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The HprK serine kinase is a component of the phosphoenolpyruvate phosphotransferase system (PTS) of bacteria that generally regulates catabolite repression through phosphorylation/dephosphorylation of the PTS protein PtsH at a conserved serine residue. However, many bacteria do not encode a complete PTS or even have an HprK homologue. Xanthomonas campestris pv. campestris (Xcc) is a pathogen that cause black rot disease in crucifer plants and one of the few Gram-negative bacteria that encodes a homologue of HprK protein (herein HprK Xcc ). To gain insight into the role of HprK Xcc and other PTS-related components in Xcc we individually mutated and phenotypically assessed the resulting strains. Deletion of hprK Xcc demonstrated its requirement for virulence and other diverse cellular processes associated including extracellular enzyme activity, extracellular-polysaccharide production and cell motility. Global transcriptome analyses revealed the HprK Xcc had a broad regulatory role in Xcc. Additionally, through overexpression, double gene deletion and transcriptome analysis we demonstrated that hprK Xcc shares an epistatic relationship with ptsH. Furthermore, we demonstrate that HprK Xcc is a functional serine kinase, which has the ability to phosphorylate PtsH. Taken together, the data illustrates the previously unappreciated global regulatory role of HprK Xcc and previously uncharacterized PTS components that control virulence in this pathogen., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Hyperglycemia causes mitochondrial damage renal tubular cells, which contribute to the progression of diabetic kidney disease. However, the metabolic aberration of renal tubular cells in an hyperglycemic milieu has not been fully elucidated. In this study, human proximal renal tubular cell line (HK-2 cell) are incubated in glucose and mannitol at 5 mM or 25 mM. Cellular metabolome was determined by capillary electrophoresis time of flight mass spectrometer (CE-TOF/MS) and capillary electrophoresis-triple quadrupole mass spectrometry (CE-QqQMS). A total of 116 metabolites were quantified. Principal component analysis (PCA) revealed excellent clustering of metabolomic changes for different treatment conditions, and exposure to glucose at 5 and 25 mM lead to distinct metabolomic profiles as compared to samples treated with serum-free medium or mannitol as osmotic control. Hierarchical clustering analysis showed a number of characteristic changes in metabolic profile following exposure to 5 mM or 25 mM glucose. Notably, lactate-to-pyruvate ratio was significantly increased, while cellular levels of citric acid, α-ketoglutaric acid (i.e. 2-oxoglutaric acid), and fumaric acid were significantly reduced after exposure to glucose at 25 mM but not 5 mM. Moreover, cellular levels of reduced glutathione and total glutathione were significantly decreased, and S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio was significantly increased after exposure to glucose 25 mM but not 5 mM. We conclude that in response to high glucose, HK-2 cells characteristic metabolomic changes, including increase in lactate-to-pyruvate ratio, reduction in Krebs cycle metabolites, reduction in glutathione antioxidant activity, and increase in cellular methylation potential. Our results may shed light on the pathogenesis of diabetic kidney disease, but the expression of glucose metabolism-related protein and enzyme activity in HK-2 cells after hyperglycemia condition need to be confirmed by further studies.

Although diabetic kidney disease (DKD) is the most common cause of end-stage kidney disease worldwide, the pathogenic mechanisms are poorly understood. There is increasing evidence that mitochondrial dysfunction contributes to the development and progression of DKD. Because the kidney is the organ with the second highest oxygen consumption in our body, it is distinctly sensitive to mitochondrial dysfunction. Mitochondrial dysfunction contributes to the progression of chronic kidney disease irrespective of underlying cause. More importantly, high plasma glucose directly damages renal tubular cells, resulting in a wide range of metabolic and cellular dysfunction. Overproduction of reactive oxygen species (ROS), activation of apoptotic pathway, and defective mitophagy are interlinked mechanisms that play pivotal roles in the progression of DKD. Although renal tubular cells have the highest mitochondrial content, podocytes, mesangial cells, and glomerular endothelial cells may all be affected by diabetes-induced mitochondrial injury. Urinary mitochondrial DNA (mtDNA) is readily detectable and may serve as a marker of mitochondrial damage in DKD. Unfortunately, pharmacologic modulation of mitochondrial dysfunction for the treatment of DKD is still in its infancy. Nonetheless, understanding the pathobiology of mitochondrial dysfunction in DKD would facilitate the development of novel therapeutic strategies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Mitochondrial dysfunction plays an important role in the pathogenesis and progression of chronic kidney disease (CKD). We study the relation between urinary mitochondrial DNA (mtDNA) levels and renal dysfunction in non-diabetic CKD., Methods: We recruited 32 CKD patients (20 had hypertensive nephrosclerosis, 12 had IgA nephropathy). Urinary supernatant mtDNA level was measured and compared to baseline clinical and pathological parameters. The patients were followed 57.8 ± 30.5 months for renal function decline., Results: The average urinary supernatant mtDNA level was 222.0 ± 210.3 copy/μL. There was a modest but significant correlation between urinary mtDNA level and proteinuria (Spearman's r = 0.387, p = 0.035), but not any other baseline clinical or pathological parameter. Urinary mtDNA level had a significant inverse correlation with the slope of GFR decline (r = -0.402, p = 0.023). Urinary mtDNA level is a predictor of renal survival even after adjusting for baseline proteinuria with multivariate Cox analysis. In this model, every increase in urinary mtDNA by 100 copy/μL confers a 25.0% increase in risk of doubling of serum creatinine or need of dialysis (95%CI, 0.7% to 55.1%)., Conclusion: Mitochondrial DNA is readily detectable in the urinary supernatant of non-diabetic CKD, and its level correlates with the rate of renal function decline and predicts the risk of doubling of serum creatinine or need of dialysis. Further studies are needed to determine the value of urinary supernatant mtDNA level as a prognostic indicator of non-diabetic CKD., (Copyright © 2018. Published by Elsevier B.V.)

Background: Mitochondrial dysfunction plays an important role in the pathogenesis and progression of diabetic nephropathy (DN). We study the relation between urinary and intra-renal mitochondrial deoxyribonucleic acid (mtDNA) levels and renal dysfunction in DN., Methods: We recruited 92 patients with biopsy-proven DN. Urinary sediment, urinary supernatant and intra-renal mtDNA levels were measured and compared with baseline renal biopsy, kidney scarring and renal function decline in the subsequent 24 months., Results: mtDNA could be detected in all urine supernatant, urine sediment and renal biopsy specimens. There was a modest but statistically significant inverse correlation between urinary supernatant and intra-renal mtDNA levels (r = -0.453, P = 0.012). Urinary supernatant mtDNA level had modest but statistically significant correlations, inversely with estimated glomerular filtration rate (r = -0.214, P = 0.04), and positively with interstitial fibrosis (r = 0.300, P = 0.005). Intra-renal mtDNA had significant inverse correlation with interstitial fibrosis (r = -0.537, P = 0.003). However, there was no significant relation between renal function decline and urinary supernatant, urinary sediment or intra-renal mtDNA levels., Conclusions: mtDNA is readily detectable in urinary supernatant and kidney tissue, and their levels correlate with renal function and scarring in DN. Further studies are needed to determine the accuracy of urinary supernatant mtDNA level as a prognostic indicator of DN, as well as its role in other kidney diseases.

Purpose: Repeated intravitreal injections of endothelin-1 (ET-1) lead to alterations in the visually evoked potentials (VEPs) and loss of retinal ganglion cells (RGCs) in rabbits. The purpose of this study was to determine whether kallidinogenase can offset the alterations induced by ET-1., Methods: ET-1 (2.5 x 10(-7) M, 20 microL) was injected into the vitreous of the right eye of rabbits (ET-1-treated eyes, n = 30) twice a week for 4 weeks. The vehicle for ET-1 was injected into the left eye on the same schedule (vehicle treated eyes, n = 30). During this 4 weeks period, kallidinogenase (1.0 unit/kg/day, kallidinogenase-treated group) or saline (saline-injected control group) was continuously delivered intravenously by an implanted osmotic pump. VEPs were recorded before, and 2 weeks and 4 weeks after, the first ET-1 injection, and all rabbits were sacrificed at 4 weeks. The number of RGC cells was counted in hematoxylin- and eosin-stained retinal sections. In the analyses, the ET-1 induced alterations were normalized to the values in the vehicle treated control eyes, i.e., kallidinogenase (K) + ET-1/K+ vehicle or saline (S) +ET-1/S + vehicle. Retinal sections were also examined by immunohistochemistry with antibodies to single-stranded DNA (ssDNA) or to glial fibrillary acidic protein (GFAP). The effect of kallidinogenase on the ONH blood flow was determined by a hydrogen gas clearance flowmeter., Results: The significant prolongation of the relative VEP implicit times (ITs) 4 weeks after the ET-1 injection (P < 0.01, paired t test; post-ET-1 vs. pre-ET-1) was significantly decreased by kallidinogenase (P < 0.001, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). The relative number of RGCs was decreased in the saline-injected group, and this decrease was also decreased by kallidinogenase (P < 0.05, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). ssDNA staining showed fewer apoptotic cells in the retina of kallidinogenase-treated rabbits. Intravitreal injection of ET-1 also decreased the blood flow in the optic nerve head and increased the GFAP immunostaining and axonal degeneration. These changes were also counteracted by kallidinogenase., Conclusion: These results indicate that kallidinogenase can counter the effects of ET-1 and should be considered for the treatment of ischemic retinal and optic nerve disorders related to abnormal ET-1 production.