Coronary Flow Velocity Reserve and Carotid Intima Media Thickness in Patients with Autosomal Dominant Polycystic Kidney Disease: From Impaired Tubules to Impaired Carotid and Coronary Arteries

Background and objectives: Cardiovascular problems are a major cause of morbidity and mortality in patients with autosomal dominant polycystic kidney disease. Endothelial dysfunction, an early and reversible feature in the pathogenesis of atherosclerosis, is associated with increased vascular smooth muscle tone, arterial stiffening, and increased intima-media thickness. Coronary flow velocity reserve is a noninvasive test showing endothelial function of epicardial coronary arteries and coronary microcirculatory function. The aim of the study was to investigate the carotid intima-media thickness and coronary flow velocity reserve in patients with autosomal dominant polycystic kidney disease.Design, setting, participants, & measurements: Thirty normotensive patients with autosomal dominant polycystic kidney disease (10 male, 20 female) with well-preserved renal function and 30 healthy subjects (12 male, 18 female) were included in the study. Coronary flow velocity reserve was measured at baseline and after dipyridamole infusion by echocardiography. Coronary flow velocity reserve was calculated as the ratio of hyperemic to baseline diastolic peak velocities.Results: Carotid intima-media thickness was significantly higher in patients than in control subjects (0.80 ± 0.29 versus 0.54 ± 0.14 mm, respectively; P < 0.001). Moreover, coronary flow velocity reserve was significantly lower in patients than in control subjects (1.84 ± 0.39 versus 2.65 ± 0.68, respectively; P < 0.001).Conclusions: Normotensive patients with autosomal dominant polycystic kidney disease with well-preserved renal function have significantly increased carotid intima-media thickness and significantly decreased coronary flow velocity reserve compared with healthy subjects. These findings suggest that atherosclerosis starts at an early stage in the course of their disease in patients with autosomal dominant polycystic kidney disease.Autosomal-dominant polycystic kidney disease (ADPKD) is a hereditary renal disease that occurs in 1 of 400 to 1000 individuals (1,2). Cardiovascular problems are a major cause of morbidity and mortality in patients with ADPKD (3). Hypertension, a common finding in patients with ADPKD, often occurs before the onset of renal insufficiency and is associated with faster progression to ESRD and increased cardiovascular mortality (4,5). Activation of the renin-angiotensin-aldosterone system (RAAS) caused by cyst expansion and local ischemia has been proposed to play an important role in the development of hypertension in ADPKD (6). The RAAS is stimulated at an early stage of ADPKD, even before the onset of hypertension and clinical findings (7,8). Likewise, increased left ventricular mass indexes and biventricular diastolic dysfunction have been reported before the development of hypertension in patients with ADPKD with well-preserved renal function (9–16). Moreover, endothelial dysfunction (ED), which is an early manifestation of vascular injury, occurs in both normotensive and hypertensive patients with ADPKD, before the onset of renal failure (17).Coronary flow velocity reserve (CFVR) represents the capacity of the coronary circulation to dilate after an increase in myocardial metabolic demands (18). Although CFVR was measured invasively until recently, it can be evaluated noninvasively by using Doppler and vasodilator stress, such as dipyridamole or adenosine (19). By using this method, impairment of CFVR can be assessed before development of angiographically detectable stenosis in epicardial coronary arteries. The aim of this study was to investigate the CFVR in patients with ADPKD, with well-preserved renal function.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Protective effect of mitochondria-targeted antioxidants in an acute bacterial infection

Acute pyelonephritis is a potentially life-threatening infection of the upper urinary tract. Inflammatory response and the accompanying oxidative stress can contribute to kidney tissue damage, resulting in infection-induced intoxication that can become fatal in the absence of antibiotic therapy. Here, we show that pyelonephritis was associated with oxidative stress and renal cell death. Oxidative stress observed in pyelonephritic kidney was accompanied by a reduced level of mitochondrial B-cell lymphoma 2 (Bcl-2). Importantly, renal cell death and animal mortality were both alleviated by mitochondria-targeted antioxidant 10(6′-plastoquinonyl) decylrhodamine 19 (SkQR1). These findings suggest that pyelonephritis can be treated by reducing mitochondrial reactive oxygen species and thus by protecting mitochondrial integrity and lowering kidney damage.Normally, the kidney and urinary tract are germ-free. However, during their lifetimes, about 40% of women and 12% of men experience urinary tract infections (UTIs) (1). Acute pyelonephritis (APN) is a potentially life-threatening complication of UTI that occurs when infection progresses to the upper urinary tract. The uropathogen most frequently associated with this disease is the pyelonephritogenic subset of Escherichia coli, which is responsible for up to 85% of both complicated and uncomplicated UTIs (2). This disease is frequently accompanied by bacterial invasion and stimulation of acute inflammatory response (3, 4). Toxin-induced epithelial damage and bladder hemorrhage contribute further to the pathogenicity of uropathogens in the kidney, with progression leading to renal damage including renal scarring and in extreme cases septicemia (5, 6). Ultimately, renal scarring is a cause of substantial morbidity (7, 8).Leukocyte infiltration in response to bacterial invasion is an important contributor to renal tissue damage (9, 10). Production and extracellular release of reactive oxygen species (ROS) by infiltrating leukocytes can lead to kidney injury and dysfunction (11, 12). Consequently, oxidative stress in renal cells may be a critical factor in the pathogenesis of pyelonephritis whereas pharmacological management of the oxidative stress response may provide a therapeutic effect in preventing renal pathologies (13–18). However, the issue is complicated by the diversity of ROS-generating mechanisms and their differential contribution to host defense from infection and collateral tissue damage. Mitochondria and NADPH oxidases (19) are the two principle sources of ROS although their relative contribution to inflammatory pathologies is not well-defined. A new class of antioxidants that specifically target mitochondrial ROS (hereafter referred to as SkQ1 and SkQR1) have been recently developed and shown to have a beneficial effect in a number of cell pathologies (20–28).In this study, we explored mechanisms of APN progression considering various aspects of interaction of renal cells with leukocytes and bacterial pathogens. The goal was to gain insight into the role and mechanisms of induction of oxidative stress in eukaryotic components of the system and find an approach of directed correction of the pathological oxidative changes in renal tissue. We analyzed the relevance of the strategy of protecting the kidney based on the activation of prosurvival and blockage of prodeath signaling pathways involving mitochondria. We evaluated chimeric compounds carrying an antioxidant moiety as potential agents to efficiently alleviate the deleterious consequences of APN. To facilitate the future design of directed pharmacologic interventions to normalize renal function subsequent to APN, we explored the role of mitochondria and oxidative stress in this pathology using positively charged membrane-permeable, mitochondrial-targeted compounds (29). We demonstrated that specific targeting of mitochondrial ROS by antioxidant resulted in a significant protective effect in animal models of APN. These results illustrate the role of mitochondrial ROS in renal tissue damage in the context of acute infection and suggest a therapeutic potential of mitochondrial antioxidants.     

High-Dosage Intravenous Immunoglobulin–Associated Macrovacuoles Are Associated with Chronic Tubulointerstitial Lesion Worsening in Renal Transplant Recipients

Background and objectives: Intravenous immunoglobulins (IVIg) may induce acute renal failure associated with tubular vacuolization. Although the use of IVIg is increasing in kidney transplantation, their impact on graft histology and function remains unknown.Design, setting, participants, & measurements: Twenty-seven kidney transplant recipients who had high immunologic risk and were treated with four courses of IVIg after transplantation were studied retrospectively at a transplant center, and findings were compared with those of 27 control subjects. Protocol kidney biopsies were performed at time of transplantation and at 3 mo and 1 yr after transplantation.Results: No episode of IVIg-related acute renal failure occurred. Nevertheless, screening biopsies revealed the presence of “microvacuoles” and “macrovacuoles.” Widespread microvacuolizations were often detected (70%) on preimplantation biopsy and not associated with IVIg. Macrovacuoles, which were absent on preimplantation biopsies, were observed exclusively in IVIg-treated patients. Macrovacuoles among IVIg-treated patients were seen in kidneys from older donors and were associated with chronic tubulointerstitial changes at 3 mo, with similar trends at 1 yr. Macrovacuoles were associated with lower creatinine clearance at last follow-up in IVIg-treated patients.Conclusions: IVIg frequently induce tubular macrovacuoles in kidney transplant recipients. These are more frequently observed in grafts from older donors, suggesting a higher vulnerability to IVIg. These data suggest a deleterious impact of IVIg-induced macrovacuoles on chronic tubulointerstitial changes and long-term renal function.Intravenous immunoglobulins (IVIg) have proved effective in the treatment of primary or secondary antibody deficiencies and various autoimmune and inflammatory disorders (1,2) and have become widely used since the 1980s. IVIg are also useful for pretransplantation desensitization of patients who have high levels of preformed anti-HLA antibodies and are awaiting renal transplantation (3) and, in combination with plasmapheresis, for the treatment of acute humoral rejection (4,5). Recently, our group also reported that the use of IVIg as a prophylactic therapy in patients at high immunologic risk was associated with good 1-yr outcome and a profound decrease in level of panel reactive antibodies (6). IVIg are usually considered to be safe and well tolerated, but the risk for IVIg-induced acute renal failure has recently been highlighted (7,8). The incidence of IVIg-related acute renal failure has been estimated to be approximately 6% in patients who are treated for autoimmune or infectious diseases (9), but because preexisting kidney disease seems to be a risk factor (10), IVIg-related renal toxicity is likely to occur more frequently in kidney transplant recipients. Although the mechanism of renal injury associated with IVIg use has not been clearly established, kidney biopsies performed in patients with IVIg-induced acute renal failure have demonstrated extensive vacuolization of proximal tubules, suggestive of osmotic nephrosis (10,11); however, the evolution of these acute histologic kidney lesions and their long-term impact on tubulointerstitial changes and renal function remain unknown. Tubular vacuolizations are commonly observed on renal transplant biopsies, including those obtained from patients who have not received IVIg. Other medications, such as calcineurin inhibitors (12–14), iodinated contrast media (15,16), and hydroxyethylstarch (17), are also potential inducers of tubular vacuolizations. To our knowledge, however, no attempt has been made to describe precisely these tubular vacuolizations and their long-term effects.At our center, protocol kidney biopsies are performed systematically at time of transplantation (day 0) and at 3 mo and at 1 yr after -transplant, allowing longitudinal follow-up and monitoring of histologic changes. We present here the results of a study undertaken with the aim of describing tubular vacuolizations observed in kidney transplant biopsies, identifying those related to IVIg, and evaluating the impact of IVIg-induced vacuolizations on tubulointerstitial damage and renal function over time.     

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56^bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α₄β₁ integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4⁺ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4⁺ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4⁺ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4⁺ T cells and the cytolytic activity of NK cells.Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) (1) and one of the major causes of neurological disability in young adults (2). MS is considered to be a primarily antigen-driven T cell-mediated disease with a complex genetic background influenced by environmental factors (1, 3) that is caused by an imbalanced immune-regulatory network (4). Among other well-known players of this network such as regulatory T cells and tolerogenic dendritic cells (DCs) (1), natural killer (NK) cells have been recently identified as additional factors in controlling homeostasis of antigen-activated T cells (5, 6).Originally discovered as antigen receptor-negative innate lymphocytes that play an important role in controlling virus-infected and tumor cells (7), NK cells have also been shown to suppress activated T cells through secretion of anti-inflammatory cytokines and/or cytolytic function (5, 6, 8–12). NK cells lyse target cells in a complex process depending on cell surface expression of certain inhibitory and activating receptors on NK cells and the corresponding ligands on target cells (13). Several activating NK-cell receptors–in particular, NKG2D (CD314) (5, 8, 9, 11, 14), the receptor for MIC-A/B and ULBP1-6, and DNAM-1 (DNAX accessory molecule, CD226) (6, 12, 15), the receptor for Nectin-2 (CD112) and poliovirus receptor (PVR/CD155)−have been proposed to be involved in NK cell-mediated lysis of activated T cells. Of note, polymorphisms in the gene encoding for DNAM-1 have been consistently found in MS-association studies (16–18). Both major NK-cell subsets, namely the CD56^brightCD16^dim/− and the CD56^dimCD16⁺ subsets (here referred to as CD56^bright and CD56^dim, respectively), seem to be capable of killing activated T cells (19). CD56^dim NK cells are the major NK-cell subset in the peripheral blood (PB) (90% of NK cells) and kill target cells without prior sensitization but only secrete low levels of cytokines (7, 20, 21), whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues and inflammatory lesions (75–95% of NK cells), where they produce high amounts of immune-modulating cytokines but acquire cytolytic functions only after prolonged activation (7, 20, 21).Immune-modulating therapies targeting NK-cell frequencies and cytolytic functions among others such as IFN-β (22–24), glatiramer acetate (25), natalizumab (26, 27), fingolimod (28, 29), and daclizumab (10, 30, 31) point to an immune-protective role of both NK-cell subsets in MS. Daclizumab, a humanized antibody directed against the IL-2 receptor (IL-2R) α-chain (CD25) (reviewed in ref. 4) is a promising MS therapy, which recently showed superior efficacy compared with IFN-β in a phase III study (32). Expansion of peripheral (10, 33) as well as intrathecal (34) CD56^bright NK cells under daclizumab treatment correlated positively with therapeutic response (10, 30, 35). Nevertheless, it still remains to be elucidated whether NK-cell immune-regulatory functions are impaired as part of the disease process and whether modulation of the IL-2R with daclizumab restores these deficits or simply boosts NK-cell activity (4). Furthermore, the distribution and function of NK cells in active MS lesions is still poorly understood. Resolving the molecular basis of NK cell-mediated immune control and its potential impairment in MS is important for a better understanding of the role of NK cells in MS pathogenesis and the mechanism of action of NK cell-modulating therapies.The aim of the current study was to characterize the role of NK cells in the pathogenesis of MS by investigating the presence, distribution, and function of NK cells in three different compartments [CNS, cerebrospinal fluid (CSF), and PB]. Furthermore, a potential deficit in NK-cell immune-regulatory function, its underlying molecular mechanism, and the impact of IL-2R modulation by daclizumab high-yield process (DAC HYP) were explored by studying PB mononuclear cells (PBMCs) derived from clinically stable therapy-naïve MS patients and MS patients receiving daclizumab treatment in comparison with those derived from healthy individuals.     

Cyst formation following disruption of intracellular calcium signaling

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca²⁺) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca²⁺-activated Ca²⁺ channel of the endoplasmic reticulum. We hypothesize that Ca²⁺ signaling through PC2, or other intracellular Ca²⁺ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca²⁺ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca²⁺ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca²⁺ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca²⁺ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca²⁺ signaling lead to ADPKD cysts.The commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1–3). Altered Ca²⁺ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca²⁺ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon short-term culture of Madin-Darby canine kidney (MDCK) cells (6–12) or cells from patients (13–18) or PC1-null mice (19, 20; for review, see ref. 21). Recently, 3D tissues have been developed that incorporate mouse cells containing a shRNA-mediated knockdown of PC1 (9, 19). The benefits of this system include the use of a cell line, thus eliminating the need to isolate primary cells, and the use of cells with a stable genetic background.Ca²⁺ signaling underpins many cellular processes ranging from cell proliferation to cell death. Intracellular Ca²⁺ levels can be modified by opening of the inositol 1, 4, 5-trisphosphate receptor (InsP3R) or other intracellular Ca²⁺ release channels, including PC2. Over 99% of PC2 resides on the endoplasmic reticulum (22), where it is known to act as a modulator of the InsP3R and the ryanodine receptor (RyR) (23), with the remainder on the primary cilia. PC2 itself can function as a Ca²⁺-activated Ca²⁺ release channel (22, 24).Although it was demonstrated in 3D cultures that the knockdown of PC1 leads to cyst development (25), the effect of knocking down PC2 or other Ca²⁺-signaling proteins has not been shown. It has been hypothesized that the disruption of PC2, or the proteins that it interacts with, will result in cyst growth, as Ca²⁺ is a major signaling molecule (26, 27). Cells with decreased PC2 have been linked with decreased Ca²⁺ signaling (28), and overexpression of PC2 has been shown to act as an inhibitor of cell proliferation (29). Changing PC2 expression levels alters the uptake of Ca²⁺ into the endoplasmic reticulum, leading to liver cyst formation (30), but no direct link involving the release of Ca²⁺ from the endoplasmic reticulum has been implicated in renal cyst development. Similarly, changes in the expression of the InsP3R have been correlated with various disease conditions; for example, the InsP3R is upregulated in colorectal cancer (31), but downregulated in bile duct obstruction and cholestasis (32, 33).Here, we demonstrate that cyst formation can be followed for several weeks using a 3D culture system and that the disruption of intracellular Ca²⁺ signaling, through the knockdown of either InsP3R or PC2, leads to cyst development.     

Natriuretic Peptides in Chronic Kidney Disease

Background and objectives: B-type natriuretic peptide (BNP) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP) are biomarkers of cardiovascular disease that is common in patients with chronic kidney disease (CKD). Conflicting data on the influence of glomerular filtration rate (GFR) on BNP and NT-proBNP levels in CKD may stem from failure to account fully for the effects of coexistent cardiac disease, dysfunction, and volume overload.Design, setting, participants, & measurements: Prospective head-to-head comparison of plasma BNP and NT-proBNP in ambulatory euvolemic CKD patients with normal LV ejection fraction and no manifest cardiac or vascular disease. GFR was estimated by the Modification of Diet in Renal Disease formula, BNP and NT-proBNP measured using Abbott AxSYM and Roche Elecsys assays, respectively, and cardiac morphology and function assessed by transthoracic echocardiography.Results: In 142 patients (42% female) of mean age 60 ± 11 yr, mean left ventricular ejection fraction was 71% ± 6%, GFR 38 ± 14 ml/min per 1.73 m², and median BNP and NT-proBNP level 59 and 311 pg/ml, respectively. Multivariate predictors of NT-proBNP level were GFR, β-blocker usage, LV mass index, and hemoglobin level. Plasma BNP was independently predicted by LV mass index and β-blocker usage but not GFR. In the 74 patients without diastolic dysfunction, there was a significant rise in NT-proBNP but not BNP as GFR declined.Conclusions: Unlike NT-proBNP, plasma BNP level is relatively independent of GFR. BNP may therefore be the more appropriate biomarker to screen for cardiac dysfunction in CKD.Patients with chronic kidney disease (CKD) are at increased risk of cardiovascular disease. Natriuretic peptides (NPs), biomarkers of myocardial dysfunction (1), offer the potential for early detection and risk stratification of cardiac disease, as evident in emergency department (2) and community (3,4) settings. This screening utility could be extended to CKD patients asymptomatic of cardiovascular disease.However, the precise influence of CKD on circulating levels of B-type natriuretic peptide (BNP) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP), the two commonly used NPs in clinical practice, continues to be debated. Dependence of plasma BNP on glomerular filtration rate (GFR) has been reported among patients with and without heart failure (HF) (5,6), but this relationship may not be independent of cardiac or volume-related factors (7,8). The data on NT-proBNP in renal dysfunction are more concordant but were derived from populations that included patients with myocardial infarction, reduced left ventricular (LV) ejection fraction (LVEF), or HF (9,10). Indeed, most studies examining the impact of renal dysfunction on NPs uniformly included such patients (5,6,8–10). Recent Doppler myocardial imaging studies have shown that even HF patients with normal LVEF have reduced LV contractility compared with controls (11,12).To limit confounding by cardiac dysfunction or volume overload, we prospectively constituted a clinically euvolemic CKD cohort without symptoms or history of cardiac disease and normal LVEF and regional function. We measured circulating levels of both NPs, hypothesizing that, in these patients, BNP can be shown to be relatively independent of GFR compared with NT-proBNP if cardiac and loading factors can be comprehensively accounted for.     

Asymmetric Dimethylarginine and Mortality in Stages 3 to 4 Chronic Kidney Disease

Background and objectives: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, reduces bioavailability of nitric oxide and induces endothelial dysfunction. This dimethylated amino acid accumulates in chronic kidney disease and may be involved in the pathophysiology of cardiovascular disease (CVD) in this population.Design, settings, participants, & methods: The Modification of Diet in Renal Disease Study was a randomized, controlled trial conducted between 1989 and 1993. We measured ADMA in frozen samples collected at baseline (n = 820) and obtained survival status, up to December 31, 2000, from the National Death Index. We examined the relationship of ADMA with prevalent CVD and performed multivariable Cox models to examine the relationship of ADMA with all-cause and CVD mortality.Results: Mean (SD) age was 52 (12) yr, GFR was 32 ± 12 ml/min per 1.73 m², and ADMA was 0.70 ± 0.25 μmol/L. A 1-SD increase in ADMA was associated with a 31% increased odds of prevalent CVD in an adjusted logistic regression model. During the 10-yr follow-up period, 202 (25%) participants died of any cause, 122 (15%) from CVD, and 545 (66%) reached kidney failure. In multivariable Cox models, a 1-SD increase in ADMA was associated with a 9% increased risk for all-cause and 19% increased risk for CVD mortality.Conclusions: In this cohort of patients with predominantly nondiabetic, stages 3 to 4 chronic kidney disease, there was a strong association of ADMA with prevalent CVD and a modest association with all-cause and CVD mortality.Endothelium-derived nitric oxide, an important mediator of vascular tone and BP regulation, is produced via a reaction catalyzed by nitric oxide synthase (1). Asymmetric dimethylarginine (ADMA), a byproduct of the breakdown of arginine methylated proteins, is an endogenous inhibitor of this reaction (2). Increased ADMA levels lead to nitric oxide depletion, impaired endothelium-dependent vasodilation, reduced free radical scavenging, and plaque rupture with thrombus formation (3–5). Plasma concentrations of ADMA are elevated in cardiovascular high-risk states such as hypertension (6,7), obesity (8), and diabetes (9) and seem to be related to endothelial dysfunction in patients with these conditions. High ADMA levels were an index of carotid intima-media thickness and were associated with future acute coronary events in general population studies (10,11).Levels of ADMA are elevated in chronic kidney disease (CKD) (12,13). This 202-Da amino acid is eliminated unchanged in the urine but is also taken up and degraded in the kidney by the enzyme dimethylarginine dimethylaminohydrolase (DDAH); ADMA accumulation in kidney failure is due to both decreased elimination and reduced DDAH activity (14). High ADMA was an independent risk factor for cardiovascular disease (CVD) and all-cause mortality in a cohort of patients who were on hemodialysis (13,15) and was associated with faster rates of kidney disease progression in patients in the earlier stages of CKD (16); however, data are limited on the relationship between ADMA levels and CVD in patients with CKD before reaching kidney failure (17,18). We therefore examined the relationship of ADMA with prevalent CVD and with all-cause and CVD mortality during long-term follow-up of a cohort of patients with stages 3 to 4 CKD.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

Early Angiography in Patients with Chronic Kidney Disease: A Collaborative Systematic Review

Background and objectives: In the general population, an early invasive strategy of routine coronary angiography is superior to a conservative strategy of selective angiography in patients who are admitted with unstable angina or non–ST segment elevation myocardial infarction (MI), but the effectiveness of this strategy in individuals with chronic kidney disease (CKD) is uncertain.Design, setting, participants, & measurements: We conducted a collaborative meta-analysis with data provided by the main authors of identified trials to estimate the effectiveness of early angiography in patients with CKD. The Cochrane, Medline, and EMBASE databases were searched to identify randomized trials that compared invasive and conservative strategies in patients with unstable angina or non-ST MI. Pooled risks ratios were estimated using data from enrolled patients with estimated GFR <60 ml/min per 1.73 m².Results: Five randomized trials that enrolled 1453 patients with CKD were included. An early invasive strategy was associated with nonsignificant reductions in all-cause mortality, nonfatal MI, and a composite of death or nonfatal MI. The invasive strategy significantly reduced rehospitalization.Conclusions: This collaborative study suggests that the benefits of an early invasive strategy are preserved in patients with CKD and that an early invasive approach reduces the risk for rehospitalization and is associated with trends of reduction in the risk for death and nonfatal re-infarction in patients with CKD. Coronary angiography should be considered for patients who have CKD and are admitted with non–ST elevation acute coronary syndromes.An invasive strategy of routine coronary angiography with revascularization when indicated anatomically after non-ST elevation myocardial infarction (MI) or unstable angina may be more efficacious than a conservative strategy of selective angiography limited to patients whose medical therapy fails. Recent meta-analyses of randomized, controlled trials found that an early invasive strategy was associated with an 18% lower risk for death or nonfatal MI and a 25% lower risk for MI than a conservative strategy (1,2). Accordingly, clinical practice guidelines for the management of non-ST acute coronary syndromes (ACS) recommend an invasive strategy for patients with hemodynamic instability, refractory angina, electrical instability, or an elevated risk for clinical events (3).Although guidelines do not recommend treatment modification on the basis of renal function (3,4), the risk-to-benefit tradeoff may be different in the 11% of adults with chronic kidney disease (CKD) (5). The risk for developing de novo coronary artery disease or death after an initial MI increases markedly with even minor reductions in GFR (4,6–8). Given this high risk, patients with CKD could derive greater absolute benefits from an invasive strategy than patients without CKD; however, the risk for adverse outcomes is also high in patients with CKD. Coronary angiography is more likely to cause cholesterol embolism or acute kidney injury—an event associated with a high risk for death (9,10)—in patients with CKD (9). In addition, both percutaneous and surgical coronary revascularization may provide less durable results in patients with CKD (11–13).It is therefore unclear how clinical trials that compare conservative and invasive strategies apply to patients with CKD. Accordingly, we performed a collaborative meta-analysis in which the authors of randomized trials that compared invasive and conservative strategies in non-ST ACS prepared data on enrolled patients with CKD.     

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K⁺ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K⁺ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K⁺ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3^S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K⁺ secretion in the setting of volume depletion.Hypertension affects 1 billion people worldwide and is a major risk factor for death from stroke, myocardial infarction, and congestive heart failure. The study of Mendelian forms of hypertension has demonstrated the key role of increased renal salt reabsorption in disease pathogenesis (1–4). Observational and intervention trials (5, 6) also indicate that increased dietary K⁺ lowers blood pressure; however, the mechanism of this effect has been unclear.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man no. 145260), featuring hypertension and hyperkalemia, has revealed a previously unrecognized mechanism that regulates the balance between renal salt reabsorption and K⁺ secretion in response to aldosterone (7). Aldosterone is produced by the adrenal glomerulosa in volume depletion, in response to angiotensin II (AII), and in hyperkalemia via membrane depolarization (8). In volume depletion, aldosterone maximizes renal salt reabsorption, whereas in hyperkalemia, aldosterone promotes maximal renal K⁺ secretion. Volume depletion increases both the NaCl cotransporter (NCC) (9) and electrogenic Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC) (10). The lumen-negative potential produced by ENaC activity provides the electrical driving force for paracellular Cl⁻ reabsorption (11). In hyperkalemia, the lumen-negative potential promotes K⁺ secretion via the K⁺ channel Kir1.1 (renal outer medullary K⁺ channel ROMK), reducing plasma K⁺ level (12, 13). Additionally, recent studies have implicated aldosterone signaling in intercalated cell transcellular Cl⁻ flux (14). In these cells, hyperkalemia induces phosphorylation of the mineralocorticoid receptor (MR) ligand-binding domain, making it incapable of ligand binding and activation. AII signaling induces dephosphorylation, and activation of the MR by aldosterone then induces transcellular Cl⁻ flux, which is required for defense of intravascular volume (14, 15). Because electrogenic Cl⁻ reabsorption and K⁺ secretion both dissipate the lumen-negative potential produced by ENaC, maximal Cl⁻ reabsorption inhibits K⁺ secretion and vice versa.Patients with PHAII have constitutive reabsorption of NaCl with concomitant inhibition of K⁺ secretion, resulting in hypertension and hyperkalemia, despite normal levels of aldosterone (7). Dominant mutations in the serine–threonine kinases with-no-lysine 4 (WNK4) or WNK1, or in CUL3 or KLHL3, elements of a ubiquitin ligase complex, cause this disease (2, 4). WNK4 modulates the activities of NCC, ENaC, Kir1.1, and MR (14, 16–21), and WNK4 function can be modulated by phosphorylation (21). CUL3/KLHL3 has been shown to target WNK4 and WNK1 for ubiquitination and degradation, and disease-causing mutations impair this binding and degradation (22–24). In particular, dominant mutations in the Kelch domain of KLHL3 prevent binding to WNKs; reciprocally, disease-causing point mutations in WNK4 also prevent WNK4–KLHL3 binding.These findings suggest that regulation of WNK degradation by CUL3/KLHL3 is highly regulated and that disease-causing mutations might phenocopy a state in which WNKs are normally turned off, producing constitutive salt reabsorption and inhibited K⁺ secretion. We now demonstrate that this inference is correct and implicate AII signaling in this process.     

Screening for Fabry Disease in Patients with Chronic Kidney Disease: Limitations of Plasma α-Galactosidase Assay as a Screening Test

Background and objectives: Fabry disease is a progressive X-linked disorder of glycosphingolipid metabolism that typically presents in childhood and progresses to heart failure and renal failure in adulthood. This study sought to determine the prevalence of Fabry disease in a multiethnic male chronic kidney disease population, involving dialysis-dependent, non–dialysis-dependent, and transplant patients.Design, setting, participants, & measurements: A total of 499 patients were screened with assay of α-galactosidase activity using fluorometric enzyme assay on plasma prepared from fresh heparinized blood, followed by leukocyte α-galactosidase activity in the subset of patients with plasma α-galactosidase activity below the second percentile (corresponding to a value <3.0 nmol/h per ml plasma).Results: This study did not identify any new cases of Fabry disease; however, repeat testing of some of the study patients identified three limitations of the plasma enzyme assay that is commonly used as a high throughput screening method for Fabry disease: (1) False-negative results can occur; (2) these false-negative results are not prevented by use of inhibitors of α-galactosidase B activity; and (3) considerable intraindividual variation in plasma α-galactosidase levels reduces the discriminatory power of the screening test.Conclusion: Clinicians need to be aware that screening using plasma will fail to detect some patients with Fabry disease.Fabry disease (FD) is a progressive X-linked disorder of glycosphingolipid metabolism caused by a deficiency of the α-galactosidase lysosomal enzyme. The deficiency of this enzyme leads to progressive accumulation of neutral glycosphingolipids (chiefly globotriaosylceramide) in many tissues throughout the body, particularly the vascular endothelium, heart, and kidney (1–5). Absence of α-galactosidase activity results in cutaneous angiokeratomas, periodic febrile acroparesthesia, decreased or absent sweating, and corneal opacities during childhood. By the second or third decade, patients often manifest progressive proteinuria and renal failure. Eventually patients succumb to nephrologic, cardiac, and cerebrovascular complications in their fourth to fifth decade (3).Typically the diagnosis of FD is made in male adolescents, but it may be missed or delayed, especially if the classic manifestations are subtle or absent. For example, alternate phenotypes have been described in heterozygous female individuals (3,6) as have cardiac (2,4,7) and renal (5) variants. These “variant” phenotypes usually have a low level of residual α-galactosidase activity that somewhat protects patients from developing microvascular glycosphingolipid accumulation, resulting in a lack of the classic phenotype. Such patients may present solely with progressive end-organ failure in their 40s (1). Because safe and effective enzyme replacement therapy is now available for FD, it is important to identify all patients with the disease earlier, when it is potentially treatable, so that appropriate family screening and genetic counseling can be provided and, if appropriate, enzyme replacement therapy can be started (8,9).Overall, the prevalence of FD has been estimated to be 1 in 40,000 to 117,000 male individuals (1,3,10); however, several recent studies suggested that the prevalence may be higher in the hemodialysis (HD) population, in which values up to 1.2% have been reported (5,11–17). Most of the available data are from Europe, the United States, and Japan (5,11–13,17); therefore, many ethnic groups are unrepresented in these estimates, and it is possible that the frequency of unrecognized FD will vary by racial, ethnic, and demographic group. This may account in part for the discrepancy in prevalence of FD that has been seen in studies in which screening of dialysis patients was done (13); however, issues of laboratory methods and the choice of appropriate screening cutoff values may also have an impact on the apparent incidence of disease. The widespread assumption that screening for FD by assay of α-galactosidase in plasma is “reliable, relatively simple, and inexpensive” (5) has not been adequately evaluated.We sought to assess the prevalence of unrecognized FD in our ethnically diverse kidney disease population through a screening program of male patients with chronic kidney disease (CKD). We also sought to validate published screening methods for FD.     

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.Hypertension is a complex disease that is characterized by increased blood pressure, renal damage, and vascular dysfunction which collectively increase risk of atherosclerosis, stroke, heart disease, and renal failure in one-quarter of all adults worldwide (1–3). Because there is strong evidence of heritability in hypertension (2, 4, 5), considerable effort has been put toward identifying novel candidate genes and their molecular mechanisms. Genome-wide association studies (GWAS) have identified many potential hypertension loci, which shed light on the genetic complexity of this disease (5–8) but have provided little mechanistic insight. As such, validation and elucidation of the functional roles and disease mechanisms for these gene candidates are the next important challenges (4).Because hypertension is a complex disease (i.e., multiple variants of small effect sizes contributing to disease risk), we hypothesized candidate gene targeting on a genetically sensitized background would reveal functional role(s) of genetic disease modifiers. The Dahl salt-sensitive (SS) rat is an inbred genetic model of salt-sensitive hypertension that displays hypertension-induced renal damage, cardiac hypertrophy and vascular dysfunction (9–11). These phenotypes are induced by exposing SS rats to a high-salt diet, which results in rapid induction of hypertensive phenotypes that closely resemble salt-induced hypertension seen in humans (12–15). Knockout of specific genes in this disease model using zinc-finger nuclease (ZFN) technology have revealed the importance of key mechanisms contributing to hypertension risk, such as the protection from salt-induced hypertension and renal injury by selective ablation of adaptive immune cells in the SS-Rag1^em1Mcwi and SS-Cd247^em1Mcwi knockout rats (16, 17) and reduced hypertension and renal injury in the SS-Ncf2^em1Mcwi (p67phox) null model exhibiting reduced medullary oxidative stress (18). Additionally, we have recently demonstrated multiple genes at a single hypertension GWAS-nominated locus (Agtrap-Plod1 locus) can have additive or subtractive effects on blood pressure and renal function when mutated in the SS rat (19). These previous studies highlight the utility of this model system for testing the roles of GWAS candidate human disease genes by disrupting their specific rat orthologs using ZFN technology (20).A single-nucleotide polymorphism (SNP) (rs381815, minor allele frequency 0.26) in intron 1 of the pleckstrin homology domain containing family A member 7 (PLEKHA7) gene, was identified by five independent GWAS to be associated with elevated systolic blood pressure and hypertension in multiple populations (5, 6, 8, 21, 22). The associated locus contains only the PLEKHA7 gene (5); however, the genetic mechanism(s) underlying this locus have not yet been functionally characterized. PLEKHA7 is highly expressed in the kidney and heart, where it may be involved in formation and maintenance of the apical junction complex of epithelial cells (23). However, limited data on PLEKHA7 function are available to extrapolate its potential role(s) in the pathogenesis of hypertension. Here we used ZFN mutagenesis to obtain the first evidence to our knowledge in any model system that Plekha7 has a functional role in several hypertension-associated phenotypes in the rat. We found that mutation of Plekha7 in the SS rat attenuated salt-induced hypertension, reduced renal damage, and improved cardiac function. We also show that Plekha7 modulates calcium handling and nitric oxide (NO) bioavailability, both of which are required for normal vascular health. Collectively, these studies provide significant mechanistic insight to the role of Plekha7 in salt-sensitive hypertension.     

Coronary Artery Calcification,ADMA, and Insulin Resistance in CKD Patients

Background and objectives: It is known that coronary artery calcification (CAC) develops in chronic kidney disease (CKD) before initiation of renal replacement therapy, and factors associated with CKD mineral and bone disorders (CKD-MBDs) are involved. However, little information is available about any association between plasma levels of asymmetric dimethylarginine (ADMA), insulin resistance, and CAC.Design, setting, participants, & measurements: A total of 111 CKD patients (79 men, 32 women; glomerular filtration rate [GFR] median, 33.7 ml/min per 1.73 m²), free of cardiovascular disease, were consecutively recruited along with 30 age-matched healthy subjects. Coronary artery calcification scores (CACS) were measured by multidetector-row CT according to Agatston score.Results: In CKD patients, CACS was distributed widely from 0 to 2901, while in age-matched, healthy control subjects (n = 30), CACS showed a range from 0 to 307. GFR had a significant negative correlation with CACS. Plasma ADMA levels were negatively correlated with GFR and positively correlated with CACS. When CACS was divided into quartiles (<50, n = 56; 50 to 300, n = 24; 300 to 600, n = 14; >600, n = 17), the patients with CACS >600 had significantly higher values of HOMA-IR, plasma ADMA levels, and fibrinogen along with serum levels of phosphorus, compared with those in patients having CACS <50. Multivariate regression analysis determined HOMA-IR as an independent contributing factor to CACS.Conclusions: CAC becomes more prevalent and severe with a decline in GFR, and plasma ADMA levels and insulin resistance, independent of factors associated with CKD-MBD, are correlated with CAC.Coronary artery calcification (CAC) is regarded as an index of the severity of atherosclerotic vascular disease and may predict future adverse cardiovascular events in patients on dialysis (1–3). In patients with chronic kidney disease (CKD) before initiation of renal replacement therapy, CAC is already present in the early phase of CKD (4–6) and among diabetic nephropathy (7). Associated factors with CAC besides age, calcium, phosphorus, iPTH, and inflammation have not been fully elucidated.Insulin resistance is known to play an important role for atherosclerosis (8) and to develop at an early stage of nondiabetic CKD in U.S. general populations (9). We report a similar result using a hyperinsulinemic euglycemic glucose clamp method and also showed that acidemia and dyslipidemia are independently associated with insulin resistance in CKD (10). In progressive renal disease, the relationship between hyperinsulinemia and hypertension is well documented (11). Arad et al. have reported that asymptomatic individuals with insulin resistance have elevated coronary calcification in general populations (12). However, it remains unknown whether insulin resistance is also correlated with CAC in CKD patients. Insulin resistance leading to atherosclerosis may be explained by increased plasma levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor (13). Concentrations of ADMA are related to endothelial dysfunction (14,15) because increased ADMA may impair blood flow, accelerate atherogenesis, and interfere with angiogenesis by inhibiting the production of nitric oxide (16). Of note, ADMA concentrations are higher in dialysis patients with clinically manifest atherosclerosis than in those without atherosclerotic disease (17), which suggests that accumulation of ADMA might be an important cardiovascular risk factor in end-stage renal disease. Moreover, the clinical importance of ADMA in coronary artery disease is highlighted in a recent trial by Meinitzer et al. (18) in which ADMA predicted cardiovascular events in 3200 patients. In addition, ADMA predicts coronary events in middle-aged white men (19).With this background in mind, we aimed to study the prevalence and associated factors of CAC in CKD patients before initiation of renal replacement therapy. Particularly, we wanted to know whether insulin resistance and/or plasma levels of ADMA would be correlated with CAC.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.