Julie Klein

LPA1 Receptor Activation Promotes Renal Interstitial Fibrosis

Tubulointerstitial fibrosis in chronic renal disease is strongly associated with progressive loss of renal function. We studied the potential involvement of lysophosphatidic acid (LPA), a growth factor-like phospholipid, and its receptors LPA(1-4) in the development of tubulointerstitial fibrosis (TIF). Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) for up to 8 d, and kidney explants were prepared from the distal poles to measure LPA release into conditioned media. After obstruction, the extracellular release of LPA increased approximately 3-fold. Real-time reverse transcription PCR (RT-PCR) analysis demonstrated significant upregulation in the expression of the LPA(1) receptor subtype, downregulation of LPA3, and no change of LPA2 or LPA4. TIF was significantly attenuated in LPA1 (-/-) mice compared to wild-type littermates, as measured by expression of collagen III, alpha-smooth muscle actin (alpha-SMA), and F4/80. Furthermore, treatment of wild-type mice with the LPA1 antagonist Ki16425 similarly reduced fibrosis and significantly attenuated renal expression of the profibrotic cytokines connective tissue growth factor (CTGF) and transforming growth factor beta (TGFbeta). In vitro, LPA induced a rapid, dose-dependent increase in CTGF expression that was inhibited by Ki16425. In conclusion, LPA, likely acting through LPA1, is involved in obstruction-induced TIF. Therefore, the LPA1 receptor might be a pharmaceutical target to treat renal fibrosis.

Lysophosphatidic acid and renal fibrosis.

The development of fibrosis involves a multitude of events and molecules. Until now the majority of these molecules were found to be proteins or peptides. But recent data show significant involvement of the phospholipid lysophosphatidic acid (LPA) in the development of pulmonary, liver and renal fibrosis. The latest data on the role of LPA and the G-protein-coupled LPA1 receptor in the development of renal fibrosis will be discussed. LPA1-receptor activation was found to be associated with increased vascular leakage and increased fibroblast recruitment in pulmonary fibrosis. Furthermore, in renal fibrosis LPA1-receptor activation stimulates macrophage recruitment and connective tissue growth factor expression. The observations make this receptor an interesting alternative and new therapeutic target in fibrotic diseases.

Urinary Proteomics Based on Capillary Electrophoresis-Coupled Mass Spectrometry in Kidney Disease: Discovery and Validation of Biomarkers, and Clinical Application

Use of capillary electrophoresis coupled to mass spectrometry (CE-MS) technology in proteome analysis has increased, with a focus on the identification of biomarker peptides in clinical proteomics. Among the reported applications, the main focus is on the urinary biomarkers for kidney disease. In this review, we discuss the principal, theoretical, and practical obstacles that are encountered when using CE-MS for the analysis of body fluids for biomarker discovery. We present several examples of a successful application of CE-MS for biomarker discovery in kidney disease, implications for disease diagnosis, prognosis, and therapy evaluation, and will also discuss current challenges and possible future improvements.

Assessment of metabolomic and proteomic biomarkers in detection and prognosis of progression of renal function in chronic kidney disease

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m2; n = 10) or advanced (8.9±4.5 mL/min/1.73 m2; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = −0.8031; p<0.0001 and ρ = −0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = −0.6557; p = 0.0001 and ρ = −0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = −0.7752; p<0.0001 and ρ = −0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.

Lysophosphatidic acid-1-receptor targeting agents for fibrosis.

Introduction: The presence of fibrosis is associated with alterations in organ architecture and is responsible for the morbidity of diseases including pneumopathies, systemic sclerosis, liver cirrhosis, chronic cardiovascular diseases, progressive kidney diseases and diabetes. Although a growing number of pro-fibrotic molecules, mediators and other pathways have been reported, there are currently very few antifibrotic molecules being evaluated in clinical trials. Areas covered: Current knowledge about the contribution of lysophosphatidic acid (LPA), a bioactive mediator acting via specific G-protein coupled receptors (LPAR), in the etiology of fibrosis. In a number of organs, fibrosis is associated with increased LPA production as well as with increased expression of some LPAR subtypes (mainly LPA1R). LPAR–/– knockout mice and treatment of animal models with specific antagonists clearly demonstrate the contribution of LPA1R subtype to the development of kidney, lung, vascular and dermal fibrosis. The involvement of LPA in liver fibrosis is also strongly suspected but still unproven. Expert opinion: Experiments in animal models clearly demonstrate that LPA1R antagonists have interesting anti-fibrotic potencies. This reveals promising perspectives for the design of new therapeutic approaches to prevent fibrosis-associated diseases. Nevertheless, the number of efficient LPA1R antagonists currently available is still low, and none of them has been used in clinical trials so far.

Comparison of CE-MS/MS and LC-MS/MS sequencing demonstrates significant complementarity in natural peptide identification in human urine.

Clinical proteomics has led to the identification of biomarkers specifically associated with a clinical condition that can serve for diagnostic or prognostic purposes. Learning more about the origin of these protein fragments would lead to a better insight in the pathology, and this requires improved identification of the peptide sequences. The aim of this study is to assess the complementarity of LC‐MS/MS and CE‐MS/MS as techniques in peptide sequence identification of the urinary low‐molecular weight proteome. A male standard human urine sample was analyzed using LC‐ and CE‐MS/MS (n = 10 per technique), identifying 905 unique peptide sequences with high confidence, 50% of those were identified only with LC, 20% only with CE and 30% with both techniques. Higher LC coverage might be due in part to the higher amount of sample that can be loaded onto an LC column. Peptides uniquely identified in CE are generally small and highly charged, likely unable to bind to the LC column In conclusion, we showed that LC‐MS/MS and CE‐MS/MS are highly complementary in identifying peptide sequences. The combination of both technologies results in significantly increased sequence coverage.

Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)

Peptides found in fetal urine predict end-stage renal disease in patients with a congenital abnormality of the kidney and urinary tract. Seeking a Signature of Renal Disease Being able to predict—in utero—whether a fetus will have severe kidney disease would help anxious parents make informed treatment decisions earlier. To this end, Klein and colleagues searched through thousands of peptides in fetal urine to find potential markers of end-stage renal disease (ESRD). The authors obtained urine samples from fetuses with posterior urethral valves (PUV), which is an abnormality of the urinary tract that can lead to ESRD. They knew the outcome of these patients with PUV, that is, whether or not they had ESRD before the age of 2. Out of >4000 peptides, Klein et al. identified a handful that were linked to ESRD, but were not present in the fetal urine of patients who did not progress to ESRD. In an independent set of samples, this peptide-based signature, called 12PUV, was similarly able to predict postnatal renal function, outperforming routine clinical procedures such as ultrasound and biochemical tests. Although many more fetal urine samples are needed for validation, this peptide-based predictor shows promise as a much needed test to help clinicians and families make prenatal treatment decisions for fetuses with PUV. Bilateral congenital abnormalities of the kidney and urinary tract (CAKUT), although are individually rare diseases, remain the main cause of chronic kidney disease in infants worldwide. Bilateral CAKUT display a wide spectrum of pre- and postnatal outcomes ranging from death in utero to normal postnatal renal function. Methods to predict these outcomes in utero are controversial and, in several cases, lead to unjustified termination of pregnancy. Using capillary electrophoresis coupled with mass spectrometry, we have analyzed the urinary proteome of fetuses with posterior urethral valves (PUV), the prototypic bilateral CAKUT, for the presence of biomarkers predicting postnatal renal function. Among more than 4000 fetal urinary peptide candidates, 26 peptides were identified that were specifically associated with PUV in 13 patients with early end-stage renal disease (ESRD) compared to 15 patients with absence of ESRD before the age of 2. A classifier based on these peptides correctly predicted postnatal renal function with 88% sensitivity and 95% specificity in an independent blinded validation cohort of 38 PUV patients, outperforming classical methods, including fetal urine biochemistry and fetal ultrasound. This study demonstrates that fetal urine is an important pool of peptides that can predict postnatal renal function and thus be used to make clinical decisions regarding pregnancy.

Congenital ureteropelvic junction obstruction: human disease and animal models

Ureteropelvic junction (UPJ) obstruction is the most frequently observed cause of obstructive nephropathy in children. Neonatal and foetal animal models have been developed that mimic closely what is observed in human disease. The purpose of this review is to discuss how obstructive nephropathy alters kidney histology and function and describe the molecular mechanisms involved in the progression of the lesions, including inflammation, proliferation/apoptosis, renin–angiotensin system activation and fibrosis, based on both human and animal data. Also we propose that during obstructive nephropathy, hydrodynamic modifications are early inducers of the tubular lesions, which are potentially at the origin of the pathology. Finally, an important observation in animal models is that relief of obstruction during kidney development has important effects on renal function later in adult life. A major short‐coming is the absence of data on the impact of UPJ obstruction on long‐term adult renal function to elucidate whether these animal data are also valid in humans.

Proteomics of Vitreous Humor of Patients with Exudative Age-Related Macular Degeneration

Background There is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD). Methods and Findings Eighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity. Conclusions Proteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.

The application of multi-omics and systems biology to identify therapeutic targets in chronic kidney disease

The quest for the ideal therapeutic target in chronic kidney disease (CKD) has been riddled with many obstacles stemming from the molecular complexity of the disease and its co-morbidities. Recent advances in omics technologies and the resulting amount of available data encompassing genomics, proteomics, peptidomics, transcriptomics and metabolomics has created an opportunity for integrating omics datasets to build a comprehensive and dynamic model of the molecular changes in CKD for the purpose of biomarker and drug discovery. This article reviews relevant concepts in omics data integration using systems biology, a mathematical modelling method that globally describes a biological system on the basis of its modules and the functional connections that govern their behaviour. The review describes key databases and bioinformatics tools, as well as the challenges and limitations of the current state of the art, along with practical application to CKD therapeutic target discovery. Moreover, it describes how systems biology and visualization tools can be used to generate clinically relevant molecular models with the capability to identify specific disease pathways, recognize key events in disease development and track disease progression.