MicroRNA-21 in Glomerular Injury

TGF-β₁ is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-β₁, and miR-21 inhibits apoptosis in cancer cells. TGF-β₁–transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-β₁ and is higher in kidneys of TGF-β₁–transgenic mice than wild-type mice. miR-21–deficient TGF-β₁–transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-β₁–transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-β/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-β1 and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21–deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-β signaling and functions.     

Significance of Screening Tests and the Incidence of New Delhi Metallo-beta-lactamase-Producing Gram-negative Bacilli in the Surgery and Transplantation Wards of a Warsaw Medical Center During the Period From April 2014 to May 2017

Background

The first New Delhi metallo-beta-lactamase (NDM)-producing bacteria were isolated in 2008 in the world, and in 2011 in Poland. Due to the high clonal diversity (17 types) of their blaNDM gene, encoded on (Tn125-like) mobile genetic elements, these strains usually exhibit resistance to nearly all available antibiotics, which is particularly dangerous for organ transplant recipients.

Imipenem-resistant Pseudomonas aeruginosa strains carry metallo-β-lactamase gene blaVIM in a level I Iranian burn hospital

Introduction

In this study, we aimed to determine the distribution of bla_VIM and bla_IMP transferable genes in Pseudomonas aeruginosa isolates from infected burn wounds in an Iranian level I burn care center. These genes confer imipenem resistance and increase the mortality rate of burn patients.

Methods

P. aeruginosa isolates from burn patients were tested for antibiotic susceptibility with Kirby-Bauer disk diffusion method and for production of metallo-β-lactamase (MBL) by EDTA disk method. DNA was purified from isolates with positive MBL results and underwent PCR for detection of bla_VIM and bla_IMP genes.

Results

MBL was produced by 23 imipenem-resistant isolates and bla_VIM gene was detected in all of these isolates. None of the isolates carried bla_IMP gene. Mortality rate of infection with MBL-producing Pseudomonas strains was 82.6% in this hospital while the mortality rate for non-MBL-producing Pseudomonas was 22.7%.

Conclusion

We found that all MBL-producing isolates in this hospital carry bla_VIM gene. This result is similar to the previous Iranian study and emphasizes the importance of VIM family of MBLs in Iran. Timely identification of these strains and strict isolation methods can prevent spread of this transferable gene to other Gram-negative bacteria and prevent the subsequent outbreak of high mortality.     

Reduced Renal Calcium Excretion in the Absence of Sclerostin Expression: Evidence for a Novel Calcium-Regulating Bone Kidney Axis

The kidneys contribute to calcium homeostasis by adjusting the reabsorption and excretion of filtered calcium through processes that are regulated by parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D₃ (1α,25[OH]₂D₃). Most of the filtered calcium is reabsorbed in the proximal tubule, primarily by paracellular mechanisms that are not sensitive to calcium-regulating hormones in physiologically relevant ways. In the distal tubule, however, calcium is reabsorbed by channels and transporters, the activity or expression of which is highly regulated and increased by PTH and 1α,25(OH)₂D₃. Recent research suggests that other, heretofore unrecognized factors, such as the osteocyte-specific protein sclerostin, also regulate renal calcium excretion. Clues in this regard have come from the study of humans and mice with inactivating mutations of the sclerostin gene that both have increased skeletal density, which would necessitate an increase in intestinal absorption and/or renal reabsorption of calcium. Deletion of the sclerostin gene in mice significantly diminishes urinary calcium excretion and increases fractional renal calcium reabsorption. This is associated with increased circulating 1α,25(OH)₂D₃ levels, whereas sclerostin directly suppresses 1α-hydroxylase in immortalized proximal tubular cells. Thus, evidence is accumulating that sclerostin directly or indirectly reduces renal calcium reabsorption, suggesting the presence of a novel calcium-excreting bone-kidney axis.     

Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII^endo+/−) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII^endo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII^endo+/− mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII^endo+/+ counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β–induced canonical Smad2 signaling was reduced in TβRII^+/− ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TβRII^+/− ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII^+/− ECs compared with TβRII^+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.     

Detection of VEB-1, OXA-10 and PER-1 genotypes in extended-spectrum β-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients

Resistance of Pseudomonas aeruginosa strains to the broad-spectrum cephalosporins may be mediated by the extended-spectrum β-lactamases (ESBLs). These enzymes are encoded by different genes located on either chromosomes or plasmids. This study aimed to investigate the prevalence of ESBLs and antimicrobial susceptibilities of P. aeruginosa isolated from burn patients in Tehran, Iran. Antimicrobial susceptibility of 170 isolates to cefpodoxime, aztreonam, ciprofloxacin, ofloxacin, ceftazidime, cefepime, imipenem, meropenem, cefotaxime, levofloxacin, piperacillin–tazobactam and ceftriaxone was determined by disc agar diffusion test. Polymerase chain reaction (PCR) amplification of the genes encoding OXA-10, PER-1 and VEB-1 was also performed. All isolates (100%) were resistant to ceftazidime, cefotaxime, cefepime and aztreonam. Imipenem and meropenem were the most effective anti-pseudomonal agents. The results revealed that 148 (87.05%) of the isolates were multidrug resistant and 67 (39.41%) of the isolates were ESBL positive. Fifty (74.62%), 33 (49.25%) and 21 (31.34%) strains among 67 ESBL-producing strains amplified bla_OXA-10, bla_PER-1 and bla_VEB-1 respectively.     

Inhibition of Ca2+-Independent Phospholipase A2β (iPLA2β) Ameliorates Islet Infiltration and Incidence of Diabetes in NOD Mice

Autoimmune β-cell death leads to type 1 diabetes, and with findings that Ca²⁺-independent phospholipase A₂β (iPLA₂β) activation contributes to β-cell death, we assessed the effects of iPLA₂β inhibition on diabetes development. Administration of FKGK18, a reversible iPLA₂β inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4⁺ T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) β-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4⁺ T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA₂β-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4⁺ T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4⁺ T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA₂β-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA₂β may be beneficial in ameliorating autoimmune destruction of β-cells and mitigating type 1 diabetes development.     

Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ₃ integrin-dependent migration in vitro. Furthermore, αVβ₃ integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ₃ integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ₃ integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.     

Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes

Apoptosis is the major cause of death of insulin-producing β-cells in type 1 diabetes mellitus (T1DM). Klotho is a recently discovered antiaging gene. We found that the Klotho gene is expressed in pancreatic β-cells. Interestingly, halplodeficiency of Klotho (KL^+/−) exacerbated streptozotocin (STZ)-induced diabetes (a model of T1DM), including hyperglycemia, glucose intolerance, diminished islet insulin storage, and increased apoptotic β-cells. Conversely, in vivo β-cell–specific expression of mouse Klotho gene (mKL) attenuated β-cell apoptosis and prevented STZ-induced diabetes. mKL promoted cell adhesion to collagen IV, increased FAK and Akt phosphorylation, and inhibited caspase 3 cleavage in cultured MIN6 β-cells. mKL abolished STZ- and TNFα-induced inhibition of FAK and Akt phosphorylation, caspase 3 cleavage, and β-cell apoptosis. These promoting effects of Klotho can be abolished by blocking integrin β1. Therefore, these cell-based studies indicated that Klotho protected β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage. In an autoimmune T1DM model (NOD), we showed that in vivo β-cell–specific expression of mKL improved glucose tolerance, attenuated β-cell apoptosis, enhanced insulin storage in β-cells, and increased plasma insulin levels. The beneficial effect of Klotho gene delivery is likely due to attenuation of T-cell infiltration in pancreatic islets in NOD mice. Overall, our results demonstrate for the first time that Klotho protected β-cells in T1DM via attenuating apoptosis.     

Inter- and intra-observer agreement of the AO classification for operatively treated distal radius fractures

The reproducibility of the AO classification for distal radius fractures remains a topic of debate. Previous studies showed variable reproducibility results. Important treatment decisions depend on correct classification, especially in comminuted, intra-articular fractures. Therefore, reliable reproducibility results need to be undisputedly determined. Hence, the study objective was to assess inter- and intra-observer agreement of the AO classification for operatively treated distal radius fractures. A database of 54 radiographs of all AO types (A, B and C) and groups (A_2-3, B_1-3, and C_1-3) of distal radius fractures was assessed in twofold. Likewise, a subset of 152 radiographs of solely C-type groups (C_1-3) was assessed. All fractures were classified by six observers with different experience levels: three consultant trauma surgeons, one sixth-year trauma surgery resident, a consultant trauma radiologist, and an intern with limited experienced. The inter-observer agreement of both main types and groups was moderate (κ = 0.49 resp. κ = 0.48) in combination with a good intra-observer agreement (κ = 0.68 resp. κ = 0.70). The inter-observer agreement of the subset C-type fractures group was fair (κ = 0.27) with moderate intra-observer agreement (κ = 0.43). According to these results, the reproducibility of the AO classification of main types and groups of distal radius fractures based on conventional radiographs is insufficient (κ < 0.50), especially at group level of C-type fractures.     

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA₄) attenuated TGF-β1–induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA₄, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA₄ analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA₄-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA₄ in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA₄. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA₄-mediated upregulation of let-7c suppresses TGF-β1–induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.There is a growing appreciation of the role of endogenously produced lipid mediators including lipoxins, resolvins, and PGD synthase metabolites in promoting the resolution of inflammatory responses.^1–4 We and others recently described distinct proresolution and antifibrotic properties of lipoxins in renal fibrosis.^5,6 TGF-β1 is implicated in numerous fibrotic conditions including tubulointerstitial fibrosis and diabetic kidney disease. The development of fibrosis in this context may reflect activation of parenchymal fibroblasts, recruitment of circulating fibrocytes, and de-differentiation of epithelia and pericytes.^7,8 In this study, we investigated the effect of lipoxin A4 (LXA₄) on TGF-β1–induced fibrotic responses of renal epithelial cells and the mechanism underlying attenuation of this fibrotic injury pattern by lipoxins.MicroRNAs (miRNAs) comprise a class of small noncoding RNAs that negatively regulate gene expression by base-pairing to partially complementary sites in the 3′ untranslated regions (UTRs) of target mRNA, preventing translation. miRNAs are implicated in the development and progression of a wide range of complex human diseases,^9–12 including diabetic nephropathy.^13–15 Here we report that LXA₄ upregulates expression of the miRNA let-7c in HK-2 cells and attenuates response to TGF-β1. Lipoxins are protective in unilateral ureteral obstruction (UUO)–induced renal fibrosis and we report that this is associated with increased let-7c expression. Conversely, TGF-β1 decreases let-7c expression and this is associated with increased expression of let-7c targets, including TGFβ receptor type 1 (TGFβR1), collagens (COL1A1, COL1A2), and thrombospondin (THBS1). Overexpression of let-7c mimics the fibrosuppressant effects of LXA₄, whereas suppression of let-7c mimics responses to TGF-β1. The importance of regulation of let-7c targets is further supported by evidence from human renal biopsy material where we report upregulation of several let-7c targets in CKD.     

Molecular characterization of multidrug-resistant (MDR) Pseudomonas aeruginosa isolated in a burn center

Objective

The aim of this study was to characterize molecularly multidrug-resistant (MDR) Pseudomonas aeruginosa isolates collected from burn center (BC) patients and environment in a hospital localized in Rio de Janeiro city, RJ, Brazil.

The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAb_inhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAb_prostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved–to–full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved–to–full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAb_inhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.