Dietary Advanced Glycation End Products in an Elderly Population with Diabetic Nephropathy: An Exploratory Investigation.

Advanced glycation end products (AGEs) are important in pathophysiology of type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD). Dietary AGEs (dAGEs) contribute to the overall AGE pool in the body. Forty elderly T2DM patients with DKD were randomly allocated to a low-AGE (n = 20) or regular diabetic (n = 20) diet group. A three-day meal questionnaire was used to estimate average quantity of dAGEs. AGE accumulation was measured using skin autofluorescence and urine spectroscopy. sRAGE (soluble receptor AGE) was quantified using ELISA. After 8 weeks, the mean consumption of dAGEs was considerably reduced, both in the low-AGE diet (p = 0.004) and the control (p = 0.019) group. The expected urinary emission peak at 490 nm was shifted to 520 nm in some spectra. dAGEs did not correspond with urine AGE output. An AGE-limited diet for two months did not affect AGE content in skin and urine, or sRAGE concentration in the blood. The role of glycemia is likely to be greater than the impact of dAGE consumption. The unique observation of a fluorescence pattern at 520 nm warrants further examination, since it might point to genetic differences in AGE regulation, which could have clinical consequences, as AGE content depends on its formation and elimination.

The Role of Advanced Glycation End Products and Its Soluble Receptor in Kidney Diseases.

Patients with chronic kidney disease (CKD) are more prone to oxidative stress and chronic inflammation, which may lead to an increase in the synthesis of advanced glycation end products (AGEs). Because AGEs are mostly removed by healthy kidneys, AGE accumulation is a result of both increased production and decreased kidney clearance. On the other hand, AGEs may potentially hasten decreasing kidney function in CKD patients, and are independently related to all-cause mortality. They are one of the non-traditional risk factors that play a significant role in the underlying processes that lead to excessive cardiovascular disease in CKD patients. When AGEs interact with their cell-bound receptor (RAGE), cell dysfunction is initiated by activating nuclear factor kappa-B (NF-κB), increasing the production and release of inflammatory cytokines. Alterations in the AGE-RAGE system have been related to the development of several chronic kidney diseases. Soluble RAGE (sRAGE) is a decoy receptor that suppresses membrane-bound RAGE activation and AGE-RAGE-related toxicity. sRAGE, and more specifically, the AGE/sRAGE ratio, may be promising tools for predicting the prognosis of kidney diseases. In the present review, we discuss the potential role of AGEs and sRAGE as biomarkers in different kidney pathologies.

On the protein content of kidney stones: an explorative study.

OBJECTIVES: Kidney stone formation is complex; urinary protein inhibitors play a major role in natural defense against stone formation. Using attenuated total-reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of kidney stones, proteins are usually not quantified and often reported as 'organic matrix', for which there is little attention: treatment of urolithiasis is based on the nature of the major organic/inorganic stone compound. Literature no longer regards urinary proteins as innocent bystander, but highlights the role of proteins as urolithiasis modulators. We explored the potential significance of the protein content of kidney stones. METHODS: 800 stones were analyzed using ATR-FTIR spectroscopy; spectra were corrected for protein content. The ratio of the amide I peak (1655 cm-1) divided by the maximum peak was calculated. A subgroup of stones (n = 43) was weighed; protein concentration was assayed. Kidney stone composition was taken into account when calculating protein concentration. Electrophoresis was implemented to investigate the protein bands. Multiple regression analysis was carried out to study the influence of various demographic variables (age, gender, stone type) on protein concentration. RESULTS: Protein concentration showed a marked variation according to the stone composition. High relative protein content (>0.4% stone mass) was found in mixed calcium apatite/calcium oxalate dihydrate stones, mixed calcium oxalate dihydrate/calcium oxalate monohydrate/calcium apatite stones, and mixed calcium oxalate monohydrate/brushite stones, whereas lower protein percentages were found in cystine, urate, and calcium oxalate monohydrate stones. Protein concentration was dependent of the patient's age. CONCLUSION: ATR-FTIR is a practical way for assessing protein concentration in kidney stones. LIST OF ABBREVIATIONS: A: absorbance; as, asymmetric vibrations; ATR-FTIR, attenuated total-reflectance Fourier-transform infrared; ß, standardized regression coefficient; CAP, calcium apatite; COD, calcium oxalate dihydrate; COM, calcium oxalate monohydrate; CV, coefficient of variation; δ, bending vibrations; ELISA, enzyme-linked immunosorbent assay; IQR, interquartile range; IR, infrared; LOD, limit of detection; LOQ, limit of quantification; MIR, mid-infrared; N or n, amount; r, correlation; r2, coefficient of determination; s, symmetric vibrations; SD, standard deviation; SE, standard error; THP, Tamm-Horsfall protein; UA, uric acid; V, stretching vibrations; VIF: variance inflation factor; ZnSe, zinc selenide.

Gut Microbiota and Their Derived Metabolites, a Search for Potential Targets to Limit Accumulation of Protein-Bound Uremic Toxins in Chronic Kidney Disease.

Chronic kidney disease (CKD) is characterized by gut dysbiosis with a decrease in short-chain fatty acid (SCFA)-producing bacteria. Levels of protein-bound uremic toxins (PBUTs) and post-translational modifications (PTMs) of albumin increase with CKD, both risk factors for cardiovascular morbidity and mortality. The relationship between fecal metabolites and plasma concentrations of PBUTs in different stages of CKD (n = 103) was explored. Estimated GFR tends to correlate with fecal butyric acid (BA) concentrations (rs = 0.212; p = 0.032), which, in its turn, correlates with the abundance of SCFA-producing bacteria. Specific SCFAs correlate with concentrations of PBUT precursors in feces. Fecal levels of p-cresol correlate with its derived plasma UTs (p-cresyl sulfate: rs = 0.342, p < 0.001; p-cresyl glucuronide: rs = 0.268, p = 0.006), whereas an association was found between fecal and plasma levels of indole acetic acid (rs = 0.306; p = 0.002). Finally, the albumin symmetry factor correlates positively with eGFR (rs = 0.274; p = 0.005). The decreased abundance of SCFA-producing gut bacteria in parallel with the fecal concentration of BA and indole could compromise the intestinal barrier function in CKD. It is currently not known if this contributes to increased plasma levels of PBUTs, potentially playing a role in the PTMs of albumin. Further evaluation of SCFA-producing bacteria and SCFAs as potential targets to restore both gut dysbiosis and uremia is needed.

The role of soluble receptor for advanced glycation end-products (sRAGE) in the general population and patients with diabetes mellitus with a focus on renal function and overall outcome.

Isoforms of the receptor for advanced glycation end-product (RAGE) protein, which lack the transmembrane and the signaling (soluble RAGE or sRAGE) domains are hypothesized to counteract the detrimental action of the full-length receptor by acting as a decoy, and they provide a potential tool to treat RAGE-associated diseases. Multiple studies have explored the relationship between sRAGE and endogenous secretory RAGE and its polymorphism and obesity, metabolic syndrome, atherosclerosis, kidney function, and increased mortality in the general population. In addition, sRAGE may be a key player in the pathogenesis of diabetes mellitus and its microvascular (e.g. kidney disease) as well as macrovascular (e.g. cardiovascular disease) complications. In this review, we focus on the role of sRAGE as a biomarker in these specific areas. As there is a lack of an underlying unifying hypothesis about how sRAGE changes according to the disease condition or risk factor, there is a call to incorporate all three players of the AGE-RAGE axis into a new universal biomarker/risk marker: (AGE + RAGE)/sRAGE. However, the measurement of RAGE in humans is not practical as it is a cell-bound receptor for which tissue is required for analysis. A high AGE/sRAGE ratio may be a valuable alternative and practical universal biomarker/risk marker for diseases associated with the AGE-RAGE axis, irrespective of low or high serum sRAGE concentrations.

Exploring the possibilities of infrared spectroscopy for urine sediment examination and detection of pathogenic bacteria in urinary tract infections.

Objectives In this study, the possibilities of Fourier-transformed infrared spectroscopy (FTIR) for analysis of urine sediments and for detection of bacteria causing urinary tract infections (UTIs) were investigated. Methods Dried urine specimens of control subjects and patients presenting with various nephrological and urological conditions were analysed using mid-infrared spectroscopy (4,000-400 cm-1). Urine samples from patients with a UTI were inoculated on a blood agar plate. After drying of the pure bacterial colonies, FTIR was applied and compared with the results obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chemometric data analysis was used to classify the different species. Results Due to the typical molecular assignments of lipids, proteins, nucleic acids and carbohydrates, FTIR was able to identify bacteria and showed promising results in the detection of proteins, lipids, white and red blood cells, as well as in the identification of crystals. Principal component analysis (PCA) allowed to differentiate between Gram-negative and Gram-positive species and soft independent modelling of class analogy (SIMCA) revealed promising classification ratios between the different pathogens. Conclusions FTIR can be considered as a supplementary method for urine sediment examination and for detection of pathogenic bacteria in UTI.

UV Fluorescence-Based Determination of Urinary Advanced Glycation End Products in Patients with Chronic Kidney Disease.

Advanced glycation end products (AGEs) are a class of proteins or lipids that are non-enzymatically glycated and oxidized after contact with aldose sugars. The accumulation of AGEs results in carbonyl stress, which is characteristic for diabetes mellitus, uremia, atherosclerosis and vascular dysfunction. In recent decades, several innovative methods have been developed to measure the concentration of AGEs in blood or urine. In the present study, we evaluated the use of UV fluorescence as an alternative tool to detect urinary AGEs in four groups of well characterized chronic kidney disease (CKD) patients over a wide range of kidney insufficiency and in a group of healthy subjects. Using an excitation wavelength of 365 nm, the fluorescence spectra of urinary AGEs were recorded in the 400-620 nm emission range. When considering the emission peaks at 440 nm and 490 nm, a significantly higher AGE-specific fluorescence intensity was detected in CKD patients compared to healthy subjects (p < 0.0001 and p = 0.0001, respectively). The urinary creatinine adjusted fluorescence emission spectra in the group of CKD patients with diabetes mellitus were comparable with those of CKD patients without diabetes mellitus. Creatinine-adjusted fluorescence emission spectra were highest in CKD patients with proteinuria, moderate in CKD patients without proteinuria and lowest in healthy controls (p < 0.0001 at both emission wavelengths). In a multiple regression analysis, age, CRP and insulin treatment were predictors of fluorescence intensity at the emission wavelength of 440 nm. Age and insulin treatment were predictors at 490 nm. The presented method is a simple, cheap, alternative method to monitor the AGE-load in the CKD population.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development.

Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine.

Antibody-drug conjugates might be the magic bullets referred to by Paul Ehrlich over 100 years ago. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry to its limits. The use of multiple heart-cutting (mLC-LC) and comprehensive (LC×LC) multidimensional LC in combination with high resolution mass spectrometry for the characterization of the lysine conjugated antibody-drug conjugate ado-trastuzumab emtansine, commercialized as Kadcyla, is presented. By combining protein and peptide measurements, attributes such as drug loading, drug distribution and drug conjugation sites can be assessed in an elegant manner.