The paradox of bardoxolone methyl: a call for every witness on the stand?

People with type 2 diabetes and chronic kidney disease (CKD) remain an extremely vulnerable population with increased cardiovascular morbidity, mortality and mounting societal costs. As such, any effort to improve their dismal outcome is heavily supported. Yet, most drugs fail to replicate the promising signals of early experiments in humans in large and methodologically sound trials. As a recent example, an independent data and safety committee advised the termination of a phase 3 trial due to excessive cardiovascular disease and especially heart failure in patients allocated to the antioxidant synthetic triterpenoid bardoxolone methyl versus placebo. We evaluate the reasons why this outcome in hindsight was possibly not totally unexpected and develop a mechanistic model that shows that the consistent drop in serum magnesium concentration in patients exposed to bardoxolone methyl might have contributed to the development of heart failure. As such, this trial, despite its negative outcome, might provide additional pieces of the puzzle enabling us to get a better grip on diseases that share increased inflammation and oxidative stress, such as type 2 diabetes, metabolic syndrome, heart failure and CKD.

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.

Chitinase-like proteins are candidate biomarkers for sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (AKI) is a frequent complication of critically ill patients and leads to high mortality rates. The specificity of currently available urinary biomarkers for AKI in the context of sepsis is questioned. This study aimed to discover urinary biomarkers for septic AKI by contemporary shotgun proteomics in a mouse model for sepsis and to validate these in individual urine samples of mice and human septic patients with and without AKI. At 48 h after uterine ligation and inoculation of Escherichia coli, aged mice (48 weeks) became septic. A subgroup developed AKI, defined by serum creatinine, blood urea nitrogen, and renal histology. Separate pools of urine from septic mice with and without AKI mice were collected during 12 h before and between 36-48 h after infection, and their proteome compositions were quantitatively compared. Candidate biomarkers were validated by Western blot analysis of urine, plasma, and renal tissue homogenates from individual mice, and a limited number of urine samples from human septic patients with and without AKI. Urinary neutrophil gelatinase-associated lipocalin, thioredoxin, gelsolin, chitinase 3-like protein 1 and -3 (CHI3L3) and acidic mammalian chitinase were the most distinctive candidate biomarkers selected for septic AKI. Both neutrophil gelatinase-associated lipocalin and thioredoxin were detected in urine of septic mice and increased with severity of AKI. Acidic mammalian chitinase was only present in urine of septic mice with AKI. Both urinary chitinase 3-like protein 1 and -3 were only detected in septic mice with severe AKI. The human homologue chitinase 3-like protein 1 was found to be more excreted in urine from septic patients with AKI than without. In summary, urinary chitinase 3-like protein 1 and -3 and acidic mammalian chitinase discriminated sepsis from sepsis-induced AKI in mice. Further studies of human chitinase proteins are likely to lead to additional insights in septic AKI.

Review of protein-bound toxins, possibility for blood purification therapy.

Protein-bound uremic retention solutes, i.e. phenolic compounds, such as p-cresylsulfate, and indolic compounds, such as indoxyl sulfate, have been intensively studied in recent years and have been shown to be associated especially with cardiovascular toxicity and adverse outcomes in chronic kidney disease. In this review, we will focus on their toxicity and their removal by dialysis strategies, which is hampered due to their protein binding. Hemodiafiltration slightly improves the removal of protein-bound solutes as compared to hemodialysis, although the clinical benefit on outcomes still needs to be demonstrated. Removal by means of absorption and interference with intestinal generation or renal tubular excretion are interesting alternative strategies under investigation.

Kidney transplantation in emerging countries: do we know all issues?

Although it seems that end stage renal disease (ESRD) therapies gradually become more accessible in the developing world, yet, the vast majority of people living in those areas do not have access to dialysis and especially transplantation because of the economic and technological inequality as compared with the developed world. Despite the great advantage in survival and considerable socioeconomic advantages of transplantation vs. dialysis, there is a widespread recognition that the growing gap between organ supply and demand will continue into the foreseeable future. Several reasons might be considered in this regard as: insufficient data on the topic in the public domain, inadequate governmental financial resources, lack of public awareness, education and motivation for organ donation as well as the low number of organized teams of transplant surgeons and nephrologists, and lack of organizational infrastructure, i.e. coordinators. The defined priorities for the future in terms of improving living donor transplantation, composition of the official waiting lists and registries of transplant recipients and living donors and the role of transplant professionals have been discussed. In conclusion, whatever the governmental support is, as professionals, we should just reinforce our efforts to help our patients as best as we can in the current situation.

When the earth trembles in the Americas: the experience of Haiti and Chile 2010.

The response of the nephrological community to the Haiti and Chile earthquakes which occurred in the first months of 2010 is described. In Haiti, renal support was organized by the Renal Disaster Relief Task Force (RDRTF) of the International Society of Nephrology (ISN) in close collaboration with Médecins Sans Frontières (MSF), and covered both patients with acute kidney injury (AKI) and patients with chronic kidney disease (CKD). The majority of AKI patients (19/27) suffered from crush syndrome and recovered their kidney function. The remaining 8 patients with AKI showed acute-to-chronic renal failure with very low recovery rates. The intervention of the RDRTF-ISN involved 25 volunteers of 9 nationalities, lasted exactly 2 months, and was characterized by major organizational difficulties and problems to create awareness among other rescue teams regarding the availability of dialysis possibilities. Part of the Haitian patients with AKI reached the Dominican Republic (DR) and received their therapy there. The nephrological community in the DR was able to cope with this extra patient load. In both Haiti and the DR, dialysis treatment was able to be prevented in at least 40 patients by screening and adequate fluid administration. Since laboratory facilities were destroyed in Port-au-Prince and were thus lacking during the first weeks of the intervention, the use from the very beginning on of a point-of-care device (i-STAT®) was very efficient for the detection of aberrant kidney function and electrolyte parameters. In Chile, nephrological problems were essentially related to difficulties delivering dialysis treatment to CKD patients, due to the damage to several units. This necessitated the reallocation of patients and the adaptation of their schedules. The problems could be handled by the local nephrologists. These observations illustrate that local and international preparedness might be life-saving if renal problems occur in earthquake circumstances.

Immediate posttransplantation cotrimoxazole-induced immune thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) can be caused by numerous drugs. When this condition develops, platelet destruction results from binding of antibodies to normal platelets only in the presence of a sensitizing drug. A recently proposed model suggests that these drug-dependent antibodies are derived from a pool of naturally occurring antibodies with weak affinity for specific epitopes on certain platelet membrane glycoproteins. We describe here a case of DITP secondary to cotrimoxazole exposure in the immediate posttransplantation phase in a renal transplant recipient. Apart from heparin-induced thrombocytopenia, DITP posttransplantation has to the best of our knowledge never been described, perhaps because of its immune-mediated origin. Our case demonstrates that DITP can occur posttransplantation, that cotrimoxazole due to its intensive use in the transplanted population is one of the most likely causative agents and that a timely recognition and treatment might have important consequences for both graft and patient.

Advantages of new hemodialysis membranes and equipment.

Chronic kidney disease is characterized by the progressive retention of a number of compounds, several of which have the potential to cause cardiovascular damage. Many of these are difficult to remove by standard dialysis strategies. Removal of the larger middle molecules (mostly larger peptidic compounds) can be obtained by increasing dialyzer pore size and/or by applying convective strategies. For protein-bound solutes, convection (essentially hemodiafiltration) positively affects removal. The HEMO study demonstrated outcome superiority for the large-pore high-flux hemodialysis membranes in a number of subgroup analyses. Likewise, the Membrane Permeability Outcome study showed outcome superiority for high flux in patients with serum albumin <4 g/dl, the group for which the study had originally been designed. Apart from a small controlled trial, data suggesting superiority for convective strategies are all observational.

Posttransplantation hypomagnesemia and its relation with immunosuppression as predictors of new-onset diabetes after transplantation.

New-onset diabetes after transplantation (NODAT) is a frequent complication and has an impact on patient and graft survival. Hypomagnesemia is common in both renal transplant recipients and in diabetics. This study examines the relationship between hypomagnesemia, NODAT and the type of immunosuppression in renal transplant recipients. We conducted a retrospective single-center analysis (2002-2008) in order to assess NODAT the first year posttransplantation as defined by American Diabetes Association criteria. Serum magnesium (Mg) levels were defined as the median of all Mg levels registered during the first month posttransplantation. Patients with NODAT (N = 75; 29.5%) versus non-NODAT had lower Mg levels (p < 0.001). Patients with an Mg level < versus > or = 1.9 mg/dL showed a faster development of NODAT (log-rank p < 0.001). Mg levels were lower in patients on calcineurin inhibitors (CNI) versus no CNI patients (p < 0.001). Mg levels, albumin, BMI, triglycerides, posttransplantation hyperglycemia, tacrolimus levels and the use of sirolimus were predictors of NODAT in the multivariate analysis. Hypomagnesemia was an independent predictor of NODAT in renal transplant recipients. We confirm that the use of CNI is associated with NODAT, but, to a large extent, this effect seems attributable to the induction of hypomagnesemia. After adjustment for Mg, sirolimus was also associated with NODAT.

Treating potassium disturbances: kill the killers but avoid overkill.

OBJECTIVES: In this publication, we review the definitions, symptoms, causes, differential diagnoses and therapies of hypokalemia and hyperkalemia. METHODS: Comprehensive tables and diagnostic algorithms are provided when appropriate. RESULTS AND CONCLUSIONS: Although both hypokalemia and hyperkalemia may be life-threatening, this is essentially the case with severe changes (serum potassium < 2.5 or > 6.5 mmol/L), the presence of symptoms or electrocardiographic deviations, the association with aggravating factors (e.g. digitalis intake) and/or rapid acute changes. Only these truly need an emergency therapeutic approach. In all other cases, a careful consideration of the causes and their correction should prevail over additional approaches to modify serum potassium concentration. Although most therapeutic approaches to both hypokalemia and hyperkalemia have been well established since many years, recently two new intestinal potassium binders have been introduced on the market. It remains to be elucidated whether these drugs truly have an additional role on top of the existing treatments.

Impact of hemodialysis duration on the removal of uremic retention solutes.

Several studies have stressed the importance of dialysis time in the removal of uremic retention solutes. To further investigate this, nine stable chronic hemodialysis patients were dialyzed for 4, 6, or 8 h processing the same total blood and dialysate volume by the Genius system and high-flux FX80 dialyzers. Inlet blood and outlet dialysate were analyzed for urea, creatinine, phosphorus, and beta2-microglobulin at various times. Total solute removal, dialyzer extraction ratios, and total cleared volumes were significantly larger during prolonged dialysis for urea, creatinine, phosphorus, and beta2-microglobulin. Reduction ratios increased progressively, except for phosphate and beta2-microglobulin, where the ratios remained constant after 2 h. In contrast, no significant difference was found for the reduction ratios of all solutes and Kt/V(urea) between the three different sessions. With longer dialyses, solutes are efficiently removed from the deeper compartments of the patient's body. Our study shows that care must be taken when using Kt/Vurea or reduction ratios as the only parameters to quantify dialysis adequacy.

Uremic toxins: do we know enough to explain uremia?

BACKGROUND: The uremic syndrome is characterized by a complex clinical picture, whereby the function of multiple organ systems is affected by the retention of a host of solutes. Recent research of the last decade has helped to unravel multiple pathophysiologic mechanisms and to identify as yet unknown responsible compounds. METHODS: The literature was screened to appreciate which compounds play the most important pathophysiologic role. RESULTS: The picture that ensues is that the main role is played by molecules which are so-called 'difficult to remove by dialysis'. The knowledge of uremic toxicity is still far from complete and we need extra information about responsible compounds and mechanisms, eventually leading to a classification of the most important culprits, to allow the development of efficient removal strategies and of pharmacologic methods to counteract pathophysiologic mechanisms. CONCLUSIONS: Uremic retention is a complex phenomenon and the most toxic compounds are difficult to remove by dialysis. Furthermore, our knowledge of the responsible pathways is still incomplete, and needs to be extended to develop new and more efficient treatment strategies.

Inhibition of calcitriol receptor binding to vitamin D response elements by uremic toxins.

The genomic action of calcitriol (1,25-dihydroxy-vitamin D3) is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs). Although renal failure is associated with resistance to the action of calcitriol, the mechanism of this resistance is not well understood. Therefore, we used the electrophoretic mobility shift assay to compare the ability of VDRs from normal and renal failure rats to bind to the osteocalcin gene VDRE. The results indicate that VDRs from renal failure rats have only half the DNA binding capacity as VDRs from control rats, despite identical calcitriol binding. Furthermore, incubation of normal VDRs with a uremic plasma ultrafiltrate resulted in a loss of > 50% of the binding sites for the osteocalcin VDRE. When VDRs bound to DNA as heterodimers with retinoid X receptors, the inhibitory effect of the uremic ultrafiltrate was due to a specific interaction with the VDR, not retinoid X receptors. In addition, uremic ultrafiltrate blocked calcitriol-induced reporter gene activity in transfected JEG-3 cells. Taken together, the results indicate that an inhibitory effect of a uremic toxin(s) on VDR-VDRE binding could underlie the calcitriol resistance of renal failure.

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns.

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.

Right atrial thrombus in an asymptomatic hemodialysis patient with malfunctioning catheter and patent foramen ovale.

The creation of an accurate functioning arteriovenous fistula has been a long-lasting problem in the hemodialysis setting. In spite of recent guidelines and largely because of the old age of the current dialysis population and a high incidence of diabetes mellitus, atherosclerosis, and related vascular problems, it is not always possible to create an adequate fistula. In that case, long-term tunneled indwelling central vein catheters are a frequently used alternative. Of the many possible complications related to venous access in hemodialysis patients, catheter dysfunction is the most prevalent. We report a 23-year-old female hemodialysis patient in whom such malfunctioning was followed by echocardiography that revealed a large right atrial thrombus (RAT) in close contact to the tip of a long-term indwelling catheter in the presence of a patent foramen ovale. Although RAT is a rare complication in hemodialysis patients, it has very specific therapeutic implications. The present patient underwent a successful surgical atrial thrombectomy. Our experience underscores that in cases of malfunctioning catheter, echocardiographic screening is mandatory.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method to measure creatinine in human urine.

Despite decades of creatinine measurement in biological fluids using a large variety of analytical methods, an accurate determination of this compound remains challenging. Especially with the novel trend to assess biomarkers on large sample sets preserved in biobanks, a simple and fast method that could cope with both a high sample throughput and a low volume of sample is still of interest. In answer to these challenges, a fast and accurate ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to measure creatinine in small volumes of human urine. In this method, urine samples are simply diluted with a basic mobile phase and injected directly under positive electrospray ionization (ESI) conditions, without further purification steps. The combination of an important diluting factor (10(4) times) due to the use of a very sensitive triple quadrupole mass spectrometer (XEVO TQ) and the addition of creatinine-d3 as internal standard completely eliminates matrix effects coming from the urine. The method was validated in-house in 2012 according to the EMA guideline on bioanalytical method validation using Certified Reference samples from the German External Quality Assessment Scheme (G-Equas) proficiency test. All obtained results for accuracy and recovery are within the authorized tolerance ranges defined by G-Equas. The method is linear between 0 and 5 g/L, with LOD and LOQ of 5 × 10(-3) g/L and 10(-2) g/L, respectively. The repeatability (CV(r) = 1.03-2.07%) and intra-laboratory reproducibility (CV(RW) = 1.97-2.40%) satisfy the EMA 2012 guideline. The validated method was firstly applied to perform the German G-Equas proficiency test rounds 51 and 53, in 2013 and 2014, respectively. The obtained results were again all within the accepted tolerance ranges and very close to the reference values defined by the organizers of the proficiency test scheme, demonstrating an excellent accuracy of the developed method. The method was finally applied to measure the creatinine concentration in 210 urine samples, coming from 190 patients with a chronic kidney disease (CKD) and 20 healthy subjects. The obtained creatinine concentrations (ranging from 0.12 g/L up to 3.84 g/L) were compared, by means of a Passing Bablok regression, with the creatinine contents obtained for the same samples measured using a traditional compensated Jaffé method. The UHPLC-MS/MS method described in this paper can be used to normalize the concentration of biomarkers in urine for the extent of dilution.

Life-threatening hyperkalemia during combined therapy with angiotensin-converting enzyme inhibitors and spironolactone: an analysis of 25 cases.

PURPOSE: The beneficial effects of spironolactone are additive to those of ACE inhibitors among patients with heart failure and/or hypertension; however, it is essential to identify patients prone to develop serious hyperkalemia during combined treatment and to evaluate the associated morbidity and mortality. SUBJECTS AND METHODS: We studied 25 patients treated with ACE inhibitors and spironolactone who were admitted to the emergency room with a serum potassium level > 6 mmol/L. Patients were followed up for at least one month after admission. RESULTS: The mean age of the patients (11 males, 14 females) was 74 +/- 13 years. Five patients were diabetics. On admission, the serum potassium was 7.7 +/- 0.7 mmol/L and the serum creatinine was 3.8 +/- 1.8 mg/dL; these values were significantly higher than the most recent follow-up laboratory measurements (4.6 +/- 0.5 mmol/L and 1.9 +/- 1.2 mg/dL, respectively) obtained at 13 +/- 5 weeks before admission. The arterial pH on admission was 7.3 +/- 0.1 and the plasma bicarbonate was 18 +/- 5 mmol/L. The main causes for acute renal failure were dehydration (n = 12) and worsening heart failure (n = 9). The mean daily dose of spironolactone was 57 +/- 32 mg and 12 patients were concomitantly treated with other drugs that may cause hyperkalemia. Two patients died, and 2 patients were resuscitated but survived. Hemodialysis was necessary in 17 patients; 12 patients were admitted to the intensive care unit. The mean duration of hospitalization was 12 +/- 6 days. Two patients needed to be started on maintenance hemodialysis therapy. CONCLUSION: A combination of ACE inhibitors and spironolactone should be considered with caution and monitored closely in patients with renal insufficiency, diabetes, older age, worsening heart failure, a risk for dehydration, and in combination with other medications that may cause hyperkalemia. A daily spironolactone dose of 25 mg should not be exceeded.