Unbiased kidney-centric molecular categorization of chronic kidney disease as a step towards precision medicine.

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD.

Antisense oligonucleotide treatment ameliorates IFN-γ-induced proteinuria in APOL1-transgenic mice.

African Americans develop end-stage renal disease at a higher rate compared with European Americans due to 2 polymorphisms (G1 and G2 risk variants) in the apolipoprotein L1 (APOL1) gene common in people of African ancestry. Although this compelling genetic evidence provides an exciting opportunity for personalized medicine in chronic kidney disease, drug discovery efforts have been greatly hindered by the fact that APOL1 expression is lacking in rodents. Here, we describe a potentially novel physiologically relevant genomic mouse model of APOL1-associated renal disease that expresses human APOL1 from the endogenous human promoter, resulting in expression in similar tissues and at similar relative levels as humans. While naive APOL1-transgenic mice did not exhibit a renal disease phenotype, administration of IFN-γ was sufficient to robustly induce proteinuria only in APOL1 G1 mice, despite inducing kidney APOL1 expression in both G0 and G1 mice, serving as a clinically relevant "second hit." Treatment of APOL1 G1 mice with IONIS-APOL1Rx, an antisense oligonucleotide (ASO) targeting APOL1 mRNA, prior to IFN-γ challenge robustly and dose-dependently inhibited kidney and liver APOL1 expression and protected against IFN-γ-induced proteinuria, indicating that the disease-relevant cell types are sensitive to ASO treatment. Therefore, IONIS-APOL1Rx may be an effective therapeutic for APOL1 nephropathies and warrants further development.

Translational strategy: humanized mini-organs.

Mini-organs engineered from decellularized organs repopulated with human stem cells can transform preclinical model strategies in target validation and biomarker discovery. Recellularized organs are whole humanized organs with preserved native architecture, conformity of the organ, composition of extracellular matrix and vascular matrix structures. With mini-organ models further understanding of developmental biology and assessment of potential therapeutic targets can be elucidated utilizing human induced pluripotent stem cells. As a next step, co-cultured mini-organ models could simulate pharmacokinetics and pharmacodynamics in physiological and pathological conditions. By overcoming key challenges, the development of humanized mini-organs as integrated biotechnology can address the translational gaps between in vitro, ex vivo and in vivo systems for an elevated human target validation model.

Renal Pre-Competitive Consortium (RPC2): discovering therapeutic targets together.

Despite significant effort, patients with kidney disease have not seen their outcomes improved significantly over the past two decades. This has motivated clinicians and researchers to consider alternative methods to identifying risk factors, disease progression markers, and effective therapies. Genome-scale data sets from patients with renal disease can be used to establish a platform to improve understanding of the molecular basis of disease; however, such studies require expertise and resources. To overcome these challenges, we formed an academic-industry consortium to share molecular target identification efforts and expertise across academia and the pharmaceutical industry. The Renal Pre-Competitive Consortium (RPC2) aims to accelerate novel drug development for kidney diseases through a systems biology approach. Here, we describe the rationale, philosophy, establishment, and initial results of this strategy.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization.

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-ßH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.

Long noncoding RNAs are dynamically regulated during ß-cell mass expansion in mouse pregnancy and control ß-cell proliferation in vitro.

Pregnancy is associated with increased ß-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. AIMS/HYPOTHESIS: This study tests the hypothesis that lncRNAs regulate ß-cell proliferation in response to prolactin in the context of ß-cell mass compensation in pregnancy. METHODS: The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 ß-cell line by EdU incorporation and cell count. RESULTS: We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary ß-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates ß-cell growth. CONCLUSIONS/INTERPRETATION: During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse ß-cell proliferation and may be a crucial component of ß-cell proliferation in ß-cell mass adaptation in both health and disease.

Myeloperoxidase Levels and Its Product 3-Chlorotyrosine Predict Chronic Kidney Disease Severity and Associated Coronary Artery Disease.

BACKGROUND: The role of myeloperoxidase in chronic kidney disease (CKD) and its association with coronary artery disease (CAD) is controversial. In this study, we compared myeloperoxidase and protein-bound 3-chlorotyrosine (ClY) levels in subjects with varying degrees of CKD and tested their associations with CAD. METHODS: From Clinical Phenotyping Resource and Biobank Core, 111 patients were selected from CKD stages 1 to 5. Plasma myeloperoxidase level was measured using enzyme-linked-immunosorbent assay. Plasma protein-bound 3-ClY, a specific product of hypochlorous acid generated by myeloperoxidase was measured by liquid chromatography mass spectrometry. RESULTS: We selected 29, 20, 24, 22, and 16 patients from stages 1 to 5 CKD, respectively. In a sex-adjusted general linear model, mean ± SD of myeloperoxidase levels decreased from 18.1 ± 12.3 pmol in stage 1 to 10.9 ± 4.7 pmol in stage 5 (p = 0.011). In patients with and without CAD, the levels were 19.1 ± 10.1 and 14.8 ± 8.7 pmol (p = 0.036). There was an increase in 3-ClY mean from 0.81 ± 0.36 mmol/mol-tyrosine in stage 1 to 1.42 ± 0.41 mmol/mol-tyrosine in stage 5 (p < 0.001). The mean 3-ClY levels in patients with and without CAD were 1.25 ± 0.44 and 1.04 ± 0.42 mmol/mol-tyrosine (p = 0.023), respectively. C-statistic of ClY when added to myeloperoxidase level to predict CKD stage 5 was 0.86, compared to 0.79 for the myeloperoxidase level alone (p = 0.0097). CONCLUSION: The myeloperoxidase levels decrease from stages 1 to 5, whereas activity increases. In contrast, both myeloperoxidase and ClY levels rise in the presence of CAD at various stages of CKD. Measuring both plasma myeloperoxidase and 3-CLY levels provide added value to determine the burden of myeloperoxidase-mediated oxidative stress.

Growth Differentiation Factor-15 and Risk of CKD Progression.

Growth differentiation factor-15 (GDF-15) is a member of the TGF-ß cytokine superfamily that is widely expressed and may be induced in response to tissue injury. Elevations in GDF-15 may identify a novel pathway involved in loss of kidney function among patients with CKD. Among participants in the Clinical Phenotyping and Resource Biobank (C-PROBE) study and the Seattle Kidney Study (SKS), we tested whether kidney tissue expression of GDF15 mRNA correlates with circulating levels of GDF-15 and whether elevations in circulating GDF-15 are associated with decline in kidney function. In matching samples of 24 patients with CKD from the C-PROBE study, circulating GDF-15 levels significantly correlated with intrarenal GDF15 transcript levels (r=0.54, P=0.01). Among the 224 C-PROBE and 297 SKS participants, 72 (32.1%) and 94 (32.0%) patients, respectively, reached a composite end point of 30% decline in eGFR or progression to ESRD over a median of 1.8 and 2.0 years of follow up, respectively. In multivariable models, after adjusting for potential confounders, every doubling of GDF-15 level associated with a 72% higher (95% confidence interval, 1.21 to 4.45; P=0.003) and 65% higher (95% confidence interval, 1.08 to 2.50; P=0.02) risk of progression of kidney disease in C-PROBE and SKS participants, respectively. These results show that circulating GDF-15 levels strongly correlated with intrarenal expression of GDF15 and significantly associated with increased risk of CKD progression in two independent cohorts. Circulating GDF-15 may be a marker for intrarenal GDF15-related signaling pathways associated with CKD and CKD progression.

Short-term Effects of Laparoscopic Adjustable Gastric Banding Versus Roux-en-Y Gastric Bypass.

OBJECTIVE: Bariatric surgery has been shown to have important long-term metabolic effects resulting in enhanced insulin sensitivity and improved glucose tolerance in patients with type 2 diabetes. The contribution of reduced caloric intake to these beneficial effects of surgery remains unclear. The aim of this study was to compare the short-term effects (1 week) of bariatric surgical procedures with a very low caloric intake (VLCI) on insulin sensitivity (IS) and insulin secretion (ISR) in nondiabetic obese subjects. RESEARCH DESIGN AND METHODS: Twenty obese patients without diabetes (BMI 44.2 ± 0.7 kg/m2) were admitted to the clinic for 1 week. At baseline and 1 week after VLCI (600 kcal/day), subjects received a hyperinsulinemic-euglycemic clamp with tracer infusion to quantify endogenous glucose production (EGP), lipolysis (rate of appearance of glycerol [RaGlycerol]), peripheral insulin sensitivity (insulin-stimulated glucose disposal [M value] divided by the steady-state plasma insulin concentration [M/I]), hepatic insulin sensitivity (Hep-IS [= 1/(EGP ⋅ insulin)]), and adipose insulin sensitivity (Adipo-IS [= 1/(RaGlycerol ⋅ insulin)]). An intravenous glucose bolus was administered at the end of the insulin clamp to measure ISR and ß-cell function (disposition index [DI]). Approximately 3 months later, patients were admitted for laparoscopic adjustable gastric banding (LAGB) (n = 10) or Roux-en-Y gastric bypass (RYGB) (n = 10), and were restudied 1 week after surgery under the same caloric regimen (600 kcal/day). RESULTS: After 1 week of VLCI, patients lost 2.1 kg without significant changes in Hep-IS, Adipo-IS, M/I, or DI. RYGB and LAGB led to greater weight loss (5.5 and 5.2 kg, respectively) and to significant improvement in Hep-IS, EGP, and lipolysis. Only RYGB improved Adipo-IS and M/I. No change in ISR or DI was observed in either surgical group. CONCLUSIONS: Bariatric surgery improves IS within 1 week. These metabolic effects were independent of caloric intake and more pronounced after RYGB compared with LAGB.

Generation of functional podocytes from human induced pluripotent stem cells.

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

The Mammalian circadian clock gene per2 modulates cell death in response to oxidative stress.

Living in the earth's oxygenated environment forced organisms to develop strategies to cope with the damaging effects of molecular oxygen known as reactive oxygen species (ROS). Here, we show that Per2, a molecular component of the mammalian circadian clock, is involved in regulating a cell's response to oxidative stress. Mouse embryonic fibroblasts (MEFs) containing a mutation in the Per2 gene are more resistant to cytotoxic effects mediated by ROS than wild-type cells, which is paralleled by an altered regulation of bcl-2 expression in Per2 mutant MEFs. The elevated survival rate and alteration of NADH/NAD(+) ratio in the mutant cells is reversed by introduction of the wild-type Per2 gene. Interestingly, clock synchronized cells display a time dependent sensitivity to paraquat, a ROS inducing agent. Our observations indicate that the circadian clock is involved in regulating the fate of a cell to survive or to die in response to oxidative stress, which could have implications for cancer development and the aging process.

Growth differentiation factor 15 predicts future insulin resistance and impaired glucose control in obese nondiabetic individuals: results from the XENDOS trial.

OBJECTIVE: Growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine that is increased in obesity and established type 2 diabetes. We assessed whether GDF-15 can predict future insulin resistance and impaired glucose control in obese nondiabetic individuals. DESIGN AND METHODS: Plasma GDF-15 concentrations were measured with an automated electrochemiluminescent immunoassay at baseline and after 4 years in 496 obese nondiabetic individuals (52% men, median age 48 years, median body mass index (BMI) 37.6 kg/m(2)) enrolled in the XENical in the prevention of Diabetes in Obese subjects (XENDOS) trial. RESULTS: The median GDF-15 concentration at baseline was 869 ng/l (interquartile range 723-1064 ng/l). GDF-15 was related to body weight, BMI, waist-to-hip ratio, and insulin resistance (homeostasis model assessment of insulin resistance (HOMA-IR)) (all P < 0.01). Changes in GDF-15 from baseline to 4 years were related to changes in body weight, BMI, waist-to-hip ratio, and HOMA-IR (all P < 0.05). Baseline GDF-15 was associated with the risk to have prediabetes or diabetes at 4 years by univariate analysis (odds ratio (OR) FOR 1 unit increase in ln GDF-15, 3.2; 95% confidence interval (CI): 1.7-6.1; P<0.001), and after multivariate adjustment for age, gender, treatment allocation (orlistat vs placebo), BMI, waist-to-hip ratio, and glucose control at baseline (OR 2.2; 95% CI: 1.1-4.7; P=0.026). Similarly, baseline GDF-15 was independently associated with HOMA-IR at 4 years (P=0.024). CONCLUSIONS: This first longitudinal study of GDF-15 in a large cohort of obese individuals indicates that GDF-15 is related to abdominal obesity and insulin resistance and independently associated with future insulin resistance and abnormal glucose control.

Consensus Guidelines for CSF and Blood Biobanking for CNS Biomarker Studies.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease.

Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition.

BACKGROUND: The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions. METHODS: We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions. RESULTS: In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. CONCLUSION: These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth.

Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo. We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.

Lack of food anticipation in Per2 mutant mice.

Predicting time of food availability is key for survival in most animals. Under restricted feeding conditions, this prediction is manifested in anticipatory bouts of locomotor activity and body temperature. This process seems to be driven by a food-entrainable oscillator independent of the main, light-entrainable clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus . Although the SCN clockwork involves self-sustaining transcriptional and translational feedback loops based on rhythmic expression of mRNA and proteins of clock genes , the molecular mechanisms responsible for food anticipation are not well understood. Period genes Per1 and Per2 are crucial for the SCN's resetting to light . Here, we investigated the role of these genes in circadian anticipatory behavior by studying rest-activity and body-temperature rhythms of Per1 and Per2 mutant mice under restricted feeding conditions. We also monitored expression of clock genes in the SCN and peripheral tissues. Whereas wild-type and Per1 mutant mice expressed regular food-anticipatory activity, Per2 mutant mice did not show food anticipation. In peripheral tissues, however, phase shifts of clock-gene expression in response to timed food restriction were comparable in all genotypes. In conclusion, a mutation in Per2 abolishes anticipation of mealtime, without interfering with peripheral synchronization by feeding cycles.

The opioid fentanyl affects light input, electrical activity and Per gene expression in the hamster suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) contain a major circadian pacemaker, which is regulated by photic and nonphotic stimuli. Although enkephalins are present in the SCN, their role in phase regulation of the pacemaker is largely unknown. The opioid agonist fentanyl, a homologue of morphine, is an addictive drug that induces phase shifts of circadian rhythms in hamsters. We observed that these phase shifts are blocked by naloxone, which is a critical test for true opioid receptor involvement, and conclude that opioid receptors are the sole mediators of the actions of fentanyl on the circadian timing system. A strong interaction between opioids and light input was shown by the ability of fentanyl and light to completely block each other's phase shifts of behavioural activity rhythms. Neuronal ensemble recordings in vitro provide first evidence that SCN cells show direct responses to fentanyl and react with a suppression of firing rate. Moreover, we show that fentanyl induces a strong attenuation of light-induced Syrian hamster Period 1 (shPer1) gene expression during the night. During the subjective day, we found no evidence for a role of shPer1 in mediation of fentanyl-induced phase shifts. Based on the present results, however, we cannot exclude the involvement of shPer2. Our data indicate that opioids can strongly modify the photic responsiveness of the circadian pacemaker and may do so via direct effects on SCN electrical activity and regulation of Per genes. This suggests that the pathways regulating addictive behaviour and the circadian clock intersect.