 Dietary Guidelines for Americans,⁴⁹ and the recommended Dietary Reference Intakes and the Acceptable Macronutrient Distribution Range.^47,48However, unlike in the general population, the relationship between dietary intake and obesity and CVD risk factors is unknown in the chronic SCI population. If a dietary pattern consisting of higher intake of whole grains and dietary fiber is favorably associated with obesity and CVD risk factors in individuals with chronic SCI, then trials of increased intake of whole grains and fiber intake could be conducted to document health benefits and inform recommendations. The purpose of this pilot study is to investigate the association between selected food group intake and CVD risk factors in individuals with chronic SCI as compared to age-, gender-, and race-matched able-bodied individuals enrolled in the Coronary Artery Risk Development in Young Adults (CARDIA) study. Data will also be used to plan future studies in the relatively understudied field of CVD and nutrition in individuals with SCI.     

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC γ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.Nephrotic syndrome is characterized by proteinuria, sodium retention, and edema. Increased renal sodium reabsorption occurs in the cortical collecting duct (CCD),^1,2 where a rate-limiting step in transepithelial sodium transport is the epithelial sodium channel (ENaC), which is composed of the three homologous subunits: α, β, γ.³ENaC activity is regulated by hormones, such as aldosterone and vasopressin (AVP)^4,5; however, adrenalectomized rats and AVP-deficient Brattleboro rats are capable of developing nephrotic syndrome,^1,6 and nephrotic patients do not consistently display elevated levels of sodium-retaining hormones,^7,8 suggesting that renal sodium hyper-reabsorption is independent of systemic factors. Consistent with this, sodium retention is confined to the proteinuric kidney in the unilateral puromycin aminonucleoside (PAN) nephrotic model.^2,9,10There is evidence that proteases contribute to ENaC activation by cleaving the extracellular loops of the α- and γ-subunits.^11–13 Proteolytic activation of ENaC by extracellular proteases critically involves the cleavage of the γ subunit,^14–16 which probably leads to the release of a 43-residue inhibitory peptide from the ectodomain.¹⁷ Both cleaved and noncleaved channels are present in the plasma membrane,^18,19 allowing proteases such as channel activating protease 1 (CAP1/prostasin),²⁰ trypsin,²⁰ chymotrypsin,²¹ and neutrophil elastase²² to activate noncleaved channels from the extracellular side.^23,24 We hypothesized that the defective glomerular filtration barrier in nephrotic syndrome allows the filtration of ENaC-activating proteins into the tubular fluid, leading to stimulation of ENaC. The hypothesis was tested in the PAN nephrotic model in rats and with urine from patients with nephrotic syndrome.     

PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis

PDGF-C is a potent mitogen for fibroblasts in vitro. Transgenic PDGF-C overexpression in the heart or liver induces organ fibrosis, and PDGF-C expression is upregulated at sites of interstitial fibrosis in human and rat kidneys; however, the effect of inhibiting PDGF-C on the development of renal fibrosis in vivo is unknown. Renal fibrosis was induced in C57BL/6 mice by unilateral ureteral obstruction (UUO), and then mice were treated with neutralizing anti–PDGF-C antiserum or nonspecific IgG. An increase in PDGF-C expression was observed in fibrotic areas after UUO, contributed in large part by infiltrating macrophages. Treatment with anti–PDGF-C reduced renal fibrosis by 30% at day 5 and reduced interstitial myofibroblast accumulation by 57%. In vitro, PDGF-C was a potent mitogen for renal fibroblasts and induced chemokine expression. In vivo, anti–PDGF-C treatment produced a decrease in the expression of the renal chemokines CCL2 and CCL5 (85 and 67% reductions, respectively), accompanied by a significant decrease in leukocyte infiltration and CCR2 mRNA expression. Further supporting a role of PDGF-C in renal fibrosis, PDGF-C^−/− mice demonstrated a reduction in fibrosis and leukocyte infiltration in response to UUO compared with wild-type littermates. In conclusion, specific neutralization or lack of PDGF-C reduces the development of renal inflammation and fibrosis in obstructed mouse kidneys. Leukocyte-derived PDGF-C induces chemokine expression, which may lead to the recruitment of additional leukocytes, creating an amplification loop for renal inflammation and fibrosis.PDGF-C is a recently identified cytokine that acts via the PDGF-α receptor and is a potent mitogen for human fibroblasts and vascular smooth muscle cells in vitro.^1,2 Observations in different organs suggest that PDGF-C plays an important role in the regulation of fibrosis. First evidence was derived from a mouse model in which transgenic overexpression of PDGF-C in the heart induced fibroblast proliferation, resulting in cardiac fibrosis, hypertrophy, and ultimately cardiomyopathy.^1,3 Transgenic mice with liver-specific PDGF-C overexpression of the transgene developed liver fibrosis and hepatocellular carcinoma.⁴ Finally, transgenic mice with a lung-specific PDGF-C overexpression developed massive mesenchymal cell hyperplasia and died from respiratory insufficiency immediately after birth.⁵ Increased PDGF-C expression has additionally been observed in different experimental models of organ fibrosis in heart and lung tissues.^6,7Additional functions of PDGF-C include the stimulation of angiogenesis^8,9 and acceleration of wound-healing processes.² A dynamic expression of PDGF-C during organogenesis in mice and humans points to a critical role of this growth factor in organ development,^10,11 which is further substantiated by studies in PDGF-C^−/− mice that die from feeding and respiratory difficulties in the perinatal period when grown on an SV129 background.¹² The phenotype of these PDGF-C^−/− mice is characterized by defective tube formation (facial clefts, spina bifida occulta), subepidermal blisters, and deficiency of connective tissues in the dorsal midline.¹²Little is known about the functional role of PDGF-C within the kidney. We recently identified a constitutive expression of PDGF-C within arterial vascular smooth muscle cells and within collecting duct cells in rat and human kidney.^13,14 We detected a de novo expression and/or overexpression of PDGF-C in fibrotic interstitial areas in different rat models of renal disease as well as in diseased human renal biopsy tissues.^13,14 We therefore hypothesized that PDGF-C plays a functional role in the pathogenesis of tubulointerstitial fibrosis. We subsequently studied the potential profibrotic role of PDGF-C both by using a neutralizing anti–PDGF-C antiserum in murine renal fibrosis induced by unilateral ureteral obstruction (UUO) and by inducing renal fibrosis in PDGF-C^−/− mice.     

Decreased cardiac glutathione peroxidase levels and enhanced mandibular apoptosis in malformed embryos of diabetic rats

OBJECTIVE— To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes.RESEARCH DESIGN AND METHODS— Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry.RESULTS— Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos.CONCLUSIONS— Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.The cellular and molecular mechanisms of diabetic embryopathy are not completely clear. Previous experimental studies have suggested that the teratological impact of a diabetic environment partly depends on excess of reactive oxygen species (ROS) in the embryo (1) as a consequence of either increased free oxygen radical formation (2–4), decreased capacity of ROS-scavenging enzymes (5–9), or both. Furthermore, previous work has demonstrated that supplementation of antioxidative agents such as copper zinc superoxide dismutase (CuZnSOD) (1,2), N-acetylcysteine (10), vitamins E and C (8), and folic acid (11) in vitro, as well as butylated hydroxytoluene (12), vitamin E (13–19), vitamin C (18,20), N-acetylcysteine (21), and folic acid (11) in vivo, attenuate malformation rate and diminish markers of oxidative stress (e.g., by normalizing tissue levels of thiobarbituric acid reactive substances [15], isoprostane 8-iso-PGF_2α [22,23], and carbonylated proteins [24]).The driving cellular force behind diabetes-induced oxidative stress is likely associated with enhanced glucose metabolism (25–27) in the embryonic/fetal cells exposed to increased ambient levels of glucose. One putative primary source of reactive radical compounds is mitochondria receiving a high influx of pyruvate and oxygen and, subsequently, producing a large amount of ROS (mainly superoxide) (3) in the oxidative processes of the electron transport chain. The ensuing leakage of superoxide into other compartments of the mitochondria and the cytosol, and the further formation of hydrogen peroxide and hydroxyl radicals, should yield mitochondrial alterations (28) as well as lipid peroxidation (22) and DNA damage (29) in the embryo. There are several observations in support of this notion. The structural alterations, mainly high-amplitude swelling of the embryonic mitochondria (28), are diminished by maternal antioxidative treatment (16), thereby supporting the notion of a ROS-related etiology of the structural changes. Enhanced lipid peroxidation, as evidenced by increased levels of the isoprostane 8-iso-PGF_2α (22,23,30), may induce several teratogenic pathways in addition to the developmental disturbance caused by peroxidation of structural lipids in mitochondrial and cellular membranes. For instance, it has been demonstrated that 8-iso-PGF_2α, which is produced nonenzymatically by ROS-mediated oxidation of arachidonic acid in the offspring (30), has its own teratogenic potency (23). In addition, an excessive peroxidation of arachidonic acid may hamper prostaglandin biosynthesis by depleting precursor pools and, in particular, yield decreased concentration of prostaglandin E2 (31), which could obstruct neural tube closure (22).ROS-induced DNA damage (29) may directly disrupt development via altered expression of key genes. In addition, cellular DNA repair processes may activate poly(ADP-ribose) polymerase (PARP), which may cause glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition by poly(ADP-ribosylation) (32). The net result would be diminished embryonic GAPDH activity, which has been demonstrated in rat embryos subjected to diabetes in vivo and high glucose in vitro (33). Furthermore, decreased glycolytic flux proximal to GAPDH (32) and the presence of increased ambient glucose levels will yield enhanced flux in the polyol (34,35) and hexosamine (36) pathways. An increased availability of proximal glycolytic intermediaries would increase diacylglycerol production and cause activation of several protein kinase C isoforms (37,38), as well as enhance the flux in the advanced glycosylation end product pathway (39). All of the consequences of inhibited GAPDH activity may thus contribute to the teratogenic outcome of diabetic pregnancy. Evidently, there are multiple ways for a diabetes-induced state of oxidative stress in the embryo to disturb embryonic/fetal development, several of which enjoy considerable experimental support.Another consequence of a state of oxidative stress would be enhanced apoptosis in embryonic/fetal tissue (40), which has been described (26,41–43) and has been suggested to be mediated by enhanced Jun-amino-terminal kinase-1 and -2 activity (19,44). It has also been suggested that maternal diabetes induces an inflammatory state in the embryo, where proinflammatory cytokines (i.e., tumor necrosis factor-α [TNF-α] [45,46]) act to downregulate the principal ROS-scavenging enzymes via increased activity of mitogen-activated protein kinases (47). The exact relation between enhanced apoptosis and induction of malformations is still unclear, mainly since we do not fully comprehend the specific transmission of a general increase in programmed cell death into precisely restricted developmental damage to embryonic organs or organ systems (42).Based on earlier studies (7,48,49) and on a question that has been raised several times—which genes are involved in diabetes-induced embryonic dysmorphogenesis? (5,8,26,42,50)—we wanted to add to the teratological knowledge by identifying differences in gene expression between the malformed and nonmalformed offspring within the same litters of diabetic animals.Our working hypothesis was that genes of the malformed offspring with an expression pattern different from that of genes in the nonmalformed offspring within the same litter may be associated, either directly or indirectly, with the teratogenic process. We thus decided to compare gene expression of embryos that were morphologically normal and those that were malformed in litters of normal and diabetic rats. By comparing gene expression and protein distribution in embryos of the same age and with exposure to an identical intrauterine milieu, we aimed to control for possible confounding factors of the teratogenic process. From earlier work, we knew that the two tissues of the rat fetuses that are particularly vulnerable to the diabetic state are the mandible and the heart (17) (Fig. 1). We also knew that a susceptible period in diabetic rat pregnancy occurs during gestational days 6–10 (51), which corresponds to weeks 2–4 in human pregnancy. Bearing in mind that the size and relative immaturity of day-10 embryos makes them difficult to evaluate for developmental defects, we chose to interrupt the rat pregnancies on gestational day 11 in order to be able to clearly distinguish between malformed and nonmalformed embryos. We decided to relate embryonic (mal)development to expression of the major oxidative defense genes (SODs, glutathione peroxidase [Gpx]-1, Gpx-2, and catalase), to key genes of glucose metabolism (aldose reductase [AR] and GAPDH), to developmental/teratological genes [poly(ADP-ribose) polymerase (PARP)], to tumor protein 53 (p53), to bone morphogenetic protein-4 (Bmp-4), to Ret proto-oncogene (Ret), to sonic hedgehog homolog (Shh), to vascular endothelial growth factor-A (VEGF-A), to TNF-α, to interleukin-6 (IL-6), and to the tissue distribution of activated caspase-3 (denoting apoptotic rate) and Gpx-1.Open in a separate windowFIG. 1.A: Fetuses displaying micrognathia (left fetus) and normal morphology (right fetus). B: Outcome of pregnancy in the control (N) and manifestly diabetic (MD) groups, distributed as normal (□), malformed (▪), and resorbed (▒) embryos on gestational day 11. *P < 0.05 vs. N (χ² statistics). (Please see http://dx.doi.org/10.2337/db08-0830 for a high-quality digital representation of this figure.)     

Relationship Between Neurological Injury and Patterns of Upright Mobility in Children With Spinal Cord Injury

Background:

The predictors and patterns of upright mobility in children with a spinal cord injury (SCI) are poorly understood.

Objective:

The objective of this study was to develop a classification system that measures children’s ability to integrate ambulation into activities of daily living (ADLs) and to examine upright mobility patterns as a function of their score and classification on the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) exam.

Methods:

This is a cross-sectional, multicenter study that used a convenience sample of subjects who were participating in a larger study on the reliability of the ISNCSCI. A total of 183 patients between 5 and 21 years old were included in this study. Patients were asked if they had participated in upright mobility in the last month and, if so, in what environment and with what type of bracing. Patients were then categorized into 4 groups: primary ambulators (PrimA), unplanned ambulators (UnPA), planned ambulators (PlanA), and nonambulators.

Results:

Multivariate analyses found that only lower extremity strength predicted being a PrimA, whereas being an UnPA was predicted by both lower extremity strength and lack of preservation of S45 pinprick sensation. PlanA was only associated with upper extremity strength.

Conclusions:

This study introduced a classification system based on the ability of children with SCI to integrate upright mobility into their ADLs. Similar to adults, lower extremity strength was a strong predictor of independent mobility (PrimA and UnPA). Lack of pinprick predicted unplanned ambulation, but not being a PrimA. Finally, upper extremity strength was a predictor for planned ambulation.Key words: ambulation, ISNCSCI, pediatrics, spinal cord injuryAfter a spinal cord injury (SCI), learning to walk often becomes the focus of rehabilitation for children and their families.^1,2 Although the majority of children with SCI do not return to full-time functional ambulation, those who accomplish some level of walking report positive outcomes such as feeling “normal” again, being eye-to-eye with peers, and having easier social interactions.³ Although not frequently reported by patients, there is some evidence of physiological benefits as well.^3–9 Regardless of age, upright mobility has been positively associated with community participation and life satisfaction.^10–12 For children, upright mobility allows them to explore their physical environment, which facilitates independence and learning as part of the typical developmental process.^13,14With the use of standers, walkers, and other assistive devices, as well as a variety of lower extremity orthoses, it is a reasonable expectation that some children with spinal injuries achieve upright stance and mobility.^7,9,13–21 However, there are 2 main challenges for clinicians and patients: understanding the factors that either encourage or discourage upright activities, and identifying how best to determine whether upright mobility is successful and meaningful. The literature on adults suggests that upright mobility is dependent on physiological and psychosocial factors. Physiological factors include the patient’s current age, neurological level, muscle strength, and comorbidities.^14,22–27 Psychosocial factors include satisfaction with the appearance of the gait pattern, cosmesis, social support for donning/doffing braces, and assistance with transfer and during ambulation.^{3,9,19,28–32}The identification of outcome measures that provide a meaningful indication of successful upright mobility has been difficult. The World Health Organization (WHO) describes 2 constructs for considering outcomes – capacity and performance.³³ Capacity refers to maximal capability in a laboratory setting. An example of a capacity measure is the Walking Index for Spinal Cord Injury (WISCI), which is an ordinal scale used to quantify walking capacity based on assistive device, type of orthosis, and amount of assistance required.^34,35 Other capacity measures include the Timed Up and Go test and the 6-minute walk test.^36,37 On the other hand, performance refers to actual activity during a patient’s daily activities in typical, real-life environments.³³ For example, the FIM is an observation scale that scores the patient’s typical daily performance.^36,38–40 The FIM is considered a burden of care measure that determines the amount of actual assistance provided to a patient during typical routines and environments, which may or may not reflect maximal ability or capacity. Performance measures provide an adequate clinical snap-shot of a patients’ daily function (evaluates what they do), whereas capacity measures are better research tools, as they are able to detect subtle changes in ambulation (evaluates what they can do).In children, no capacity outcome measures of ambulation have been tested for validity or reliability. Availability of reliable and valid performance measures is also lacking. The WeeFIM is a performance measure for children, but it is not SCI specific. It is scored on the child’s burden of care, that is, on the maximal assistance required rather than the child’s maximal independence or the highest capacity of performance during a typical day. For children, another commonly used scale is the Hoffer Scale, which relies on the physician’s or therapist’s subjective determination of the purpose of the upright mobility activities (for function or for exercise).^41,42 Because parents and school systems are encouraged to integrate “exercise” ambulation into daily activities, it may not be possible to distinguish between therapeutic and functional ambulation in the home, school, or community environments. In the schools, a teacher/therapist should incorporate upright mobility into the classroom setting by donning a child’s braces and then having her/him ambulate a short distance to stand at an easel in art class or to stand upright when talking to friends during recess. In this situation, walking serves the dual purpose of being functional and therapeutic.For this study, it was decided not to rely on a subjective determination of therapeutic versus functional ambulation as the main outcome measure. Instead, we were interested in the children and adolescents who have successfully integrated independent mobility into their daily activities, regardless of frequency, distance, or purpose. Recent literature in studies of children and adolescents suggests that spontaneity is important for participation in functional and social activities. For example, a survey of patients using functional electrical stimulation for hand function found a reduction in the dependence on others for donning splints, which facilitated independence with activities of daily living (ADLs) in adolescents.^43–45 In a more recent study, Mulcahey et al⁴⁶ found that a reduction of spontaneity in adolescents was a barrier for social activity; during cognitive interviews, children reported not participating in sleepovers due to planning their bowel/bladder programs.To date, there are no measures that integrate spontaneity of standing and/or upright mobility into the daily activities of children. Toward that aim, this study introduces a new scale that attempts to categorize children into 4 mutually exclusive groups: primary ambulators, unplanned ambulators, planned ambulators, and nonambulators. The purpose of this study was to examine ambulation patterns among children and adolescents with SCI as a function of neurological level, motor level, and injury severity, as defined by the motor, sensory, and anorectal examinations of the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI). A secondary aim of the study was to determine how performance on the ISNCSCI exam was associated with the ability of children to independently integrate ambulation into their daily routines.     

CTGF Promotes Inflammatory Cell Infiltration of the Renal Interstitium by Activating NF-κB

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-κB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-κB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-γ, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-κB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-κB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-κB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-κB pathway.Connective tissue growth factor (CTGF) is a member of the C-terminal cystein-rich proteins (CCN) family of early response genes. CTGF is a 38-kD cystein-rich secreted protein that is up-regulated in proliferative disorders or fibrotic lesions in several human diseases, including skin disorders, atherosclerosis, pulmonary fibrosis, and kidney diseases.^1,2 In human biopsies of different renal pathologies and in experimental models of kidney injury, renal CTGF overexpression was correlated with cellular proliferation and extracellular matrix (ECM) accumulation, both at glomerular and interstitial areas.^2–4 In the diabetic kidney, elevated CTGF expression co-localizes with sites of epithelial-to-mesenchymal transition (EMT) on the tubular epithelium.⁵ In cultured renal cells, recombinant CTGF significantly increases ECM production and induces transition of tubuloepithelial cells to myofibroblasts.^6–8 In experimental diabetic nephropathy in mice, the blockade on endogenous CTGF, by antisense oligonucleotides, has beneficial effects on renal damage progression.⁹ In cultured renal cells, CTGF blockade inhibits ECM accumulation and EMT caused by angiotensin II and transforming growth factor-β (TGF-β).^3,10 These data suggest that CTGF could be an important target for the treatment of renal fibrosis.CTGF also induces other cellular responses. Depending on the cell type, CTGF regulates cell growth, proliferation, and apoptosis. CTGF is a downstream mediator of TGF-β-induced apoptosis of mesothelial cells,¹¹ but contributes to the survival of hepatic stellate cells.¹² CTGF may play a role as a secreted tumor suppressor protein¹³ or contribute to promote tumor cell growth and invasion.¹⁴ Some studies suggested that CTGF could also be involved in the inflammatory response. CTGF is a chemotactic factor for monocytes¹⁵ and regulates cellular adhesion and migration in mesangial cells.¹⁶ Moreover, in cultured mesangial cells, CTGF enhances the production of proinflammatory factors, including chemotactic molecules, and activates nuclear factor-kappa B (NF-κB).¹⁷ However, there is no data about the in vivo effect of CTGF on the renal inflammatory process.The molecular mechanisms involved in CTGF signaling are far from being understood. CTGF interacts with tyrosine kinase receptors and integrins that activate multiple signaling systems including NF-κB and mitogen-activated protein kinase (MAPK) pathways.^12,17–19 Although the regulation of the inflammatory response in the kidney is a complex process, the activation of NF-κB plays a pivotal role. Experimental studies have shown that NF-κB blockade by different methods, including I-κB overexpression, NF-κB decoy oligonucleotides, NF-κB inhibitors (parthenolide among others), or indirectly by statins, glucocorticoids, and antioxidants, prevents renal damage.^20–23 Activation of renal NF-κB has been described in human kidney diseases, associated to proinflammatory factors overexpression.^24,25 We have now investigated whether CTGF could modulate the inflammatory response in the kidney and the mechanisms underlying this process, evaluating the involvement of the NF-κB signaling pathway.     

Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation

Connexins are important in vascular development and function. Connexin 40 (Cx40), which plays a predominant role in the formation of gap junctions in the vasculature, participates in the autoregulation of renal blood flow (RBF), but the underlying mechanisms are unknown. Here, Cx40-deficient mice (Cx40-ko) had impaired steady-state autoregulation to a sudden step increase in renal perfusion pressure. Analysis of the mechanisms underlying this derangement suggested that a marked reduction in tubuloglomerular feedback (TGF) in Cx40-ko mice was responsible. In transgenic mice with Cx40 replaced by Cx45, steady-state autoregulation and TGF were weaker than those in wild-type mice but stronger than those in Cx40-ko mice. Nω-Nitro-L-arginine-methyl-ester (L-NAME) augmented the myogenic response similarly in all genotypes, leaving autoregulation impaired in transgenic animals. The responses of renovascular resistance and arterial pressure to norepinephrine and acetylcholine were similar in all groups before or after L-NAME inhibition. Systemic and renal vasoconstrictor responses to L-NAME were also similar in all genotypes. We conclude that Cx40 contributes to RBF autoregulation by transducing TGF-mediated signals to the afferent arteriole, a function that is independent of nitric oxide (NO). However, Cx40 is not required for the modulation of the renal myogenic response by NO, norepinephrine-induced renal vasoconstriction, and acetylcholine- or NO-induced vasodilation.Connexins (Cx''s) are important in vascular development, cardiovascular function, and arterial pressure (AP) control.^1,2 Four Cx isoforms (Cx37, Cx40, Cx43, and Cx45) form gap junctions to facilitate intercellular communication in the vasculature.^1,2 Among these, Cx40 plays a prominent role. Cx40 is abundantly expressed in endothelial cells in most vascular beds.^1,2 Genetic ablation of Cx40 causes severe impairment of conducted vasodilator responses in arterioles,^3,4 uncoordinated vasomotion,⁵ and hypertension.^5–7 Furthermore, vascular expression of Cx40 is reduced in genetically hypertensive rats.⁸Within the kidney, gap junctions are prevalent in the juxtaglomerular apparatus (JGA).⁹ The JGA is a unique structure coordinating tubular function to the regulation of preglomerular vasomotor tone and renin release. Cx40 is the predominant connexin in the JGA, with expression in endothelial and renin-producing cells of afferent arterioles and glomerular mesangial cells.^6,10–12 Cx40 is thus strategically localized for impacting GFR, tubuloglomerular feedback (TGF), and renin secretion. Indeed, deletion of Cx40 leads to increased production of renin, ectopic renin expression, and loss of pressure- and angiotensin II (Ang II)-dependent control of renin release.^6,7,13 A rise in plasma renin concentration is also seen after administration of a putative Cx40-inhibiting peptide.¹² However, Cx40 expression is increased in response to a chronic reduction of renal perfusion pressure, a common stimulus for renin synthesis.¹⁰Our knowledge of the role of Cx40 in the regulation of organ blood flow and vascular resistance in vivo is limited. In the kidney, intrarenal infusion of peptides designed to inhibit Cx37, Cx40, or both Cx40 and Cx43 reduces basal renal blood flow (RBF) and increases AP.^12,14 Steady-state autoregulation of RBF and GFR is reported to be partially inhibited by peptides directed against Cx37 or Cx40.¹² Not known, however, is which of the three mechanisms responsible for renal autoregulation (TGF, myogenic response (MR), and an undefined third mechanism^15,16) is affected. In isolated JGAs, TGF responses¹⁷ and associated calcium waves¹⁸ are inhibited by nonspecific pharmacologic gap junction disrupters (e.g., heptanol). Such interventions also attenuate the MR to changes in vascular pressure in isolated cerebral¹⁹ and mesenteric arteries,²⁰ as is the case for inhibitory peptides against Cx37 and Cx43.²⁰ However, the functional significance of genetic deletion of Cx40 for blood flow regulation and autoregulation in vivo in any vascular bed, including the kidney, is not known. We postulated that Cx40 is required for complete autoregulation and TGF activity.Also poorly understood is the importance of gap junctions in vivo in vasoconstrictor and vasodilator responses of resistance arterioles. α-Adrenergically induced vasoconstriction is blunted by pharmacologic gap junction inhibitors in isolated arteries.^20,21 Gap junctions are also implicated in vasodilation,^14,22 although it is unclear whether or not Cx40 is involved.^3,14,23 Few studies have tested the participation of connexins in vasodilation in vivo.^5,14 To our knowledge, no data exist regarding connexins in vasoconstrictor responses in an animal.Nitric oxide (NO) may interact with connexin function. NO is thought to modulate connexin conductance acutely²⁴ and expression chronically.²⁵ NO attenuates the magnitude of acute vasoconstriction elicited by receptor agonists such as Ang II and NE.²⁶ In addition, NO blunts the strength of the pressure-induced MR in RBF autoregulation, an effect specific to the kidney and dependent on JGA function and TGF.^27,28 Considering the possible involvement of Cx40 in JGA function, we postulated that Cx40 is critical for NO modulation of agonist-induced renal vascular responses and pressure-induced RBF autoregulation.The present study tested the hypotheses that (a) Cx40 is essential to RBF autoregulation and is involved in MR, TGF, or both responses to an acute change in renal perfusion pressure, (b) Cx40 contributes to agonist-induced vasodilator and constrictor responses in the renal and systemic circulation, and (c) Cx40 is involved in the signaling pathway of NO that blunts the renal MR. To this end, we conducted RBF studies on mice with genetic ablation of Cx40. Parallel studies were performed on mice with the coding region for Cx40 replaced by Cx45 regulated by the Cx40 promotor. In the latter, we postulated that Cx45 can assume Cx40 function, at least in part.     

Muscle Density and Bone Quality of the Distal Lower Extremity Among Individuals with Chronic Spinal Cord Injury

Background:

Understanding the related fates of muscle density and bone quality after chronic spinal cord injury (SCI) is an important initial step in determining endocrine-metabolic risk.

Objective:

To examine the associations between muscle density and indices of bone quality at the distal lower extremity of adults with chronic SCI.

Methods:

A secondary data analysis was conducted in 70 adults with chronic SCI (C2-T12; American Spinal Injury Association Impairment Scale [AIS] A-D; ≥2 years post injury). Muscle density and cross-sectional area (CSA) and bone quality indices (trabecular bone mineral density [TbBMD] at the distal tibia [4% site] and cortical thickness [CtTh], cortical area [CtAr], cortical BMD [CtBMD], and polar moment of inertia [PMI] at the tibial shaft [66% site]) were measured using peripheral quantitative computed tomography. Calf lower extremity motor score (cLEMS) was used as a clinical measure of muscle function. Multivariable linear regression analyses were performed to determine the strength of the muscle-bone associations after adjusting for confounding variables (sex, impairment severity [AIS A/B vs AIS C/D], duration of injury, and wheelchair use).

Results:

Muscle density was positively associated with TbBMD (b = 0.85 [0.04, 1.66]), CtTh (b = 0.02 [0.001, 0.034]), and CtBMD (b = 1.70 [0.71, 2.69]) (P < .05). Muscle CSA was most strongly associated with CtAr (b = 2.50 [0.12, 4.88]) and PMI (b = 731.8 [161.7, 1301.9]) (P < .05), whereas cLEMS was most strongly associated with TbBMD (b = 7.69 [4.63, 10.76]) (P < .001).

Conclusion:

Muscle density and function were most strongly associated with TbBMD at the distal tibia in adults with chronic SCI, whereas muscle size was most strongly associated with bone size and geometry at the tibial shaft.Key words: bone mineral density, bone quality, muscle density, muscle size, osteoporosis, peripheral quantitative computed tomography, spinal cord injurySpinal cord injury (SCI) is associated with sublesional muscle atrophy,^1–3 changes in muscle fiber type,^4,5 reductions in hip and knee region bone mineral density (BMD),^6–8 and increased central and regional adiposity after injury.^9,10 Adverse changes in muscle and bone health in individuals with SCI contribute to an increased risk of osteoporosis,^11–13 fragility fractures,¹⁴ and endocrine-metabolic disease (eg, diabetes, dyslipidemia, heart disease).^15–17 Crosssectional studies have shown a higher prevalence of lower extremity fragility fractures among individuals with SCI ranging from 1% to 34%.^18–20 Fragility fractures are associated with negative health and functional outcomes, including an increased risk of morbidity and hospitalization,^21,22 mobility limitations,²³ and a reduced quality of life.²⁴ Notably, individuals with SCI have a normal life expectancy, yet fracture rates increase annually from 1% per year in the first year to 4.6% per year in individuals greater than 20 years post injury.^25,26Muscle and bone are thought to function as a muscle-bone unit, wherein muscle contractions impose loading forces on bone that produce changes in bone geometry and structure.^27,28 A growing body of evidence has shown that individuals with SCI (predominantly those with motor complete injury) exhibit similar patterns of decline in muscle cross-sectional area (CSA) and BMD in the acute and subacute stages following injury.^4,11,29 Prospective studies have exhibited a decrease in BMD of 1.1% to 47% per year^6,7,30 and up to 73% in the 2 to 7 years following SCI.^8,14,31,32 Decreases in muscle CSA have been well-documented following SCI, with greater disuse atrophy observed after complete SCI versus incomplete SCI, presumably due to the absence of voluntary muscle contractions and associated mobility limitations.^1,2,16 Muscle quality is also compromised early after SCI, resulting in sublesional accumulation of adipose tissue in the chronic stage of injury^3,33,34; the exact time course of this event has been poorly elucidated to date. Adipose tissue deposition within and between skeletal muscle is linked to an increase in noncontractile muscle tissue and a reduction in muscle force-generating capacity on bone.^35,36 Skeletal muscle fat infiltration is up to 4 times more likely to occur in individuals with SCI,^1,16,37 contributing to metabolic complications (eg, glucose intolerance),¹⁶ reduced muscle strength and function,³⁸ and mobility limitations³ – all factors that may be associated with a deterioration in bone quality after SCI.The association between lean tissue mass and bone size (eg, BMD and bone mineral content) in individuals with SCI has been wellestablished using dual energy x-ray absorptiometry (DXA).^9,10,29,34 However, DXA is unable to measure true volumetric BMD (vBMD), bone geometry, and bone structure. Peripheral quantitative computed tomography (pQCT) is an imaging technique that improves our capacity to measure indices of bone quality and muscle density and CSA at fracture-prone sites (eg, tibia).^3,39 Recent evidence from cross-sectional pQCT studies has shown that muscle CSA and calf lower extremity motor score (cLEMS) were associated with indices of bone quality at the tibia in individuals with SCI.^13,40 However, neither study measured muscle density (a surrogate of fatty infiltration when evaluating the functional muscle-bone unit). Fatty infiltration of muscle is common after SCI^1,16,37 and may affect muscle function or the muscle-bone unit, but the association between muscle density and bone quality indices at the tibia in individuals with chronic SCI is unclear. Muscle density measured using pQCT may be an acceptable surrogate of muscle quality when it is difficult to assess muscle strength due to paralysis.^3,39 Additionally, investigating which muscle outcome (muscle density, CSA, cLEMS) is most strongly associated with vBMD and bone structure may inform modifiable targets for improving bone quality and reducing fracture risk after chronic SCI.The primary objective of this secondary analysis was to examine the associations between pQCTderived calf muscle density and trabecular vBMD at the tibia among adults with chronic SCI. The secondary objective was to examine the associations between calf muscle density, CSA, and function and tibial vBMD, cortical CSA and thickness, and polar moment of inertia (PMI). First, we hypothesize that calf muscle density will be a positive correlate of trabecular and cortical vBMD, cortical CSA and thickness, and PMI at the tibia in individuals with chronic SCI. Second, we hypothesize that of the key muscle variables (cLEMS, CSA and density), calf muscle density and cLEMS will be most strongly associated with trabecular vBMD, whereas calf muscle CSA will be most strongly associated with cortical CSA and PMI.     

Activation of p53 Promotes Renal Injury in Acute Aristolochic Acid Nephropathy

Ingestion of aristolochic acid (AA) can cause AA nephropathy (AAN), in which excessive death of tubular epithelial cells (TECs) characterize the acute phase. AA forms adducts with DNA, which may lead to TEC apoptosis via p53-mediated signaling. We tested this hypothesis both by studying p53-deficient mice and by blocking p53 in TECs with its inhibitor pifithrin-α. AA induced acute AAN in wild-type mice, resulting in massive apoptotic and necrotic TEC death and acute renal failure; p53 deficiency or pharmacologic inhibition attenuated this injury. In vitro, AA induced apoptotic and necrotic death of TEC in a time- and dosage-dependent manner, with apoptosis marked by a 10-fold increase in cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling–positive/Annexin V-positive propidium iodide–negative TECs (all P < 0.001). AA induced dephosphorylation of STAT3 and the subsequent activation of p53 and TEC apoptosis. In contrast, overexpression of STAT3, p53 inhibition, or p53 knockdown with small interfering RNA all attenuated AA-induced TEC apoptosis. Taken together, these results suggest that AA induces TEC death via apoptosis by dephosphorylation of STAT3 and posttranslational activation of p53, supporting the hypothesis that p53 promotes renal injury in acute AAN.Chinese herb nephropathy was first reported in Belgium in patients with prolonged intake of Chinese herbs during a slimming regimen and is recognized as one of the most severe complications caused by traditional Chinese medicine.^1–3 It is now clear that the major substance that causes Chinese herb nephropathy is the plant nephrotoxin aristolochic acid and its metabolism products.^4–6 Thus, the term aristolochic acid nephropathy (AAN), instead of Chinese herbal nephropathy, is used today.^7,8 AAN has emerged as an important cause of drug-associated renal failure worldwide.⁹Patients with AAN exhibit a rapidly progressive renal deterioration, resulting in acute renal failure that could lead to ESRD.^1–3,10,11 A similar clinical course was observed in experimental animals treated with AA.^12,13 Pathologically, chronic AAN is characterized by extensive interstitial fibrosis with atrophy and loss of renal tubules.^{1–3,10–13} The lesions of chronic AAN are mainly in the cortex involving proximal tubular epithelial cells (TECs)^10–13; glomeruli are relatively spared with minimal inflammation.^9–12 In contrast, progressive TEC death occurs early in the clinical course with an absence of renal fibrosis and inflammation in experimental models and patients with acute AAN.^10,14,15 Although apoptosis is an important pathologic feature in in vivo and in vitro studies of acute AAN,^16–18 the underlying mechanisms remain unclear.In considering the genotoxic effect of AA with the formation of AA-DNA adducts and the importance of the p53 signaling pathway in DNA damage and cell apoptosis,^19–21 we hypothesized that TEC apoptosis in acute AAN is dependent on p53 signaling. We investigated this by inducing acute AAN in p53 knockout (KO) and p53 wild-type (WT) mice and by blocking the p53 activities with a pharmacologic inhibitor. We further studied the toxicity of AA on TEC apoptosis by examining a panel of apoptotic biomarkers. The mechanism that AA induced TEC apoptosis by activating p53 via a STAT3-dependent posttranslational modification was identified.     

Depression,Pain Intensity,and Interference in Acute Spinal Cord Injury

Background:

The high prevalence of pain and depression in persons with spinal cord injury (SCI) is well known. However the link between pain intensity, interference, and depression, particularly in the acute period of injury, has not received sufficient attention in the literature.

Objective:

To investigate the relationship of depression, pain intensity, and pain interference in individuals undergoing acute inpatient rehabilitation for traumatic SCI.

Methods:

Participants completed a survey that included measures of depression (PHQ-9), pain intensity (“right now”), and pain interference (Brief Pain Inventory: general activity, mood, mobility, relations with others, sleep, and enjoyment of life). Demographic and injury characteristics and information about current use of antidepressants and pre-injury binge drinking also were collected. Hierarchical multiple regression was used to test depression models in 3 steps: (1) age, gender, days since injury, injury level, antidepressant use, and pre-injury binge drinking (controlling variables); (2) pain intensity; and (3) pain interference (each tested separately).

Results:

With one exception, pain interference was the only statistically significant independent variable in each of the final models. Although pain intensity accounted for only 0.2% to 1.2% of the depression variance, pain interference accounted for 13% to 26% of the variance in depression.

Conclusion:

Our results suggest that pain intensity alone is insufficient for understanding the relationship of pain and depression in acute SCI. Instead, the ways in which pain interferes with daily life appear to have a much greater bearing on depression than pain intensity alone in the acute setting.Key words: depression, pain, spinal cord injuriesThe high incidence and prevalence of pain following spinal cord injury (SCI) is well established^1–6 and associated with numerous poor health outcomes and low quality of life (QOL).^1,7,8 Although much of the literature on pain in SCI focuses on pain intensity, there is emerging interest in the role of pain interference or the extent to which pain interferes with daily activities of life.^7,9 With prevalence as high as 77% in SCI, pain interference impacts life activities such as exercise, sleep, work, and household chores.^2,7,10–13 Pain interference also has been associated with disease management self-efficacy in SCI.¹⁴ There is a significant relationship between pain intensity and interference in persons with SCI.⁷ Like pain, the high prevalence of depression after SCI is well-established.^15–17 Depression and pain often co-occur,^18,19 and their overlap ranges from 30% to 60%.¹⁹ Pain is also associated with greater duration of depressed mood.²⁰ Pain and depression share common biological pathways and neurotransmitter mechanisms,¹⁹ and pain has been shown to attenuate the response to depression treatment.^21,22Despite the interest in pain and depression after SCI and implications for the treatment of depression, their co-occurrence has received far less attention in the literature.²³ Greater pain has been associated with higher levels of depression in persons with SCI,^16,24 although this is not a consistent finding.²⁵ Similarly, depression in persons with SCI who also have pain appears to be worse than for persons with non-SCI pain, suggesting that the link between pain and depression may be more intense in the context of SCI.²⁶ In one of the few studies of pain intensity and depression in an acute SCI rehabilitation setting, Cairns et al ²⁷ found a co-occurrence of pain and depression in 22% to 35% of patients. This work also suggested an evolution of the relationship between pain and depression over the course of the inpatient stay, such that they become associated by discharge. Craig et al²⁸ found that pain levels at discharge from acute rehabilitation predicted depression at 2-year follow-up. Pain interference also has been associated with emotional functioning and QOL in persons with SCI^1,7,29,30 and appears to mediate the relationship between ambulation and depression.³¹Studies of pain and depression in person with SCI are often limited methodologically to examine the independent contributions of pain intensity and interference to depression in an acute setting. For example, they include only pain intensity^{16,23,25,28,30}; classify subjects by either pain plus depression²³ or pain versus no pain^8,28,30; use pain intensity and interference as predictor and outcome, respectively¹; collapse pain interference domains into a single score¹; or use only univariate tests (eg, correlations).^7,8,25,30 In addition, the vast majority focus on the chronic period of injury. To fill a gap in knowledge, we examined the independent contributions of pain intensity and pain interference to depression, while accounting for injury and demographic characteristics, antidepressant treatment, and pre-injury binge drinking in a sample of persons with acute SCI. We hypothesized that when accounting for both pain intensity and interference in the model, interference would have an independent and significant relationship with depression, above and beyond pain intensity.     

ABCB1 Genotypes Predict Cyclosporine-Related Adverse Events and Kidney Allograft Outcome

Cyclosporine A (CsA) is a substrate of P-glycoprotein, an efflux transporter encoded by the ABCB1 gene. Compared with carriers of the wild-type gene, carriers of T allelic variants in exons 21 or 26 have reduced P-glycoprotein activity and, secondarily, increased intracellular concentration of CsA; therefore, carriers of T variants might be at increased risk for CsA-related adverse events. We evaluated the associations between ABCB1 genotypes (in exons 12, 21, and 26) and CsA-related outcomes in 147 renal transplant recipients who were receiving CsA-based immunosuppression and were included in the Mycophenolate Steroids Sparing study. During a median of 65.5 mo follow-up, carriers of T allelic variants in exons 21 or 26 had a three-fold risk for delayed graft function (DGF), a trend to slower recovery of renal function and lower GFR at study end, and significantly higher incidences of new-onset diabetes and cytomegalovirus reactivation compared with carriers of the wild-type genotype. T variants in both exons 21 and 26 were independently associated with 3.8- and 3.5-fold higher risk for DGF, respectively (P = 0.022 and P = 0.034). The incidence of acute rejection and the mean CsA dose and blood levels were comparable in genotype groups. In conclusion, renal transplant recipients with T allelic variants in ABCB1 exons 21 or 26 are at increased risk for CsA-related adverse events. Genetic evaluation may help to identify patients at risk and to modulate CsA therapy to optimize graft and patient outcomes.The introduction of cyclosporine A (CsA) therapy in the early 1980s opened a new era in organ transplantation. Compared with steroid- and azathioprine-based regimens, immunosuppressive protocols including this inhibitor of calcineurin—a key enzyme involved in T cell activation¹—decreased the incidence of acute rejections from 40% to 50% to 20% to 30% and increased one-year survival rates of the grafts from 60% to between 80% and 90%.² Thirty years later, CsA remains a cornerstone of immunosuppressive therapy for recipients of both renal and nonrenal transplants worldwide. However, standard recommended doses are associated with nephrotoxicity, resulting in delayed graft function (DGF) and progressive renal function deterioration in the long term.^3,4 Moreover, CsA worsens glucose tolerance and lipid profile; increases systemic BP; and, similarly to other immunosuppressants, enhances the risk of opportunistic infections, lymphoproliferative disorders, and cancer.^1,5To minimize side effects without increasing the risk of rejection, treatment is titrated to target CsA blood levels according to well established guidelines.⁶ However, the therapeutic index remains narrow, whereas the frequency and severity of CsA-related adverse effects are considerably variable among patients, even at comparable CsA levels.^1,6 This suggests the possibility that a heterogeneous individual susceptibility may result in increased risk in some patients despite exposure to CsA levels that in the majority of cases are devoid of significant toxicity.⁶ Thus, identifying markers or predictors of individual response to CsA therapy might help to tailor CsA therapy and optimize the risk/benefit profile of CsA-based immunosuppression.Drug efficacy and tolerability are influenced by several factors, including the activity of proteins and enzymes involved in drug transport and metabolism.⁷ CsA is a substrate of an efflux transporter—the P-glycoprotein (P-gp) encoded by the multidrug resistance-1 gene (now referred as ABCB1)—which actively transports lipophilic drugs and other xenobiotics from the intracellular to the extracellular domain.⁸ This transporter is expressed in lymphocytes,⁹ and in other leukocytes,¹⁰ as well as in hepatocytes and on the brush border of enterocytes and proximal tubular cells.⁸ Reduced expression or functional inhibition of this efflux-pump invariably results in increased intracellular and tissue drug concentrations,^8,9 but may have unpredictable effects on CsA blood levels. Indeed, CsA blood levels increased, decreased, or did not change in different settings, likely because of different balances between enhanced distribution into the tissue compartment and decreased excretion into the gastrointestinal lumen or urinary tract.⁷ Increased CsA concentrations in lymphocytes,¹¹ polymorphonuclear cells,¹² and other circulating leukocytes¹³ have been associated with increased production and release of reactive oxygen species (ROS). ROS production by leukocytes is the primary defense against invading micro-organisms, but has also been involved in the pathogenesis of ischemia-reperfusion damage of engrafted tissues or organs.^3,14 Thus, increased intracellular CsA disposition with enhanced ROS production might amplify oxidative stress and tissue damage to the graft after reperfusion.¹⁵ Increased intralymphocyte drug concentration is also associated with more effective inhibition of lymphocyte proliferation by CsA in vitro,¹⁶ an effect that, in vivo, might translate into more effective protection against graft rejection,¹⁷ but also into excess risk of opportunistic infections, lymphoproliferative disorders, or cancer.P-glycoprotein expression and activity are reduced in carriers of one or two T allelic variants in exons 12, 21, and 26 as compared with carriers of the wild-type ABCB1 gene.^8,18,19 Thus, at comparable CsA exposure, carriers of the allelic variants are expected to have higher intracellular and tissue CsA levels than wild-type carriers and, conceivably, should be exposed to more, and more severe, CsA-related events. We formally tested this hypothesis in a large cohort of renal transplant recipients prospectively monitored in the setting of a randomized, controlled, clinical trial, the Mycophenolate Steroids Sparing (MYSS) study, which aimed to compare the risk/benefit profile of mycophenolate mofetil and azathioprine therapy in immunosuppressive regimens including the CsA microemulsion Neoral.²⁰     

Dynamic Three-Dimensional Ultrasound to Evaluate Scapular Movement Among Manual Wheelchair Users and Healthy Controls

Background:

A large percentage of individuals with spinal cord injury (SCI) report shoulder pain that can limit independence and quality of life. The pain is likely related to the demands placed on the shoulder by transfers and propulsion. Shoulder pathology has been linked to altered scapular mechanics; however, current methods to evaluate scapular movement are invasive, require ionizing radiation, are subject to skin-based motion artifacts, or require static postures.

Objective:

To investigate the feasibility of applying 3-dimensional ultrasound methods, previously used to look at scapular position in static postures, to evaluate dynamic scapular movement.

Method:

This study evaluated the feasibility of the novel application of a method combining 2-dimensional ultrasound and a motion capture system to determine 3-dimensional scapular position during dynamic arm elevation in the scapular plane with and without loading.

Results:

Incremental increases in scapular rotations were noted for extracted angles of 30°, 45°, 60°, and 75° of humeral elevation. Group differences were evaluated between a group of 16 manual wheelchair users (MWUs) and a group of age- and gender-matched able-bodied controls. MWUs had greater scapular external rotation and baseline pathology on clinical exam. MWUs also had greater anterior tilting, with this difference further accentuated during loading. The relationship between demographics and scapular positioning was also investigated, revealing that increased age, pathology on clinical exam, years since injury, and body mass index were correlated with scapular rotations associated with impingement (internal rotation, downward rotation, and anterior tilting).

Conclusion:

Individuals with SCI, as well as other populations who are susceptible to shoulder pathology, may benefit from the application of this imaging modality to quantitatively evaluate scapular positioning and effectively target therapeutic interventions.Key words: kinematics, scapula, ultrasound, wheelchair userThe shoulder is a common site of injury across many populations. Because it is the most mobile joint in the body, the high prevalence of disorders is not surprising. Individuals are at increased risk for shoulder pathology when exposed to high forces, sustained postures, and repetitive movements.¹ Wheelchair users are exposed to all of these factors in activities of daily living. Among manual wheelchair users (MWUs), 35% to 67% report shoulder pain.^2–7 In this population, the presence of shoulder dysfunction significantly affects function and decreases quality of life.^8,9 With altered scapular kinematics being linked to a multitude of shoulder problems, the identification of changes in kinematics may allow for earlier detection of pathology and targeting of appropriate interventions.^10–25 However, evaluation of dynamic scapular movement is a challenging task, as the scapula rotates about 3 axes while also gliding underneath overlying tissue. Direct visualization of the bone is ideal but is often limited by cost, availability, and exposure to radiation, and skin-based systems are prone to error.^26–33The overall goal of this study was to investigate the feasibility of applying 3-dimensional ultrasound methods, previously used to look at scapular position in static postures, to evaluate dynamic scapular movement.³⁴ The specific goals were as follows:

1. Evaluate intermediate angles of functional elevation during dynamic movement (30°, 45°, 60°, and 75°). We hypothesize that we will see incremental increases in external rotation, upward rotation, and posterior tipping throughout the movement to maintain the distance between the acromion and humerus.
2. Compare dynamic scapular movement between MWUs and able-bodied controls (ABs). We anticipate that the nature of wheelchair propulsion and demands of activities of daily living will elucidate differences between this population and ABs with comparably lower daily demands on the shoulder.
3. Evaluate the effect of loading on scapular movement, as other studies have suggested that differences in kinematics are clearer in the presence of loading.^10,35,36
4. Investigate the relationship between shoulder pathology, age, years since injury, and body mass index (BMI) and scapular positioning.

     

Portal vein glucose sensors do not play a major role in modulating physiological responses to insulin-induced hypoglycemia in humans

OBJECTIVE—Experimental data from animal studies indicate that portal vein glucose sensors play a key role in the responses to slow-fall hypoglycemia. However, their role in modulating these responses in humans is not well understood. The aim of the present study was to examine in humans the potential role of portal vein glucose sensors in physiological responses to insulin-induced hypoglycemia mimicking the slow fall of insulin-treated diabetic subjects.RESEARCH DESIGN AND METHODS—Ten nondiabetic subjects were studied on two different occasions during intravenous insulin (2 mU · kg⁻¹ · min⁻¹) plus variable glucose for 160 minutes. In both studies, after 60 min of normal plasma glucose concentrations, hypoglycemia (47 mg/dl) was induced slowly (60 min) and maintained for 60 min. Hypoglycemia was preceded by the ingestion of either oral placebo or glucose (28 g) given at 30 min.RESULTS—Plasma glucose and insulin were not different with either placebo or glucose (P > 0.2). Similarly, counterregulatory hormones, substrates, and symptoms were not different with either placebo or glucose. The Stroop color and colored words subtest of the Stroop test deteriorated less (P < 0.05) with glucose than placebo.CONCLUSIONS—In contrast to animals, in humans, prevention of portal hypoglycemia with oral glucose from the beginning of insulin-induced slow-fall hypoglycemia has no effect on sympathoadrenal and symptomatic responses to hypoglycemia.It has been suggested that glucose sensors in the portal area are necessary to monitor glucose derived from the gut (1). In fact, when exogenous glucose is infused directly in the portal vein (2,3) or in the duodenum (4) or ingested orally as glucose load (5–7), a portal-arterial glucose gradient is generated with glucose concentrations higher in the portal vein than in arterial circulation. Such portal-arterial glucose gradient generates a portal signal that is probably dependent on glucose-sensitive nerves in the portal veins, the firing rate of which is inversely proportional to the portal glucose concentration (8). The signal then moves through the hepatic vagal afferences to modulate the function of different tissues (e.g., liver, pancreatic β-cells) involved in the control of glucose homeostasis (9). In addition, signals enter the central nervous system to regulate hypothalamic functions, such as feeding and satiety (10). Recent evidence indicates that GLUT2 transporter is essential for glucose sensing by the portal glucose sensor (11,12) and also that glucagon-like peptide 1 (GLP-1) receptor is required for the function of the portal glucose sensor in mice (9).Earlier studies in animals have shown that portal-arterial glucose gradient is involved in the control of intake of food (10) and stimulation of net hepatic glucose uptake (2,13). In addition, portal glucose sensors modulate the sympathetic responses to hypoglycemia (14,15). However, how portal glucose sensors may affect sympathetic responses to hypoglycemia and how their activity integrates with that of glucose-sensitive areas in the brain is not well understood (16). In fact, several studies in animals indicate that the brain is the prominent center for the sensing of hypoglycemia. In dogs in which insulin-induced hypoglycemia was allowed to occur peripherally while brain euglycemia was maintained by glucose infusions in carotid and vertebral arteries bilaterally, the responses of counterregulatory hormones decreased nearly completely compared with dogs with brain neuroglycopenia (17,18). In rats, the ventromedial hypothalamus (VMH) appears to be necessary to trigger counterregulation during hypoglycemia. In fact, bilateral lesions of the VMH in conscious rats suppresses glucagon and catecholamine responses during hypoglycemia (19,20), suggesting that the VHM is one of the most important sites acting as a glucose sensor (21,22). However, there is evidence that in rats, activation of portal glucose sensors by glucose may be the most important modulators of sympathetic response to hypoglycemia, resulting in a significant suppression of this response (23–25).Recent studies in rats have established that portal vein glucose sensors, responsible for hypoglycemic detection, extend beyond the portal vein being placed also in the superior mesenteric vein and that their role is essential in detecting slow, but not fast, fall in blood glucose (26).Limited knowledge is available about the potential role of portal glucose sensors in humans. Only three studies (5–7) have addressed the question, with conflicting results. In fact, counterregulatory hormone responses to hypoglycemia have been found potentiated (5), reduced (6), or reduced in early phase and potentiated in late phase (7) after ingestion of oral glucose (5,6) or orange juice (7). It is likely that methodological differences account, at least in part, for these divergent results.So far, no study has investigated in humans the role of portal glucose sensors on counterregulation, symptoms, and cognitive function during hypoglycemia induced slowly, to mimic the hypoglycemia of the clinical situation (27). It is worthy of note that in the postprandial state, a condition characterized by glucose arriving in the portal vein from the gut, sympathetic responses and some aspects of cognitive function are affected by the rate of fall of blood glucose (27). However, in the postprandial condition, it is not only glucose that enters in the portal system but also other substrates that may suppress sympathoadrenal responses to hypoglycemia (28).The aim of the present study was to examine in humans the potential effects of portal glucose sensors on hormonal counterregulatory responses and responses of symptoms and cognitive function in a model of slow-fall insulin-induced hypoglycemia. For this purpose, healthy subjects were studied during hypoglycemia preceded by ingestion of either oral glucose to prevent portal hypoglycemia or placebo.     

A Recessive Gene for Primary Vesicoureteral Reflux Maps to Chromosome 12p11-q13

Primary vesicoureteral reflux (pVUR) is one of the most common causes of pediatric kidney failure. Linkage scans suggest that pVUR is genetically heterogeneous with two loci on chromosomes 1p13 and 2q37 under autosomal dominant inheritance. Absence of pVUR in parents of affected individuals raises the possibility of a recessive contribution to pVUR. We performed a genome-wide linkage scan in 12 large families segregating pVUR, comprising 72 affected individuals. To avoid potential misspecification of the trait locus, we performed a parametric linkage analysis using both dominant and recessive models. Analysis under the dominant model yielded no signals across the entire genome. In contrast, we identified a unique linkage peak under the recessive model on chromosome 12p11-q13 (D12S1048), which we confirmed by fine mapping. This interval achieved a peak heterogeneity LOD score of 3.6 with 60% of families linked. This heterogeneity LOD score improved to 4.5 with exclusion of two high-density pedigrees that failed to link across the entire genome. The linkage signal on chromosome 12p11-q13 originated from pedigrees of varying ethnicity, suggesting that recessive inheritance of a high frequency risk allele occurs in pVUR kindreds from many different populations. In conclusion, this study identifies a major new locus for pVUR and suggests that in addition to genetic heterogeneity, recessive contributions should be considered in all pVUR genome scans.Vesicoureteral reflux (VUR; OMIM no. 193000) is the retrograde flow of urine from the bladder to the ureters and the kidneys during micturation. Uncorrected, VUR can lead to repeated urinary tract infections, renal scarring and reflux nephropathy, accounting for up to 25% of pediatric end stage renal disease.^1,2 VUR is commonly seen as an isolated disorder (primary VUR; pVUR), but it can also present in association with complex congenital abnormalities of the kidney and urinary tract or with specific syndromic disorders, such as renal-coloboma and branchio-oto-renal syndromes.^3–8pVUR has a strong hereditary component, with monozygotic twin concordance rates of 80%.^9–12 Sibling recurrence rates of 30% to 65% have suggested segregation of a single gene or oligogenes with large effects.^9,12–14 Interestingly however, the three published genome-wide linkage scans of pVUR have strongly suggested multifactorial determination.^15–17 Two pVUR loci have been identified with genome-wide significance on chromosomes 1p13 and 2q37 under an autosomal dominant transmission with locus heterogeneity.^15,16 Multiple suggestive signals have also been reported, but remarkably, these studies show little overlap.^15–17 These data suggest that pVUR may be extremely heterogeneous, with mutations in different genes each accounting for a fraction of cases. The genes underlying pVUR loci have not yet been identified, but two recent studies have reported segregating mutations in the ROBO2 gene in up to 5% of pVUR families.^18,19Despite evidence for genetic heterogeneity and different subtypes of disease, genetic studies have all modeled pVUR as an autosomal dominant trait.^15–17,20 Recessive inheritance has generally not been considered because the absence of affected parents can be explained by spontaneous resolution of pVUR with older age. However, many pVUR cohorts are composed of affected sibships or pedigrees compatible with autosomal recessive transmission, suggesting the potential for alternative modes of inheritance.^{9–12,16,17,20–22} Systematic family screening to clarify the mode of inheritance is not feasible for pVUR because the standard diagnostic tool, the voiding cystourethrogram (VCUG), is invasive and would expose participants to radiation. Formal assessment of a recessive contribution in sporadic pVUR has also been difficult because studies have been conducted in populations with low consanguinity rates.^{9–12,16,17,20–22} However, recent studies have identified an unexpected recessive contribution to several complex traits such as ductus arteriosus or autism.^23,24 Thus, in addition to genetic heterogeneity, genes with alternative modes of transmission may segregate among pVUR families, and misspecification of the inheritance model may complicate mapping studies of this trait.Several approaches can be considered to address the difficulties imposed by complex inheritance, variable penetrance, and genetic heterogeneity. Studying large, well characterized cohorts with newer single-nucleotide polymorphism (SNP)-based technologies can maximize inheritance information across the genome and increase the power of linkage studies.²⁵ In addition, in the setting of locus heterogeneity and uncertainty about the mode of transmission, analysis under a dominant and a recessive model has greater power compared with nonparametric methods and more often results in detection of the correct mode of transmission without incurring a significant penalty for multiple testing.^26–29 We combined these approaches in this study and successfully localized a major gene for VUR, which unexpectedly demonstrates autosomal recessive transmission.     

Cycling with Functional Electrical Stimulation Before and After a Distal Femur Fracture in a Man with Paraplegia

Case Presentation:

A man with chronic paraplegia sustained a distal femur fracture following an unrelated fall while enrolled in a study examining musculoskeletal changes after 6 months of cycling with functional electrical stimulation (FES). After healing, he restarted and completed the study.

Management and Outcome:

Study measures included areal bone mineral density, trabecular bone microarchitecture, cortical bone macroarchitecture, serum bone formation/resorption markers, and muscle volume. The patient made small gains in bone- and muscle-related measures. Bone markers had not returned to baseline prior to restarting cycling, which may have impacted results.

Discussion:

This case shows that cycling with FES may be safely resumed after distal femur fracture.Key words: bone, cycling, fracture, functional electrical stimulation, spinal cord injuryCycling with functional electrical stimulation (FES) is an intervention for people with spinal cord injury (SCI) that is often used to improve overall health by targeting fitness, bone density, muscle volume, and other health indicators that impact risk for metabolic syndrome, diabetes, and cardiovascular disease.^1–6 Inclusion in FES cycling programs is often impacted by bone status; programs and studies may exclude people with bone density that is a predetermined standard deviation below normal.^7,8 Other programs base exclusion on lower extremity fracture history.^9,10Even though people are excluded due to concerns about fractures during interventions, there is no evidence to support an increased fracture risk with FES cycling, with or without a fracture history. One study reported greater shear and less compressive forces at the knee with standing versus seated electrical stimulated exercise, suggesting greater risk with the knee flexed to 90°.¹¹ However, the only fracture reported in the literature occurred during a high force flexed isometric contraction,¹² not cycling. For people with SCI, lower extremity fractures typically occur during nontraumatic activities such as transfers^13,14 and are labeled fragility fractures,¹⁵ with the majority occurring in the distal femur and proximal tibia.^13,14 These fractures are thus caused by a different mechanism than what may occur during FES cycling. Furthermore, people with a history of low bone density and fragility fractures may have the greatest benefit from interventions such as FES cycling to decrease fracture risk by improving bone health.This case report describes the outcomes for a man with chronic paraplegia who sustained an unrelated distal femur fracture while enrolled in an FES cycling study. There are 2 objectives of this case report. The first is to demonstrate that FES cycling can be safely resumed following fracture healing, and the second is to discuss the subject’s musculoskeletal outcomes after he restarted the study once medically cleared after fracture healing.     

Slit Diaphragms Contain Tight Junction Proteins

Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.Slit diaphragms are specialized cell-cell junctions located between mature podocytes that have fascinated cell biologists and nephrologists for more than 40 yr.¹ In contrast to podocytes, most other epithelial cells have junctional complexes composed of tight junctions and adherens junctions. Slit diaphragms originate from typical apical junctional complexes between primordial epithelia of the early S-shaped body. These junctional complexes migrate in a zipper-like fashion to the base of the cell where tight junctions persist as interdigitation of the foot processes begins.^2,3 Slit diaphragms appear during the capillary loop stage and gradually replace tight junctions. In many diseases associated with proteinuria and foot process loss or effacement, there is a rerun in reverse of this developmental sequence, and tight junctions reappear between adjoining foot processes.^4–6Major progress has been made recently in establishing the molecular make-up of the slit diaphragms. Several integral membrane proteins, including nephrin,⁷ podocin,⁸ and Neph1,⁹ not found in other junctions, have been identified as slit diaphragm components. Slit diaphragms are currently looked upon as signaling platforms in which nephrin and Neph1 transduce major signals that serve to maintain the filtration slits and to regulate podocyte shape through interaction of slit diaphragm proteins with the actin cytoskeleton.¹⁰ Mutations in nephrin,⁷ Neph1,⁹ and podocin⁸ have been linked to diseases associated with foot process effacement and proteinuria. In addition to these specialized slit diaphragm proteins, a number of other proteins that are associated with junctions in other locations are also concentrated at the slit diaphragms, including the adherens junction proteins P-cadherin,¹¹ FAT,¹² β-catenin,¹¹ and p120 catenin;¹³ scaffold proteins such as ZO-1,^14,15 CD2AP,¹⁶ MAGI-2,¹⁷ and CASK;¹³ and actin binding proteins, including IQGAP¹⁷ and α-actinin 4.^17,18 Because slit diaphragms share some morphologic features with adherens junctions and contain P-cadherin and catenins, slit diaphragms are assumed to represent modified adherens junctions.¹¹ However, several scaffold proteins that are often associated with tight junctions (i.e., ZO-1,^14,15 MAGI-1,¹⁹ MAGI-2,¹⁷ and CASK¹³) are present at slit diaphragms and have been shown to associate with nephrin. Based on their derivation from typical tight junctions² and the fact that they are replaced by tight junctions in nephrosis,^4,6 we reasoned that slit diaphragms might also contain membrane proteins normally associated with tight junctions.In this paper, we used morphological, biochemical, and bioinformatics techniques to investigate the expression of representative tight junction proteins in glomeruli in situ and in slit diaphragm-enriched fractions. Here, we document the presence of several tight junction proteins in slit diaphragms and demonstrate their interactions with slit diaphragm proteins in both normal and PAN nephrotic rats. The presence of tight junction proteins in slit diaphragms adds a new dimension to understanding the organization and functions of these junctions.