Molecular mechanisms attributed to colistin renal proximal tubular epithelial cytotoxicity

Modified Zhenwu Tang delays chronic renal failure progression by modulating oxidative stress and hypoxic responses in renal proximal tubular epithelial cells

Background: The global burden of multi-drug resistant gram-negative bacterial (MDR-GNB) infections has been increasing. Neonates are at a particularly high-risk and there is limited treatment option. The use of colistin has been re-introduced for this population. However, data on its use in neonates is scarce. Objectives: To determine the effectiveness and adverse effects of intravenous colistin in neonates with multidrug-resistant gram-negative infections. Design: This is a retrospective cohort study of the clinical profile and outcome of neonates with MDR-GNB infections given colistin for a minimum of 3 days conducted from April 2015 to April 2019. Results: A total of 175 pediatric patients had MDR-GNB infections. 75 (43%) neonates met the inclusion criteri a and received intravenous colistin. Of the 75 patients with MDRGNB infections- that included sepsis, pneumonia, urinary tract infection and abscess, 37 (49.3%) were alive and 38 (50.7%) patients died. Nephrotoxicity was seen in 4% if patients and 2.6% patients had hypersensitivity reaction. MDROs isolated were Acinetobacter baumanii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Conclusions: Intravenous colistin is 50% effective and is relatively safe to use in neonates.

ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-β1 Autoinduction in Proximal Tubular Epithelial Cells

BackgroundInfections caused by multi-drug resistant gram-negative bacterial infections are the principle threats to the critically ill patients of intensive care units. Increasing reports of these infections from the Nepalese intensive care unit underline the clinical importance of these pathogens. However, the impact of these infections on the patient’s clinical outcome has not yet been clearly evaluated. The objective of our study was to determine the incidence and associated clinical outcome of multi-drug resistant gram-negative bacterial infections in intensive care unit from a tertiary care center of Nepal.MethodsA prospective cohort study was conducted among adult patients admitted in intensive care unit of B. P Koirala Institute of Health Sciences from July to December 2017. Patients infected with multi-drug resistant gram-negative bacteria, non-multi-drug resistant gram-negative bacteria and those without infection were included. Identification of gram-negative bacteria and their antibiotic susceptibility pattern was performed with standard microbiological methods. Demographic, clinical profiles and outcomes (in-hospital-mortality, intensive care unit and hospital length of stay) were documented.ResultsThe incidence rate of multi-drug resistant gram-negative bacteria infections was 47 per 100 admitted patients (64/137) with 128 episodes. Acinetobacter species (41%, 52/128) was the commonest followed by Klebsiella pneumoniae (28%, 36/128) and Pseudomonas spp (21%, 27/128). Patients with multi-drug resistant gram-negative bacteria in comparison to non-multi-drug resistant gram-negative bacteria had high healthcare-associated infections (95%, 61/64 versus 20%, 2/10; p = < 0.001). In-hospital-mortality was 38% (24/64), 20% (2/10) and 10% (4/41) in multi-drug resistant, non-multi-drug resistant and uninfected group respectively (p = 0.007). After adjustment for independent risk factors, compared to uninfected patients, the odds ratio (CI) for in-hospital-mortality in multi-drug resistant and non-multi-drug resistant group was (4.7[1.4–15.5], p = 0.01) and 2.60 [0.38–17.8], p = 0.32) respectively. Multi-drug resistant patients also had longer intensive care unit and hospital stay, however, it was statistically insignificant.ConclusionThe incidence of multi-drug resistant gram-negative bacterial infections was remarkably high in our intensive care unit and showed a significant association with healthcare-associated infections and in-hospital-mortality.

Comparative biology may reveal novel therapeutic strategies against human diseases. Ischemia‑reperfusion (IR) injury induces a number of diseases. It is known that hibernating mammals survive IR since during hibernation, prolonged periods of torpor with a marked decrease in blood flow and breathing rate are interrupted by short periods of arousal. In the present study, the differences in the characteristics of endoplasmic reticulum (ER) stress and the subsequent unfolded protein response, which are induced by IR and may cause cell death among humans, mice or the native hibernator Syrian hamster were examined in vitro using renal proximal tubular epithelial cells (RPTECs) derived from these three sources. RPTECs were subjected to anoxia or reoxygenation, both at 37˚C. Cell death was measured by LDH release assay. ER stress was assessed by determining the levels of phosphorylated protein kinase RNA‑like ER kinase, ubiquitinated proteins and Bcl‑2‑associated X protein (Bax) by western blot analysis. For proteasomal activity, a specific assay was used. The results revealed that anoxia induced ER stress in all the evaluated RPTECs, from which only the hamster‑derived RPTECs recovered during reoxygenation. Anoxia and reoxygenation increased protein ubiquitination in the human‑ and mouse‑derived RPTECs, whereas this was decreased in the hamster‑derived RPTECs. Anoxia enhanced proteasomal activity in all the evaluated RPTECs. In the human‑ and mouse‑derived RPTECs, reoxygenation reduced proteasomal activity, which remained high in the hamster‑derived RPTECs. Anoxia and reoxygenation increased Bax expression and induced cell death in the human‑ and mouse‑derived RPTECs, while neither Bax overexpression nor cell death occurred in the hamster‑derived RPTECs. Thus, on the whole, the findings of this study demonstrate that compared to human‑ or mouse‑derived RPTECs, those derived from the hamster recover more rapidly from ER stress following warm anoxia‑reoxygenation, possibly due to increased proteasomal function.

Autophagy deficiency is a clinical feature of early diabetic nephropathy (EDN). This study aimed to investigate the role of REG1A in EDN. Rat renal proximal tubular epithelial cells were exposed to high glucose (HG) to establish in vitro EDN model. mRNA expression was determined using RT-qPCR. Protein expression was determined using Western blot. LC3 expression was determined using immunofluorescence. The location of REG1A was determined using RNA FISH assay. Cytokine release was determined using ELISA assay. We found that HG promoted the inflammation and fibrosis of rat renal proximal tubular epithelial cells, whereas it inhibited autophagy. REG1A was upregulated after exposed to HG. REG1A localized in cytoplasm of rat renal proximal tubular epithelial cells. REG1A knockdown inhibited the proliferation and fibrosis of rat renal proximal tubular epithelial cells. Moreover, REG1A knockdown reversed the effects of HG exposure and promoted autophagy in rat renal proximal tubular epithelial cells. Inhibition of REG1A protects against EDN. Therefore, targeting REG1A may be a promising strategy for EDN.

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

There is limited data on the prevalence and antibiotic susceptibility profile of Gram-negative bacteria in northwest Nigeria. This study thus aimed to investigate the prevalence of multidrug resistant Gram-negative bacterial infections among patients in two healthcare facilities in Sokoto, northwest Nigeria. A total of 735 non-duplicate clinical bacterial isolates were collected between January and July 2019, from among specimens processed by the diagnostic microbiological laboratory of the two hospitals. The isolates were identified using MALDI-TOF mass spectrometry and tested against a panel of sixteen (16) antibiotics using the current EUCAST guidelines. Of the 735 randomly selected bacterial isolates, 397 (54.0%) yielded Gram-negative bacteria. In the two hospitals, E. coli 104 (26.2%) and Klebsiella spp. 58 (14.6%) were the most common Gram-negative pathogens implicated in all infections. Overall, the isolates exhibited moderate to high resistance to all tested antibiotics, the lowest was observed against amikacin (7.1%). The phenotypic test for ESBL and carbapenemase enzymes showed that 48 (24.6%) and 15 (32.6%) of the isolates were positive, with 88.9% of the isolates being multidrug resistant. The study documents prevalent high multidrug resistant Gram-negative bacterial infections, predominantly caused by E. coli and K. pneumoniae in Sokoto, northwest Nigeria. The isolates were mostly MDR and exhibited ESBL and carbapenemase activities. The findings of this study call for urgent implementation of infection control measures and antibiotic stewardship in our hospitals so as to limit the spread of antibiotic-resistant bacteria in our healthcare facilities.

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22phox protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22phox protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22phox protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22phox protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22phox protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

Recent studies have identified high glucose as a potent stimulus for the intracellular synthesis of angiotensin II. However, the exact roles of angiotensin II and angiotensin II type 1 receptor blockers (ARB) in high-glucose-induced renal tubular function remain unclear. N-Acetyl-beta-glucosaminidase (NAG), angiotensin II and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in renal proximal tubular epithelial cells (RPTECs) with or without high glucose/ARB were determined using a modified commercial procedure. The changes of p22phox and cytoplasmic inhibitory kappa B (IkB) protein levels in RPTECs were measured using Western blotting. High-glucose treatment (4x10-2 mol/L) significantly increased NAG release, angiotensin II concentrations in cell lysates and 8-OHdG and p22phox protein levels compared with those in regular glucose medium (1.75x10(-2) mol/L). ARBs (candesartan, olmesartan or valsartan; 1x10(-9)-10(-7) mol/L) showed a significant reduction in high-glucose-induced NAG, 8-OHdG and p22phox protein levels in RPTECs. Significant decreases of cytoplasmic IkB protein levels were observed in the high-glucose-treated group in RPTECs. ARBs markedly reversed high-glucose-induced reduction of IkB protein levels in RPTECs. ARBs reduce high-glucose-induced oxidative stress in RPTECs possibly via blockade of intracellular as well as extracellular AT1 receptor signaling, which possibly protects renal tubular cell function during diabetic nephropathy.

Xanthine oxidase/NADPH oxidase inhibition by hydralazine attenuates acute kidney injury and prevents the transition of acute kidney injury to chronic kidney disease

India witnessed the exponential rise of antibiotic resistance due to the high burden of communicable disease. The Indian Council of Medical Research reported Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae (PEAK organisms) as the most common gram-negative isolates, constituting 65.5% of total isolates. The present study aimed to observe the demographics and clinical outcomes of patients infected with these four common gram-negative bacteria in ICUs across India. This prospective multicentric observational study was conducted in ICUs of 19 hospitals across India. The data collected for each patient included: demography, diagnosis, disease severity score, site of infection, PEAK organism, risk factors for multidrug resistance, antibiotic sensitivity, resistance pattern, total ventilator days, and 28-day mortality. Subgroup analysis of 28-day mortality was done for community-acquired vs hospital-acquired infection, appropriate empirical antibiotic, Carbapenem- and Colistin-resistant infections. A total of 936 patients were included in the analysis. Resistance to Cephalosporin, Fluroquinolones, Piperacillin Tazobactam, Carbapenem, Aminoglycosides, and Colistin was observed in 84, 68, 55, 47, 37, and 4.2% of patients, respectively. The 28-day crude mortality rate was 23.5%, which was higher in the subgroup with isolates resistant to empiric antibiotics compared to those with sensitive isolates (29.6 vs 21.4%, p > 0.05). Moreover, 32 and 27% mortality rates were observed in patients who were infected with Carbapenem-resistant and Colistin-resistant PEAK organisms, respectively. The present study observed a high prevalence of antibiotic resistance in Indian ICUs, contributing to a crude mortality rate of 23.5%. Patients with Carbapenem and Colistin resistance may exhibit higher 28-day crude mortality. Das SK, Joshi Z, Govil D, Shah MS, Jakaraddi GN, Sinha S, et al. Epidemiology and Clinical Outcome of Common Multi-drug Resistant Gram-negative Bacterial Infections in a Network of Hospitals in India (IMPRES): A Multicenter Intensive Care Unit-based Prospective Clinical Study. Indian J Crit Care Med 2025;29(6):504-509.CTRI identifier: CTRI/2023/01/049121.

Background & objectives: Antineutrophil cytoplasmic antibody (ANCA) is often used in laboratory tests to confirm paucicellular vasculitis. However, ANCA is also occasionally found in patients with infectious disorders independent of any vasculitic process. We retrospectively studied the association between perinuclear antineutrophil cytoplasmic antibody (p-ANCA) and clinical conditions, especially infectious diseases. Methods: Between 2007 and 2010, 1291 patients (118 p-ANCA-positive and 1173 p-ANCA-negative patients) were tested for ANCA. We selected the total 118 p-ANCA-positive patients, and selected 118 of the 1173 p-ANCA-negative patients randomly. They were divided into 2 equal groups according to the presence or absence of p-ANCA. Data on their medical history and hospitalization course were retrospectively analyzed using their medical records. Results: Overall, 44 p-ANCA-positive patients (37.3%) and 14 p-ANCA-negative patients (11.9%) had infections. From the former group, 36 patients (81.8%) had Staphylococcus aureus infection, 15 (34.1%) had multidrug resistant gram-negative bacterial infection, and 21 (47.7%) had Pseudomonas aeruginosa infection. Of the latter group, 6 patients (42.9%) had Staphylococcus aureus infection, 4 (28.6%) had multidrug resistant gram-negative bacterial infection, and 5 (35.7%) had Pseudomonas aeruginosa infection. Further, 21 p-ANCA-positive patients (17.8%) and 7 p-ANCA-negative patients (5.9%) were diagnosed with vasculitis. Lastly, 37 p-ANCA-positive patients (31.4%) and 16 p-ANCA-negative patients (13.8%) required intensive care unit admission. Conclusions: p-ANCA is significantly associated with some infections. Patients with severe infections may produce p-ANCA, especially those requiring ICU admission. Those who test positive for p-ANCA should be thoroughly investigated not only for vasculitis but also for infectious conditions.

Reactive oxygen species (ROS) and oxidative stress are associated with the progression of diabetic nephropathy (DN). Hydralazine is an antihypertensive agent and may act as a xanthine oxidase (XO) inhibitor to reduce uric acid levels in a mouse renal injury model. This study aimed to investigate the potential mechanisms of hydralazine in experimental DN. Streptozotocin-induced diabetic mice were fed a high-fat diet to generate DN. Human renal proximal tubular epithelial cells were used in vitro. Nitrendipine and allopurinol which can reduce blood pressure or XO activity levels, were used as two positive controls. Hydralazine downregulated NF-κB/p38 signaling pathways and reduced TNF-α/IL-6 expressions in high glucose-stimulated renal proximal tubular epithelial cells. Hydralazine reduced in vitro ROS production via XO inhibition and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated heme oxygenase (HO)-1 activation. Furthermore, hydralazine reduced high glucose-induced apoptosis by downregulating PARP/caspase-3 signaling. Hydralazine and allopurinol but not nitrendipine reduced serum uric acid levels and systemic inflammation. Hydralazine and allopurinol treatment improved renal function with decreased urinary albumin-to-creatinine ratios, glomerular hypertrophy, glomerulosclerosis, and fibrosis in the kidney of DN mice. While both hydralazine and allopurinol downregulated XO and NADPH oxidase expression, only hydralazine upregulated Nrf2/HO-1 renal expression, suggesting the additional effects of hydralazine independent of XO/ NADPH oxidase inhibition. In conclusion, hydralazine protected renal proximal tubular epithelial cells against the insults of high glucose and prevented renal damage via XO/NADPH oxidase inhibition and Nrf-2/HO-1 activation, suggesting the comprehensive antioxidation and anti-inflammation mechanisms for the management of DN.

myo-Inositol oxygenase (MIOX) catalyzes the oxidative cleavage of myo-inositol (MI) to give d-glucuronic acid, a committed step in MI catabolism. d-Glucuronic acid is further metabolized to xylitol via the glucuronate-xylulose pathway. Although accumulation of polyols such as xylitol and sorbitol is associated with MI depletion in diabetic complications, no causal relationship has been established. Therefore we are examining the role of MIOX in diabetic nephropathy. Here we present evidence that the basis for the depletion of MI in diabetes is likely to be mediated by the increased expression of MIOX, which is induced by sorbitol, mannitol, and xylitol in a porcine renal proximal tubular epithelial cell line, LLC-PK1. To understand the molecular mechanism of regulation of MIOX expression by polyols, we have cloned the human MIOX gene locus of 10 kb containing 5.6 kb of the 5' upstream sequence. Analysis of the 5' upstream sequence led to the identification of an osmotic response element (ORE) in the promoter region, which is present approximately 2 kb upstream of the translation start site. Based on luciferase reporter and electrophoretic mobility shift assays, polyols increased the ORE-dependent expression of MIOX. In addition, we demonstrate that the activity of the promoter is dependent on the binding of the transcription factor, tonicity element-binding protein, or osmotic response element-binding protein, to the ORE site. These results suggest that the expression of MIOX is up-regulated by a positive feedback mechanism where xylitol, one of the products of MI catabolism via the glucuronate-xylulose pathway, induces an overexpression of MIOX.