Challenges in primary focal segmental glomerulosclerosis diagnosis : from the diagnostic algorithm to novel biomarkers

Primary or idiopathic focal segmental glomerulosclerosis (FSGS) is a kidney entity that involves the podocytes, leading to heavy proteinuria and in many cases progresses to end-stage renal disease. Idiopathic FSGS has a bad prognosis, as it involves young individuals who, in a considerably high proportion (∼15%), are resistant to corticosteroids and other immunosuppressive treatments as well. Moreover, the disease recurs in 30-50% of patients after kidney transplantation, leading to graft function impairment. It is suspected that this relapsing disease is caused by a circulating factor(s) that would permeabilize the glomerular filtration barrier. However, the exact pathologic mechanism is an unsettled issue. Besides its poor outcome, a major concern of primary FSGS is the complexity to confirm the diagnosis, as it can be confused with other variants or secondary forms of FSGS and also with other glomerular diseases, such as minimal change disease. New efforts to optimize the diagnostic approach are arising to improve knowledge in well-defined primary FSGS cohorts of patients. Follow-up of properly classified primary FSGS patients will allow risk stratification for predicting the response to different treatments. In this review we will focus on the diagnostic algorithm used in idiopathic FSGS both in native kidneys and in disease recurrence after kidney transplantation. We will emphasize those potential confusing factors as well as their detection and prevention. In addition, we will also provide an overview of ongoing studies that recruit large cohorts of glomerulopathy patients (Nephrotic Syndrome Study Network and Cure Glomerulonephropathy, among others) and the experimental studies performed to find novel reliable biomarkers to detect primary FSGS

Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Effect of medium chain triglycerides (MCT) on jejunal mucosa mass and protein synthesis

The effects of medium chain triglycerides (MCT) on jejunal mucosa mass and protein synthesis were compared with results from previous experiments with rats fed by parenteral nutrition or enteral nutrition. Other published studies have also been analysed. Three experimental models were studied. In the traumatic model, production of a femoral fracture was followed by Kirschner pin insertion into the medullary canal of both fragments at reduction. (Forty ras were fed enteral nutrition and 93 were given parenteral nutrition.) A second model entailed resection under ether anaesthesia using the technique described by Higgins. (Fifty five rats were fed enteral nutrition and 28 with parenteral nutrition.) A third model entailed a terminolateral portocaval shunt under anaesthesia with pentobarbital. (Sixty nine rats were treated this way and then given enteral nutrition.) Proportions of medium chain/long chain triglycerides (LCT) were as follows: 0/100, 20/80, 40/60, 50/50, and 92/8 for enteral nutrition and 0/100, 30/70, 50/50, and 70/30 for parenteral nutrition. Faecal losses of alpha amino nitrogen, protein, total fats, and free fatty acids were analysed together with the quantitative intake, weight gain of the rats, jejunal mucosal mass, and protein synthesis in relation to the MCT proportion ingested or given by enteral nutrition or parenteral nutrition. From analysis of our results and those of others, several conclusions could be drawn. Firstly, the route of administration of MCT is extremely important and enterocytes might be considered one of the main target sites. Secondly, a high proportion of MCT (more than 80%) offers no advantage for jejunal mucosa and produces undesirable side effects. Thirdly, the effect of MCT on jejunal mucosal protein synthesis depends on the metabolic state. Finally, an increase in jejunal mucosal mass directly correlated with MCT concentrations, but no correlation was found between mass and protein synthesis. A positive correlation, however, between MCT proportion and enzyme activity (alkaline phosphatase and sucrase) in the brush border membrane was seen as well as a positive correlation with the concentration of phospholipids in the microvilli

Rapid and accurate method for quantifying busulfan in plasma samples by isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Objectives: Administration of busulfan is extending rapidly as a part of a conditioning regimen in patients undergoing hematopoietic stem cell transplantation (HSCT). Monitoring blood plasma levels of busulfan is recommended for identifying the optimal dose in patients and for minimizing toxicity. The aim of this research was to validate a simple, rapid, and cost-effective analytical tool for measuring busulfan in human plasma that would be suitable for routine clinical use. This novel tool was based on liquid chromatography coupled to mass spectrometry. Methods: Human plasma samples were prepared using a one-step protein precipitation protocol. These samples were then resolved by isocratic elution in a C18 column. The mobile phase consisted 2 mM ammonium acetate and 0.1% formic acid dissolved in a 30:70 ratio of methanol/water. Busulfan-d8 was used as the internal standard. Results: The run time was optimized at 1.6 min. Standard curves were linear from 0.03 to 5 mg/L. The coefficient of variation (%CV) was less than 8%. The accuracy of this method had an acceptable bias that fell within 85-115% range. No interference between busulfan and the interfering compound hemoglobin, lipemia, or bilirubin not even at the highest concentrations of compound was tested. Neither carryover nor matrix effects were observed using this method. The area under the plasma drug concentration-time curves obtained for 15 pediatric patients who received busulfan therapy prior to HSCT were analyzed and correlated properly with the administered doses. Conclusions: This method was successfully validated and was found to be robust enough for therapeutic drug monitoring in a clinical setting

Effect of medium chain triglycerides (MCT) on jejunal mucosa mass and protein synthesis

The effects of medium chain triglycerides (MCT) on jejunal mucosa mass and protein synthesis were compared with results from previous experiments with rats fed by parenteral nutrition or enteral nutrition. Other published studies have also been analysed. Three experimental models were studied. In the traumatic model, production of a femoral fracture was followed by Kirschner pin insertion into the medullary canal of both fragments at reduction. (Forty ras were fed enteral nutrition and 93 were given parenteral nutrition.) A second model entailed resection under ether anaesthesia using the technique described by Higgins. (Fifty five rats were fed enteral nutrition and 28 with parenteral nutrition.) A third model entailed a terminolateral portocaval shunt under anaesthesia with pentobarbital. (Sixty nine rats were treated this way and then given enteral nutrition.) Proportions of medium chain/long chain triglycerides (LCT) were as follows: 0/100, 20/80, 40/60, 50/50, and 92/8 for enteral nutrition and 0/100, 30/70, 50/50, and 70/30 for parenteral nutrition. Faecal losses of alpha amino nitrogen, protein, total fats, and free fatty acids were analysed together with the quantitative intake, weight gain of the rats, jejunal mucosal mass, and protein synthesis in relation to the MCT proportion ingested or given by enteral nutrition or parenteral nutrition. From analysis of our results and those of others, several conclusions could be drawn. Firstly, the route of administration of MCT is extremely important and enterocytes might be considered one of the main target sites. Secondly, a high proportion of MCT (more than 80%) offers no advantage for jejunal mucosa and produces undesirable side effects. Thirdly, the effect of MCT on jejunal mucosal protein synthesis depends on the metabolic state. Finally, an increase in jejunal mucosal mass directly correlated with MCT concentrations, but no correlation was found between mass and protein synthesis. A positive correlation, however, between MCT proportion and enzyme activity (alkaline phosphatase and sucrase) in the brush border membrane was seen as well as a positive correlation with the concentration of phospholipids in the microvilli

Interaction between CypB and Na/K-ATPase beta and alfa subunits.

<p>A: Yeast two hybrid assay. CDNA for CypB was cloned in frame into the yeast expression vector (pGBKT7) that harbours the GAL4 activation domain. The recombinant plasmids plus Human Kidney Matchmaker cDNA Library were co-transformed into AH109 yeast strain. The co-transformed cells were selected on Ade-/Leu-/His-/Trp-, 10 µM Aminotriazol dropout plates to monitor for growth. The positive control represents co-transformation of p53 and T-antigen in two-hybrid expression vectors, pGBKT7-P53 and pGADT7 (BD Biosciences, Clontech), respectively. B: GST-pull-down assays. CypB was fused in frame with the GST gene. GST and GST-HCypB products were immobilized on Sepharose 4B and incubated with COS-7 cells lysates expressing Na/K-β1 (pCMV-HA-Na/K). The bound proteins were analyzed by immunoblotting with anti-Na/K-β1 rabbit polyclonal antibody. C–E: Co-Immunoprecipitation assays. HK-2 lysates were immunoprecipitated with mouse monoclonal antibodies against Na/K-β1 or Na/K-α1 and rabbit polyclonal antibody against CypB. The immunoprecipitates were subjected to Western blotting analyses, as indicated in the figure. As control, ChromePure mouse IgG and ChromePure rabbit IgG were used. Figures 1B to 1E are representative of at least three independent experiments.</p

Na/K pump subunits distribution upon subcellular fractionation of CsA-treated or CypB-silenced HK-2 cells.

<p>Sh Luc, Sh Luc treated with CsA, and Sh CypB cells show different patterns of sedimentation on sucrose gradients of α and β Na/K ATPase and CypB. Cells treated or untreated with CsA were harvested and loaded on sucrose gradients (10 to 50% w/v) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013930#s4" target="_blank">Methods</a>. (A) Western Blot of fractions eluted from sucrose gradient of Sh Luc cells probed against protein markers of specific cell compartments: Syntaxin-6 (Golgi), Calnexin (ER), E-cadherin (Plasmatic Membrane) and S6 (Ribosomes). (B) Western Blot of fractions eluted from sucrose gradient of Sh Luc, Sh Luc + CsA, and Sh CypB cells probed against α and β Na/K ATPase and CypB. Figures are representative of at least three independent experiments.</p