Chronic kidney disease induced by an adenine rich diet upregulates integrin linked kinase (ILK) and its depletion prevents the disease progression.

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-ß1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.

Discovery of potent calpain inhibitors based on the azolo-imidazolidenone scaffold.

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1-10 µM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).

Peripheral insulin resistance in ILK-depleted mice by reduction of GLUT4 expression.

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blocked in vivo and in vitro, although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance.

Markers of endothelial damage in patients with chronic kidney disease on hemodialysis.

Patients with Stage 5 chronic kidney disease who are on hemodialysis (HD) remain in a chronic inflammatory state, characterized by the accumulation of uremic toxins that induce endothelial damage and cardiovascular disease (CVD). Our aim was to examine microvesicles (MVs), monocyte subpopulations, and angiopoietins (Ang) to identify prognostic markers in HD patients with or without diabetes mellitus (DM). A total of 160 prevalent HD patients from 10 centers across Spain were obtained from the Biobank of the Nephrology Renal Network (Madrid, Spain): 80 patients with DM and 80 patients without DM who were matched for clinical and demographic criteria. MVs from plasma and several monocyte subpopulations (CD142+/CD16+, CD14+/CD162+) were analyzed by flow cytometry, and the plasma concentrations of Ang1 and Ang2 were quantified by ELISA. Data on CVD were gathered over the 5.5 yr after these samples were obtained. MV level, monocyte subpopulations (CD14+/CD162+ and CD142+/CD16+), and Ang2-to-Ang1 ratios increased in HD patients with DM compared with non-DM patients. Moreover, MV level above the median (264 MVs/µl) was associated independently with greater mortality. MVs, monocyte subpopulations, and Ang2-to-Ang1 ratio can be used as predictors for CVD. In addition, MV level has a potential predictive value in the prevention of CVD in HD patients. These parameters undergo more extensive changes in patients with DM.

Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank.

BACKGROUND: Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory. OBJECTIVE: To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard. STUDY DESIGN: Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis. METHODS AND RESULTS: PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was >90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by ß-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of ß-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of ß-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results. CONCLUSIONS: These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

Renal Integrin-Linked Kinase Depletion Induces Kidney cGMP-Axis Upregulation: Consequences on Basal and Acutely Damaged Renal Function.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis-related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage.

Effect of uraemia on endothelial cell damage is mediated by the integrin linked kinase pathway.

KEY POINTS: Patients with chronic kidney disease have a higher risk of developing cardiovascular diseases than the general population. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which have poor clearance by conventional dialysis. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity and in this study we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. For the first time, we demonstrate the implication of ILK in the protection against endothelial cell damage (inhibition of proliferation, toxicity, oxidative stress and programed cell death) induced by uraemic serum from chronic kidney disease patients and uraemic toxins. This molecular mechanism may have clinical relevance because it highlights the importance of maintaining high levels of ILK activity to help preserve endothelial integrity, at least in early stages of chronic kidney disease. ABSTRACT: Patients with chronic kidney disease (CKD) have a higher risk of developing cardiovascular diseases. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which, mostly protein-bound compounds such as indoxyl sulfate (IS) and p-cresyl sulphate, having poor clearance by conventional dialysis, induce endothelial toxicity. However, the molecular mechanism by which uraemic toxins regulate early stages of endothelial dysfunction remains unclear. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity. In this study, we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. First, we show that incubation of EA.hy926 cells with human uraemic serum from CKD patients upregulates ILK activity. This ILK activation also occurs when the cells are exposed to IS (25-100 µg ml(-1)), p-cresol (10-100 µg ml(-1)) or both combined, compared to human serum control. Next, we observed that high doses of both toxins together induce a slight decrease in cell proliferation and increase apoptosis and reactive oxygen species production. Interestingly, these toxic effects displayed a strong increase when the ILK protein is knocked down by small interfering RNA, even at low doses of uraemic toxins. Abrogation of AKT has demonstrated the ILK/AKT signalling pathway involved in these processes. This study has demonstrated the implication of ILK in the protection against endothelial cell damage induced by uraemic toxins, a molecular mechanism that could play a protective role in the early stages of endothelial dysfunction observed in uraemic patients.

Impacto de la implementación de la norma ISO 9001: 2008 en el proceso de cesión de muestras del biobanco Red de Investigación Renal española / Impact of implementing ISO 9001: 2008 standard on the Spanish Renal Research Network biobank sample transfer process

Antecedentes: La certificación de biobancos según la norma ISO 9001:2008 pretende mejorar la gestión de los procesos realizados en estos con dos objetivos: la satisfacción del cliente y la mejora continua. En este trabajo se presenta el impacto de la certificación ISO 9001:2008 sobre los procesos de cesión de muestras de un biobanco español especializado en muestras de pacientes renales y con un gran aumento del número de estas entre los años 2009 (12 582 viales) y 2010 (37 042 viales). Métodos: El biobanco de la Red de Investigación Renal española (REDinREN) situado en la Universidad de Alcalá ha puesto en marcha la norma ISO 9001:2008 para la gestión eficaz del material humano cedido a los centros de investigación. Se han analizado mediante encuestas dos períodos en el proceso «cesión de muestras». Durante el primer período, entre las fechas 1-10-12 y 26-11-12 (8 semanas), se han realizado cambios mínimos para corregir errores puntuales. En el segundo período, entre las fechas 7-01-13 y 18-02-13 (6 semanas), se han realizado acciones correctivas generales. Resultados: La identificación de inconvenientes y la puesta en marcha de acciones correctivas para la certificación permitieron: reducir el 70 % del tiempo de ejecución del proceso, aumentar significativamente (200 %) el número de muestras procesadas y mejorar un 25 % el proceso. El aumento del número de muestras procesadas estuvo directamente relacionado con la mejora del proceso. Conclusión: La certificación de la norma ISO 9001:2008, obtenida en julio de 2013, permitió la mejora de los procesos del biobanco REDinREN, aumentando la calidad y la satisfacción del cliente (AU)

Background: Biobank certification ISO 9001:2008 aims to improve the management of processes performed. This has two objectives: customer satisfaction and continuous improvement. This paper presents the impact of certification ISO 9001:2008 on the sample transfer process in a Spanish biobank specialising in kidney patient samples. The biobank experienced a large increase in the number of samples between 2009 (12,582 vials) and 2010 (37,042 vials). Methods: The biobank of the Spanish Renal Research Network (REDinREN), located at the University of Alcalá, has implemented ISO standard 9001:2008 for the effective management of human material given to research centres. Using surveys, we analysed two periods in the "sample transfer" process. During the first period between 1-10-12 and 26-11-12 (8 weeks), minimal changes were made to correct isolated errors. In the second period, between 7-01-13 and 18-02-13 (6 weeks), we carried out general corrective actions. Results: The identification of problems and implementation of corrective actions for certification allowed: a 70% reduction in the process execution time, a significant increase (200%) in the number of samples processed and a 25% improvement in the process. The increase in the number of samples processed was directly related to process improvement. Conclusion: The certification of ISO standard 9001:2008, obtained in July 2013, allowed an improvement of the REDinREN biobank processes to be achieved, which increased quality and customer satisfaction (AU)

Impact of implementing ISO 9001:2008 standard on the Spanish Renal Research Network biobank sample transfer process.

BACKGROUND: Biobank certification ISO 9001:2008 aims to improve the management of processes performed. This has two objectives: customer satisfaction and continuous improvement. This paper presents the impact of certification ISO 9001:2008 on the sample transfer process in a Spanish biobank specialising in kidney patient samples. The biobank experienced a large increase in the number of samples between 2009 (12,582 vials) and 2010 (37,042 vials). METHODS: The biobank of the Spanish Renal Research Network (REDinREN), located at the University of Alcalá, has implemented ISO standard 9001:2008 for the effective management of human material given to research centres. Using surveys, we analysed two periods in the “sample transfer” process. During the first period between 1-10-12 and 26-11-12 (8 weeks), minimal changes were made to correct isolated errors. In the second period, between 7-01-13 and 18-02-13 (6 weeks), we carried out general corrective actions. RESULTS: The identification of problems and implementation of corrective actions for certification allowed: a 70% reduction in the process execution time, a significant increase (200%) in the number of samples processed and a 25% improvement in the process. The increase in the number of samples processed was directly related to process improvement. CONCLUSION: The certification of ISO standard 9001:2008, obtained in July 2013, allowed an improvement of the REDinREN biobank processes to be achieved, which increased quality and customer satisfaction.

Relevant role of PKG in the progression of fibrosis induced by TNF-like weak inducer of apoptosis.

TNF-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that activates the FGF-inducible 14 receptor. Both TWEAK and the FGF-inducible 14 receptor are constitutively expressed in the kidney. TWEAK has been shown to modulate several biological responses, such as inflammation, proliferation, differentiation, and apoptosis, that contribute to kidney injury. However, the role of TWEAK in fibrosis and TWEAK-activated intracellular signaling pathways remain poorly understood. We tested the hypothesis that TWEAK can be a potent inducer of renal fibrosis by increasing transforming growth factor (TGF)-ß1 expression (a well-known switch in the fibrosis process) through PKG-I downregulation. We showed that in human mesangial cells, TWEAK increased TGF-ß1 expression and activity, leading to higher levels of the extracellular matrix protein fibronectin and decreased PKG-I expression and activity via the Ras pathway. PKG-I activation with 8-bromo-cGMP, Ras inactivation with dominant negative Ras, or Ras pathway inhibition with the ERK1/2 inhibitor PD-98059 resulted in the prevention of TWEAK-induced TGF-ß1 upregulation. In vivo, exogenous administration of TWEAK to wild-type mice downregulated kidney PKG-I and increased kidney TGF-ß1 expression. These effects were blunted in H-Ras knockout mice. Together, these data demonstrate, for the first time, the key role of PKG-I in TGF-ß1 induction by TWEAK in kidney cells.