Pyridoxal-5'-phosphate: A cost-effective treatment candidate for hypertensive patients?

OBJECTIVES: Because angiotensin (Ang) II is an essential vasoconstrictive peptide, we analyzed the impact of its post-translational modification to pyruvamide-Ang II (Ang P) by pyridoxal-5'-phosphate (PLP) on blood pressure. PLP is a less expensive vitamin B6 derivative and, therefore, could be a cost-effective drug against hypertension. METHODS: Effect of Ang P on calcium ion entry into vascular smooth muscle cells (VSMCs) was analyzed. Binding affinity of Ang P to angiotensin II type 1 receptor (AT1R) was measured. Vasoconstrictive effect of Ang P was investigated using the bioassay of isolated perfused rat kidneys. Spontaneously hypertensive rats (SHR) were administered PLP. Additionally, Wistar Kyoto rats (WKY) received Ang II and PLP. Blood pressure was measured time-dependently. RESULTS: Ang II, incubated with PLP, was post-translationally modified to Ang P. Calcium ion entry in VSMCs was significantly lower with Ang P compared to Ang II. Binding affinity of Ang P to AT1R was lower compared to Ang II. Perfusion pressure of isolated perfused rat kidneys increased less by Ang P than by Ang II. Blood pressure of SHR treated with PLP decreased significantly. Blood pressure of WKY rats treated with Ang II was increased to hypertensive values, whereas blood pressure of WKY rats cotreated with Ang II and PLP was not. CONCLUSION: PLP induces a post-translational modification of Ang II decreasing blood pressure in rats. Assuming that increased PLP intake in the form of vitamin B6 might reduce blood pressure in hypertensive patients, PLP might be a cost-effective drug against hypertension.

Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury.

BACKGROUND: Coexistent CKD and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway. METHODS: To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients. RESULTS: We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 in monocytes in vitro . In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events. CONCLUSIONS: Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.

Sample preparation of formalin-fixed paraffin-embedded tissue sections for MALDI-mass spectrometry imaging.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MALDI MSI) has become a powerful tool with a high potential relevance for the analysis of biomolecules in tissue samples in the context of diseases like cancer and cardiovascular or cardiorenal diseases. In recent years, significant progress has been made in the technology of MALDI MSI. However, a more systematic optimization of sample preparation would likely achieve an increase in the molecular information derived from MALDI MSI. Therefore, we have employed a systematic approach to develop, establish and validate an optimized "standard operating protocol" (SOP) for sample preparation in MALDI MSI of formalin-fixed paraffin-embedded (FFPE) tissue sample analyses within this study. The optimized parameters regarding the impact on the resulting signal-to-noise (S/N) ratio were as follows: (i) trypsin concentration, solvents, deposition method, and incubation time; (ii) tissue washing procedures and drying processes; and (iii) spray flow rate, number of layers of trypsin deposition, and grid size. The protocol was evaluated on interday variability and its applicability for analyzing the mouse kidney, aorta, and heart FFPE tissue samples. In conclusion, an optimized SOP for MALDI MSI of FFPE tissue sections was developed to generate high sensitivity, to enhance spatial resolution and reproducibility, and to increase its applicability for various tissue types. This optimized SOP will further increase the molecular information content and intensify the use of MSI in future basic research and diagnostic applications. Graphical Abstract.

Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys.

Emerging evidence highlights cellular senescence's pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21+ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21+ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.

Lineage tracing reveals transient phenotypic adaptation of tubular cells during acute kidney injury.

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

Registration of Image Modalities for Analyses of Tissue Samples Using 3D Image Modelling.

PURPOSE: Biopsies are a diagnostic tool for the diagnosis of histopathological, molecular biological, proteomic, and imaging data, to narrow down disease patterns or identify diseases. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) provides an emerging state-of-the-art technique for molecular imaging of biological tissue. The aim of this study is the registration of MALDI MSI data sets and data acquired from different histological stainings to create a 3D model of biopsies and whole organs. EXPERIMENTAL DESIGN: The registration of the image modalities is achieved by using a variant of the authors' global, deformable Schatten-q-Norm registration approach. Utilizing a connected-component segmentation for background removal followed by a principal-axis based linear pre-registration, the images are adjusted into a homogeneous alignment. This registration approach is accompanied by the 3D reconstruction of histological and MALDI MSI data. RESULTS: With this, a system of automatic registration for cross-process evaluation, as well as for creating 3D models, is developed and established. The registration of MALDI MSI data with different histological image data is evaluated by using the established global image registration system. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, this multimodal image approach offers the possibility of molecular analyses of tissue specimens in clinical research and diagnosis.