DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function.

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain.

Cognitive impairment is common in extracerebral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. However, we lack mechanistic insights allowing development of targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single-nuclei RNA-sequencing and single-cell thallium autometallography), human samples and in vitro experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and cognition in CKD. CKD disrupts the barrier of brain endothelial cells in vitro and the blood-brain barrier in vivo, establishing that the uremic state modifies vascular permeability in the brain. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium efflux via the calcium-dependent channel KCa3.1, and induces p38-MAPK associated IL-1ß maturation in microglia. Restoring potassium homeostasis in microglia using a KCa3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1ß receptor 1 (IL-1R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Accordingly, in CKD mice, impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. Thus, our data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1ß release and IL-1R1-mediated neuronal dysfunction in CKD. Hence, our study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches.

Noncanonical microglial IL-1ß maturation in chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Organ transplantation reverses cognitive impairment in chronic kidney disease (CKD), indicating that cognitive impairment driven by CKD is therapeutically amendable. We recently demonstrated that impaired cognition in CKD is linked to IL-1ß-release from microglia and IL-1R1-signaling in neuronal cells, thereby identifying a signaling pathway that can be exploited therapeutically. However, the mechanism of IL-1ß-maturation in microglia in CKD remains unknown. We hypothesized that microglia cells require caspase-1 for CKD-driven cognitive impairment. METHODS: We used a combination of single cell analyses, in situ analyses, genetically modified mouse models (including newly generated Cre-LoxP mouse models) and in vitro models. The current study builds on a recently identified intercellular crosstalk between microglia and neurons that impairs cognition in chronic kidney disease (CKD). RESULTS: Here, we show that despite NLRP3 inflammasome activation in the brain and protection of mice with constitutive NLRP3 deficiency from CKD-induced cognitive impairment, (i) caspase-1 is not required for IL-1ß maturation in microglia and (ii) targeted caspase-1 deficiency in microglia does not improve cognition in CKD mice. These data indicate that IL-1ß maturation in microglia is independent of the NLRP3-caspase-1 interaction in CKD. Indeed, microglia activation in CKD induces noncanonical, cathepsin C-caspase-8 mediated IL-1ß maturation. Depletion of cathepsin C or caspase-8 blocks IL-1ß maturation in microglia. Preliminary analyses suggest that noncanonical microglia IL-1ß maturation occurs also in diabetes mellitus. CONCLUSION: These results identify a noncanonical IL-1ß-maturation pathway as a potential therapeutic target to combat microglia-induced neuronal dysfunction in CKD and possible other peripheral diseases.

CD248 induces a maladaptive unfolded protein response in diabetic kidney disease.

Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells in vitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing in vivo and in vitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 in vivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

The role of endoplasmic reticulum-mitochondria-associated membranes in diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide. The pathomechanisms of DKD are multifactorial, yet haemodynamic and metabolic changes in the early stages of the disease appear to predispose towards irreversible functional loss and histopathological changes. Recent studies highlight the importance of endoplasmic reticulum-mitochondria-associated membranes (ER-MAMs), structures conveying important cellular homeostatic and metabolic effects, in the pathology of DKD. Disruption of ER-MAM integrity in diabetic kidneys is associated with DKD progression, but the regulation of ER-MAMs and their pathogenic contribution remain largely unknown. Exploring the cell-specific components and dynamic changes of ER-MAMs in diabetic kidneys may lead to the identification of new approaches to detect and stratify diabetic patients with DKD. In addition, these insights may lead to novel therapeutic approaches to target and/or reverse disease progression. In this review, we discuss the association of ER-MAMs with key pathomechanisms driving DKD such as insulin resistance, dyslipidaemia, ER stress, and inflammasome activation and the importance of further exploration of ER-MAMs as diagnostic and therapeutic targets in DKD.

Fat tissue quantity, waist circumference or waist-to-hip ratio in patients with chronic kidney disease: A systematic review and meta-analysis.

The BMI predicts mortality and cardiovascular disease (CVD) in the general population, while in patients with end-stage chronic kidney disease (CKD) a high BMI is associated with improved survival, a phenomenon referred to as the "obesity paradox". While BMI is easy to determine and helps to categorize patients, it does not differentiate between fat tissue, lean tissue and bone mass. As the BMI may be altered in CKD, e.g. by muscle wasting, we determined in this meta-analysis (i) the association of mortality with fat tissue quantity in CKD and (ii) the association of mortality with abdominal obesity (as measured by waist circumference (WC) or waist-to-hip ratio (WHR)) in CKD. We systematically reviewed databases for prospective or retrospective cohort studies. In eleven studies with 23,523 patients the association between mortality and high fat tissue quantity in CKD was calculated. The pooled hazard ratio (HR) for this association in the CKD group in the dialysis group 0.91 (CI 0.84- 0.98, p = 0.01) which is comparable to the HR for the association with BMI. The HR in patients without dialysis was 0.7 (95% CI 0.53- 0.93, p = 0.01), suggesting a better risk prediction of high fat tissue content with mortality as compared to higher BMI with mortality in patients with CKD without dialysis. Importantly, both BMI and fat tissue quantity in CKD are described by the "obesity paradox": the higher the fat tissue content or BMI, the lower the mortality risk. In thirteen studies with 55,175 patients the association between mortality and high WC or WHR in CKD (with or without dialysis) was calculated. We observed, that the HR in the WHR group was 1.31 (CI 1.08-1.58, p = 0.007), whereas the overall hazard ratio of both groups was 1.09 (CI 1.01-1.18, p = 0.03), indicating that a higher abdominal obesity as measured by WHR is associated with higher mortality in CKD. Our analysis suggests gender-specific differences, which need larger study numbers for validation. This meta-analysis confirms the obesity paradox in CKD using fat tissue quantity as measure and further shows that using abdominal obesity measurements in the routine in obese CKD patients might allow better risk assessment than using BMI or fat tissue quantity. Comparable to the overall population, here, the higher the WHR, the higher the mortality risk.

Factor XII signaling via uPAR-integrin ß1 axis promotes tubular senescence in diabetic kidney disease.

Coagulation factor XII (FXII) conveys various functions as an active protease that promotes thrombosis and inflammation, and as a zymogen via surface receptors like urokinase-type plasminogen activator receptor (uPAR). While plasma levels of FXII are increased in diabetes mellitus and diabetic kidney disease (DKD), a pathogenic role of FXII in DKD remains unknown. Here we show that FXII is locally expressed in kidney tubular cells and that urinary FXII correlates with kidney dysfunction in DKD patients. F12-deficient mice (F12-/-) are protected from hyperglycemia-induced kidney injury. Mechanistically, FXII interacts with uPAR on tubular cells promoting integrin ß1-dependent signaling. This signaling axis induces oxidative stress, persistent DNA damage and senescence. Blocking uPAR or integrin ß1 ameliorates FXII-induced tubular cell injury. Our findings demonstrate that FXII-uPAR-integrin ß1 signaling on tubular cells drives senescence. These findings imply previously undescribed diagnostic and therapeutic approaches to detect or treat DKD and possibly other senescence-associated diseases.

The Extent of Lifestyle-Induced Weight Loss Determines the Risk of Prediabetes and Metabolic Syndrome Recurrence during a 5-Year Follow-Up.

It is controversial whether lifestyle-induced weight loss (LIWL) intervention provides long-term benefit. Here, we investigated whether the degree of weight loss (WL) in a controlled LIWL intervention study determined the risk of prediabetes and recurrence of metabolic syndrome (MetS) during a 5-year follow-up. Following LIWL, 58 male participants (age 45−55 years) were divided into four quartiles based on initial WL: Q1 (WL 0−8.1%, n = 15), Q2 (WL 8.1−12.8%, n = 14), Q3 (WL 12.8−16.0%, n = 14), and Q4 (WL 16.0−27.5%, n = 15). We analyzed changes in BMI, HDL cholesterol, triglycerides (TGs), blood pressure, and fasting plasma glucose (FPG) at annual follow-up visits. With a weight gain after LIWL between 1.2 (Q2) and 2.5 kg/year (Q4), the reduction in BMI was maintained for 4 (Q2, p = 0.03) or 5 (Q3, p = 0.03; Q4, p < 0.01) years, respectively, and an increase in FPG levels above baseline values was prevented in Q2−Q4. Accordingly, there was no increase in prediabetes incidence after LIWL in participants in Q2 (up to 2 years), Q3 and Q4 (up to 5 years). A sustained reduction in MetS was maintained in Q4 during the 5-year follow-up. The present data indicate that a greater initial LIWL reduces the risk of prediabetes and recurrence of MetS for up to 5 years.

ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis.

Diabetes mellitus is hallmarked by accelerated atherosclerosis, a major cause of mortality among patients with diabetes. Efficient therapies for diabetes-associated atherosclerosis are absent. Accelerated atherosclerosis in diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we directly compared the effects of high glucose and oxidized LDL, revealing that high glucose induced more pronounced responses in regard to maladaptive unfolded protein response (UPR), senescence, and vascular endothelial cell barrier disruption. Ex vivo, diabetic ApoE-/- mice displayed increased levels of senescence and UPR markers within atherosclerotic lesions compared with nondiabetic ApoE-/- mice. Activated protein C pretreatment maintained barrier permeability and prevented glucose-induced expression of senescence and UPR markers in vitro. These data suggest that high glucose-induced maladaptive UPR and associated senescence promote vascular endothelial cell dysfunction, which-however-can be reversed by aPC. Taken together, current data suggest that reversal of glucose-induced vascular endothelial cell dysfunction is feasible.

Osteocalcin Is Independently Associated with C-Reactive Protein during Lifestyle-Induced Weight Loss in Metabolic Syndrome.

Bone-derived osteocalcin has been suggested to be a metabolic regulator. To scrutinize the relation between osteocalcin and peripheral insulin sensitivity, we analyzed changes in serum osteocalcin relative to changes in insulin sensitivity, low-grade inflammation, and bone mineral density following lifestyle-induced weight loss in individuals with metabolic syndrome (MetS). Participants with MetS were randomized to a weight loss program or to a control group. Before and after the 6-month intervention period, clinical and laboratory parameters and serum osteocalcin levels were determined. Changes in body composition were analyzed by dual-energy X-ray absorptiometry (DXA). In participants of the intervention group, weight loss resulted in improved insulin sensitivity and amelioration of inflammation. Increased serum levels of osteocalcin correlated inversely with BMI (r = -0.63; p< 0.001), total fat mass (r = -0.58, p < 0.001), total lean mass (r = -0.45, p < 0.001), C-reactive protein (CRP) (r = -0.37; p < 0.01), insulin (r = -0.4; p < 0.001), leptin (r = -0.53; p < 0.001), triglycerides (r = -0.42; p < 0.001), and alanine aminotransferase (ALAT) (r = -0.52; p < 0.001). Regression analysis revealed that osteocalcin was independently associated with changes in CRP but not with changes in insulin concentration, fat mass, or bone mineral density, suggesting that weight loss-induced higher serum osteocalcin is primarily associated with reduced inflammation.

Methods to Detect Endoplasmic Reticulum Stress and Apoptosis in Diabetic Nephropathy.

A variety of pathophysiological cellular dysfunctions stress the endoplasmic reticulum (ER), promoting an accumulation of unfolded proteins in the ER lumen. The latter is sensed by intrinsic ER transmembrane proteins: IRE1α (inositol-requiring protein-1α), PERK (protein kinase RNA (PKR)-like ER kinase), and ATF6 (activating transcription factor 6) which when activated trigger the unfolded protein response (UPR), which includes an inhibition of protein translation while inducing specific transcription factors that induce genes aiming to relieve the ER stress response. Collectively, this reduces the burden of unfolded proteins within the ER, eventually restoring ER homeostasis and thus promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes cell death. Diabetic nephropathy (dNP), a leading cause of end-stage renal disease in industrialized countries, is mechanistically closely linked with ER stress and renal cell death. Here, we describe methods (both in vivo and in vitro) for monitoring ER stress, UPR signaling, and cell death in renal cells by analyzing proteins and protein-protein interactions serving as markers of ER stress or cell death. These methods include visualization of interactions of UPR regulators by proximity ligation assay on renal tissue and cells and methods to detect cell death based on DNA fragmentation or fluorochrome substrates for caspases. We include two selected in vivo models to manipulate ER stress regulators and thus the UPR in murine models of dNP. Collectively, these analyses allow assessment of the activation of ER stress-induced signaling pathways and cell death in dNP and manipulation of the UPR in vivo, enabling researchers to probe for causality.

The P5 disulfide switch: taming the aging unfolded protein response.

Aging cells are characterized by a loss of proteostasis and a decreased ability to survive under environmental stress. Regulation of the UPR in aging cells has been under much scrutiny, and studies have shown that the UPR in these cells differs considerably from younger cells with regard to the induction of apoptosis and chaperone activity. The role of IRE-1 and PERK in UPR-associated apoptosis makes the regulation of these signaling cascades an important target of study. The seemingly contradictory findings regarding the role of P5 in activating and deactivating these responses warrant further investigation and may hold the key to unlocking the role of this protein in various pathological conditions. Another important target for study with regard to P5 is the effects of the localization of this protein in the mitochondria and the consequences, if any, of these effects on the activation of the UPR.

An in-silico insight into the characteristics of ß-propeller phytase.

Phytase is an enzyme that is found extensively in the plant kingdom and in some species of bacteria and fungi. This paper identifies and analyses the available full length sequences of ß-propeller phytases (BPP). BPP was chosen due to its potential applicability in the field of aquaculture. The sequences were obtained from the Uniprot database and subject to various online bioinformatics tools to elucidate the physio-chemical characteristics, secondary structures and active site compositions of BPP. Protparam and SOPMA were used to analyse the physiochemical and secondary structure characteristics, while the Expasy online modelling tool and CASTp were used to model the 3-D structure and identify the active sites of the BPP sequences. The amino acid compositions of the four sequences were compared and composed in a graphical format to identify similarities and highlight the potentially important amino acids that form the active site of BPP. This study aims to analyse BPP and contribute to the clarification of the molecular mechanism involved in the enzyme activity of BPP and contribute in part to the possibility of constructing a synthetic version of BPP.