The Intestinal Microbiota Composition in Early and Late Stages of Diabetic Kidney Disease.

Many studies have suggested that gut microbiota dysbiosis may be one of the pathogenesis factors of diabetes mellitus (DM), while it is not clear whether it is involved in the development of diabetic kidney diseases (DKD). The objective of this study was to determine bacterial taxa biomarkers during the progression of DKD by investigating bacterial compositional changes in early and late DKD. 16S rRNA gene sequencing was performed on fecal samples, including the diabetes mellitus (DM), DNa (early DKD), and DNb (late DKD) groups. Taxonomic annotation of microbial composition was performed. Samples were sequenced on the Illumina NovaSeq platform. At the genus level, we found counts of Fusobacterium, Parabacteroides, and Ruminococcus_gnavus were significantly elevated both in the DNa group (P = 0.0001, 0.0007, and 0.0174, respectively) and the DNb group (P < 0.0001, 0.0012, and 0.0003, respectively) compared with those in the DM group. Only the level of Agathobacter was significantly decreased in the DNa group than the DM group and in the DNb group than the DNa group. Counts of Prevotella_9, Roseburia were significantly decreased in the DNa group compared with those in the DM group (P = 0.001 and 0.006, respectively) and in the DNb group compared with those in the DM group (P < 0.0001 and 0.003, respectively). Levels of Agathobacter, Prevotella_9, Lachnospira, and Roseburia were positively correlated with an estimated glomerular filtration rate (eGFR), but negatively correlated with microalbuminuria (MAU), 24 h urinary protein quantity (24hUP), and serum creatinine (Scr). Moreover, the areas under the curve (AUCs) of Agathobacter and Fusobacteria were 83.33% and 80.77%, respectively, for the DM and DNa cohorts, respectively. Notably, the largest AUC for DNa and DNb cohorts was also that of Agathobacter at 83.60%. Gut microbiota dysbiosis was found in the early and late stages of DKD, especially in the early stage. Agathobacter may be the most promising intestinal bacteria biomarker that can help distinguish different stages of DKD. IMPORTANCE It is not clear as to whether gut microbiota dysbiosis is involved in the progression of DKD. This study may be the first to explore gut microbiota compositional changes in diabetes, early-DKD, and late DKD. We identify different gut microbial characteristics during different stages of DKD. Gut microbiota dysbiosis is found in the early and late stages of DKD. Agathobacter may be the most promising intestinal bacteria biomarker that can help distinguish different stages of DKD, although further studies are warranted to illustrate these mechanisms.

The common risk factors for progression and mortality in COVID-19 patients: a meta-analysis.

Coronavirus disease 2019 (COVID-19), defined by the World Health Organization (WHO), has affected more than 50 million patients worldwide and caused a global public health emergency. Therefore, there is a recognized need to identify risk factors for COVID-19 severity and mortality. A systematic search of electronic databases (PubMed, Embase and Cochrane Library) for studies published before September 29, 2020, was performed. Studies that investigated risk factors for progression and mortality in COVID-19 patients were included. A total 344,431 participants from 34 studies were included in this meta-analysis. Regarding comorbidities, cerebrovascular disease (CVD), chronic kidney disease (CKD), coronary heart disease (CHD), and malignancy were associated with an increased risk of progression and mortality in COVID-19 patients. Regarding clinical manifestations, sputum production was associated with a dramatically increased risk of progression and mortality. Hemoptysis was a risk factor for death in COVID-19 patients. In laboratory examinations, increased neutrophil count, decreased lymphocyte count, decreased platelet count, increased C-reactive protein (CRP), coinfection with bacteria or fungi, increased alanine aminotransferase (ALT) and creatine kinase (CK), increased N-terminal pronatriuretic peptide (NT-proBNP), and bilateral pneumonia in CT/X-ray were significantly more frequent in the severe group compared with the non-severe group. Moreover, the proportion of patients with increased CRP and total bilirubin (TBIL) was also significantly higher in the deceased group than in the survival group. CVD, CKD, sputum production, increased neutrophil count, decreased lymphocyte count, decreased platelet count, increased CRP, coinfection with bacteria or fungi, increased ALT and CK, increased NT-proBNP, and bilateral pneumonia in CT/X-ray were associated with an increased risk of progression in COVID-19 patients. Moreover, the proportion of patients with increased sputum production, hemoptysis, CRP and TBIL was also significantly higher in the deceased group.

Roles of Krüppel-like factor 5 in kidney disease.

Transcription factor Krüppel-like factor 5 (KLF5) is a member of the Krüppel-like factors' (KLFs) family. KLF5 regulates a number of cellular functions, such as apoptosis, proliferation and differentiation. Therefore, KLF5 can play a role in many diseases, including, cancer, cardiovascular disease and gastrointestinal disorders. An important role for KLF5 in the kidney was recently reported, such that KLF5 regulated podocyte apoptosis, renal cell proliferation, tubulointerstitial inflammation and renal fibrosis. In this review, we have summarized the available information in the literature with a brief description on how transcriptional, post-transcriptional and post-translational modifications of KLF5 modulate its function in a variety of organs including the kidney with a focus of its importance on the pathogenesis of various kidney diseases. Furthermore, we also have outlined the current and possible mechanisms of KLF5 activation in kidney diseases. These studies suggest a need for more systemic investigations, particularly for generation of animal models with renal cell-specific deletion or overexpression of KLF5 gene to examine direct contributions of KLF5 to various kidney diseases. This will promote further experimentation in the development of therapies to prevent or treat various kidney diseases.

Endothelin receptor antagonists for the treatment of diabetic nephropathy: A meta-analysis and systematic review.

BACKGROUND: Diabetic nephropathy (DN) is the main cause of chronic kidney disease and end-stage renal disease worldwide. Although available clinical trials have shown that endothelin receptor (ER) antagonists may be a novel and beneficial drug for DN, no consistent conclusions regarding their sufficient effectiveness and safety for patients with DN have been presented. AIM: To assess the effectiveness and safety of ER antagonists among patients with DN. METHODS: The EMBASE, PubMed, MEDLINE, Cochrane, and ClinicalTrials.gov databases were searched without any language restrictions. Relative risks with 95% confidence intervals (CIs) for dichotomous data and mean differences or standardized mean difference with 95%CIs for continuous data were calculated using Review Manager 5.3 software. Publication bias was assessed using Egger's test with Stata/SE software. RESULTS: We enrolled seven studies with six data sets and 5271 participants. The ER antagonists group showed a significantly greater reduction in albuminuria and more patients with 40% reduction in urinary albumin-to-creatinine ratio than the control group (P < 0.0001 and P = 0.02, respectively). Subgroup analysis for reductions in estimated glomerular filtration rate (eGFR) showed that for the middle-dosage subgroup, the ER antagonists group exhibited lower eGFR reduction than the control group (P < 0.00001; mean difference, 0.70 95%CI: 0.66, 0.74). Moreover, significant reductions in systolic and diastolic blood pressure were observed in the invention group. CONCLUSION: ER blockades combined with angiotensin converting enzyme inhibitor /angiotensin II type 1 receptor blockers may be an effective treatment to lower blood pressure and reduce proteinuria in DN with declined eGFR. However, attention should be given to adverse events, including cardiac failure, anemia, and hypoglycemia, as well as serious adverse events.

The Role of Circulating RBP4 in the Type 2 Diabetes Patients with Kidney Diseases: A Systematic Review and Meta-Analysis.

BACKGROUND: Diabetic nephropathy is a common and serious complication of diabetes mellitus (DM) and is one of the leading causes of end-stage renal disease worldwide. Although there have been many investigations on biomarkers for DN, there is no consistent conclusion about reliable biomarkers. The purpose of this study was to perform a systematic review and meta-analysis of the role of circulating retinol-binding protein 4 (RBP4) in the type 2 diabetes mellitus (T2DM) patients with kidney diseases. MATERIALS AND METHODS: We searched the PubMed, MEDLINE, EMBASE, and Web of Science databases for publications. For the 12 cross-sectional studies that we included in the review, we calculated standard mean differences (SMD) with 95% confidence intervals (CI) for continuous data when the applied scales were different. Risk of bias of included trials was assessed by using the Newcastle-Ottawa Scale. RESULTS: RBP4 concentrations in the micro-, macro-, or micro+macroalbuminuria groups were significantly higher than those in the normal albuminuria group of T2DM patients [P = 0.001, SMD 1.07, 95% CI (0.41, 1.73)]. The estimated glomerular filtration rate (eGFR) was negatively associated with circulating RBP4 concentrations in patients with T2DM [summary Fisher's Z = -0.48, 95% CI (-0.69, -0.26), P < 0.0001]. The albumin-to-creatinine ratio (ACR) was positively associated with circulating RBP4 concentrations in patients with T2DM [summary Fisher's Z = 0.20, 95% CI (0.08, 0.32), P = 0.001]. CONCLUSION: The levels of circulating RBP4 were significantly higher both in T2DM subjects with micro/macroalbuminuria and in T2DM subjects with declined eGFR. The levels of circulating RBP4 were positively correlated with ACR but negatively correlated with eGFR. Circulating RBP4 could be a reliable biomarker for kidney diseases in T2DM.

Roles of 12-Lipoxygenase and Its Interaction with Angiotensin II on p21 and p27 Expression in Diabetic Nephropathy.

BACKGROUND: 12-Lipoxygenase (12-LO) and angiotensin II (Ang II) are involved in the development of diabetic renal hypertrophy, in which cyclin-kinase inhibitors, p21 and p27 play pivotal roles. Here, we study the effects of 12-LO and its interaction with Ang II on glomerular p21 and p27 expression in diabetic conditions. METHODS: Models used in the current study include glomerular mesangial cells (MCs); and glomeruli from (1) type 2 diabetic db/db mice; (2) type 2 diabetic rats induced by high-fat diet feeding followed by streptozotocin injection; (3) 12-LO knockout (12-LOKO) mice; and (4) normal rats infused with Ang II or 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE, arachidonic acid metabolite of 12-LO). RESULTS: The protein expression levels of p21 and p27 were increased in high glucose-stimulated MCs and in glomeruli isolated from db/db mice. In type 2 diabetic rats, cinnamyl-3,4-dihydroxy-α-cynanocinnamate (inhibitor of 12-LO) attenuated the increases in glomerular p21 and p27 protein expression, while in normal rats, 12(S)-HETE injection increased glomerular p21 and p27 expression. 12(S)-HETE and Ang II were mutually stimulated in glomeruli. Glomerular p21 and p27 expression were decreased in 12-LOKO mice compared to levels in control mice, and Ang II stimulation increased the protein expression of p27 in control but not 12-LOKO mice. Ang II stimulation had no effect on p21 protein expression in 12-LOKO mice. CONCLUSION: 12-LO is involved in diabetic renal hypertrophy via the induction of p21 and p27 protein expression and interacts with Ang II to induce p27 upregulation in diabetes. The current results suggest a potential amplifying loop in the pathogenesis of diabetic nephropathy.

Effect of peritoneal dialysis versus hemodialysis on renal anemia in renal in end-stage disease patients: a meta-analysis.

The aim of this meta-analysis was to evaluate the effect of peritoneal dialysis (PD) and hemodialysis (HD) on renal anemia (RA) in renal disease patients by a meta-analysis. Relevant studies published before June 2015 were searched. Pooled odds ratio (OR) with 95% confidence interval (CI) was used to evaluate the effect of HD and PD on RA based on five indexes: hemoglobin, ferritin, transferrin saturation index, serum albumin, and parathyroid hormone. Sensitivity analysis and publication bias assessment were conducted to evaluate the stability and reliability of our results. A total of fourteen eligible studies with 1103 cases underwent HD and 625 cases underwent PD were used for this meta-analysis. There were no significant difference for levels of hemoglobin (SMD = -0.23, 95% CI: -0.74 to 0.28), ferritin (SMD = 0.01, 95% CI: -0.59 to 0.62), parathyroid hormone (SMD = 0.11, 95% CI: -1.53 to 1.75) and transferrin saturation index (SMD = -0.06, 95% CI: -0.67 to 0.56) between HD and PD group. However, the content of serum albumin in HD group was much more than that in PD group (SMD = 1.58, 95% CI: 0.35 to 2.81). Neither of the included studies could reverse the pooled side effect and Egger's test demonstrated no publication bias. Both of the two dialysis strategies have a similar effect on RA in renal disease patients.

12-Lipoxygenase Inhibition on Microalbuminuria in Type-1 and Type-2 Diabetes Is Associated with Changes of Glomerular Angiotensin II Type 1 Receptor Related to Insulin Resistance.

(1) BACKGROUND: 12-lipoxygenase (12-LO) is involved in the development of diabetic nephropathy (DN). In the present study, we investigated whether 12-LO inhibition may ameliorate type-2 DN (T2DN) by interfering with insulin resistance (IR); (2) METHODS: Rat glomerular mesangial cells, glomeruli and skeletal muscles were isolated and used in this study. Kidney histological changes were confirmed by periodic-acid Schiff staining; mRNA expression was detected by competitive reverse transcription polymerase chain reaction; and the protein level was determined by Western blot and the enzyme-linked immunosorbent assay, respectively; (3) RESULTS: The inhibition of 12-LO attenuated microalbuminuria (MAU) increases in type-2 diabetic rats, but not in type-1 diabetic rats. Infusion of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly increased the expression of angiotensin II (Ang II) and Ang II type 1 receptor (AT1R), but decreased the expression of AT1R-associated protein (ATRAP) in rat glomeruli, compared to the control. An in vitro study revealed that both 12(S)-HETE and insulin upregulated AT1R expression in rat mesangial cells. In the presence of p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, the 12(S)-HETE-induced ATRAP reduction was significantly abolished. Interestingly, 12-LO inhibition did not influence AT1R expression in type-1 diabetic rats, but significantly abolished the increased AT1R and Ang II expression in glomeruli of type-2 diabetic rats. Furthermore, the inhibition of 12-LO significantly corrected impaired insulin sensitivity and fast serum insulin level, as well as the p-AMP-activated protein kinase (AMPK) reduction in skeletal muscle of type-2 diabetic rats; (4) CONCLUSION: The inhibition of 12-LO potentially ameliorated MAU by preventing IR through the downregulation of glomerular AT1R expression in T2DN.

Effect of angiotensin II type 1 receptor blocker on 12-lipoxygenase activity and slit diaphragm protein expression in type 2 diabetic rat glomeruli.

BACKGROUND: The 12-lipoxygenase (12-LO) and angiotensin II (Ang II) interaction plays an important role in diabetic nephropathy (DN). Proteinuria in DN is associated with decreased slit diaphragm proteins including nephrin and P-cadherin. Therefore, we investigated whether Ang II type 1 receptor (AT1) blocker (ARB) regulates 12-LO activity and slit diaphragm protein expression in diabetic rat glomeruli. METHOD: Glomeruli were isolated with the sieving method, and classified into small glomeruli (SG; 75-µm sieve) and large glomeruli (LG; 125-µm sieve). RESULTS: 12(S)-HETE, a lipid product of 12-LO, was increased by Ang II in the glomeruli. Infusion of 12(S)-HETE and Ang II significantly decreased nephrin expression in LG, but increased it in SG compared to control. Glomerular P-cadherin expression was reduced after Ang II and 12(S)-HETE treatment without differences between LG and SG. ARB did not influence glycemic levels but completely abolished the increases in 12(S)-HETE, AT1 expression, and proteinuria in diabetic rats. Nephrin expression was significantly reduced in LG but increased in SG in diabetic rats compared to control. P-cadherin expression decreased in both diabetic LG and SG. The abnormalities of nephrin and P-cadherin were partially but significantly reversed by ARB. CONCLUSION: ARB potentially ameliorates DN via the up-regulation of glomerular nephrin and P-cadherin expression through the inhibition of 12-LO activation in the glomeruli of rats with DN.

Arginase-1-dependent promotion of TH17 differentiation and disease progression by MDSCs in systemic lupus erythematosus.

Expansion of myeloid-derived suppressor cells (MDSCs) has been documented in some murine models and patients with autoimmune diseases, but the exact role of MDSCs in this process remains largely unknown. The current study investigates this question in patients with systemic lupus erythematosus (SLE). Patients with active SLE showed a significant increase in HLA-DR(-)CD11b(+)CD33(+)MDSCs, including both CD14(+)CD66b(-)monocytic and CD14(-)CD66b(+)granulocytic MDSCs, in the peripheral blood compared to healthy controls (HCs). The frequency of MDSCs was positively correlated with the levels of serum arginase-1 (Arg-1) activity, T helper 17 (TH17) responses, and disease severity in SLE patients. Consistently, in comparison with MDSCs from HCs, MDSCs from SLE patients exhibited significantly elevated Arg-1 production and increased potential to promote TH17 differentiation in vitro in an Arg-1-dependent manner. Moreover, in a humanized SLE model, MDSCs were essential for the induction of TH17 responses and the associated renal injuries, and the effect of MDSCs was Arg-1-dependent. Our data provide direct evidence demonstrating a pathogenic role for MDSCs in human SLE. This study also provides a molecular mechanism of the pathogenesis of SLE by demonstrating an Arg-1-dependent effect of MDSCs in the development of TH17 cell-associated autoimmunity, and suggests that targeting MDSCs or Arg-1 may offer potential therapeutic strategies for the treatment of SLE and other TH17 cell-mediated autoimmune diseases.

Prediction of the molecular mechanisms and potential therapeutic targets for diabetic nephropathy by bioinformatics methods.

In this study, we aimed to explore the molecular mechanisms of and genetic factors influencing diabetic nephropathy (DN). Gene expression profiles associated with DN were obtained from the GEO database (Accession no. GSE20844). The differentially expressed genes (DEGs) between diabetic mice and non-diabetic mice were screened. Subsequently, the DEGs were subjected to functional and pathway analysis. The protein-protein interaction (PPI) network was constructed and the transcription factors (TFs) were screened among the DEGs. A total of 92 upregulated and 118 downregulated genes were screened. Pathway analysis revealed that the p53 signaling pathway, the transforming growth factor (TGF)-ß signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway were significantly enriched by upregulated genes. Serpine1 (also known as plasminogen activator inhibitor-1), early growth response 1 (Egr1) and Mdk were found to be significant nodes in the PPI network by three methods. A total of 12 TFs were found to be differentially expressed, of which nuclear receptor subfamily 4, group A, member 1 (Nr4a1) and peroxisome proliferator-activated receptor gamma (Pparg) were found to have multiple interactions with other DEGs. We demonstrated that the p53 signaling pathway, the TGF-ß signaling pathway and the MAPK signaling pathway were dysregulated in the diabetic mice. The significant nodes (Serpine1, Egr1 and Mdk) and differentially expressed TFs (Nr4a1 and Pparg) may provide a novel avenue for the targeted therapy of DN.

Epigenetic Histone Modifications Involved in Profibrotic Gene Regulation by 12/15-Lipoxygenase and Its Oxidized Lipid Products in Diabetic Nephropathy.

AIMS: Epigenetic mechanisms, including histone post-translational modifications and DNA methylation, are implicated in the pathogenesis of diabetic nephropathy (DN), but the mediators are not well known. Moreover, although dyslipidemia contributes to DN, epigenetic changes triggered by lipids are unclear. In diabetes, increased expression of 12/15-lipoxygenase (12/15-LO) enhances oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which promote oxidant stress, glomerular and mesangial cell (MC) dysfunction, and fibrosis, and mediate the actions of profibrotic growth factors. We hypothesized that 12/15-LO and its oxidized lipid products can regulate epigenetic mechanisms mediating profibrotic gene expression related to DN. RESULTS: 12(S)-HETE increased profibrotic gene expression and enrichment of permissive histone lysine modifications at their promoters in MCs. 12(S)-HETE also increased protein levels of SET7, a histone H3 lysine 4 methyltransferase, and promoted its nuclear translocation and enrichment at profibrotic gene promoters. Furthermore, SET7 (Setd7) gene silencing inhibited 12(S)-HETE-induced profibrotic gene expression. 12/15-LO (Alox15) gene silencing or genetic knockout inhibited transforming growth factor-ß1 (TGF-ß1)-induced expression of Setd7 and profibrotic genes and histone modifications in MCs. Furthermore, 12/15-LO knockout in mice ameliorated key features of DN and abrogated increases in renal SET7 and profibrotic genes. Additionally, 12/15-LO siRNAs in vivo blocked increases in renal SET7 and profibrotic genes in diabetic mice. INNOVATION AND CONCLUSION: These novel results demonstrate for the first time that 12/15-LO-derived oxidized lipids regulate histone modifications associated with profibrotic gene expression in MCs, and 12/15-LO can mediate similar actions of TGF-ß1 and diabetes. Targeting 12/15-LO might be a useful strategy to inhibit key epigenetic mechanisms involved in DN.

Meta-analysis of the efficacy and safety of nucleotide/nucleoside analog monotherapy for hepatitis B virus-associated glomerulonephritis.

BACKGROUND/AIMS: Antiviral monotherapy is recommended for hepatitis B virus-associated glomerulonephritis (HBV-GN) treatment. Although considered superior to interferon-α in several respects, nucleotide/nucleoside analog (NA) monotherapy has not been studied. This metaanalysis evaluates the efficacy and safety of NA monotherapy for treating HBV-GN. METHODS: We searched for controlled clinical trials of NA monotherapy for HBVGN in the MEDLINE, Embase, Cochrane Library, Chinese BioMedical Literature on disc, Chinese National Knowledge Infrastructure, and Wanfang databases. Primary outcome measures were proteinuria remission, HBV-DNA negative conversion rate, and hepatitis B e-antigen (HBeAg) clearance. Secondary outcome measures were variations in proteinuria, serum albumin, alanine aminotransferase (ALT), and serum creatinine (Scr). RESULTS: Ten trials involving 325 patients were included: four randomized controlled trials, two cohort clinical trials, and four self-controlled studies. Based on the fixed-effects model, we found significant proteinuria remission rate improvement in the NA group (relative risk (RR): 3.60, 95% confidence interval (CI): 1.99 – 6.50), negative conversion rate of HBV-DNA (RR: 2.20, 95% CI: 1.55 – 3.13), and clearance of HBeAg (RR: 4.49, 95% CI: 1.29 – 15.67). Improvement in ALT (mean difference (MD): 56.60, 95% CI: 50.41 – 62.79) was found with the fixedeffects model, and a slight decrease in Scr (MD: 25.25, 95% CI: –17.11 – 67.61, p = 0.24) was shown. CONCLUSIONS: HBV-GN proteinuria remission with elevated serum albumin, decreased HBV replication, and improved HBeAg clearance could be achieved using NA monotherapy. Furthermore, NA monotherapy may protect renal function in HBV-GN patients by preventing Scr elevation.

p16ink4a Expression Is Increased through 12-Lipoxygenase in High Glucose-Stimulated Glomerular Mesangial Cells and Type 2 Diabetic Glomeruli.

BACKGROUND/AIMS: Arachidonic acid-metabolizing enzyme, 12-lipoxygenase (12-LO), is involved in the glomerular hypertrophy of diabetic nephropathy (DN), in which cyclin-dependent kinase inhibitors (CKIs) play important roles. However, it is unclear whether 12-LO regulates the expression of the CKI p16(ink4a) in DN. METHODS: Primary glomerular mesangial cells (MCs) and glomeruli isolated from rats were used in this study. The rats were fed a high-fat diet and given low-dose streptozotocin to induce type 2 diabetes. The 12-LO product, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), was infused through an osmotic minipump. Enzyme-linked immunosorbent assay, Western blot, and morphometric analyses were performed. RESULTS: High glucose (HG) increased the p16(ink4a) protein expression in MCs, but this increase was prevented by the 12-LO inhibitor, cinnamyl-3,​4-dihydroxy-α-cynanocinnamate (CDC). The levels of p-p38MAPK and p16(ink4a) in MCs were significantly elevated after the 12(S)-HETE treatment, whereas the p38MAPK inhibitor SB203580 prevented these increases. Compared with levels in control MCs, marked increases in p38MAPK activation and p16(ink4a) expression were observed in MCs plated on collagen IV, while the CDC treatment prevented these changes. Subcutaneous injection of CDC did not affect glucose levels, but completely attenuated the diabetes-related increases in the 12(S)-HETE content, p16(ink4a) expression, p-p38MAPK levels, glomerular volume, and the kidney/body weight ratio. Compared with levels in controls, p16(ink4a) and p-p38MAPK in the glomeruli derived from 12(S)-HETE-treated rats were significantly higher. CONCLUSIONS: 12-LO-p38MAPK mediates the upregulation of p16(ink4a) in HG-stimulated MCs and type 2-diabetic glomeruli, and new therapies aimed at 12-LO inhibition may prove beneficial in ameliorating diabetes-induced glomerular hypertrophy.

Renal protective effect of sirtuin 1.

Silent information regulator 2 (Sir2) is a nicotinamide adenine dinucleotide- (NAD(+)-) dependent deacetylase. The homology of SIRT1 and Sir2 has been extensively studied. SIRT1 deacetylates target proteins using the coenzyme NAD(+) and is therefore linked to cellular energy metabolism and the redox state through multiple signalling and survival pathways. During the past decade, investigators have reported that SIRT1 activity is essential in cancer, neurodegenerative diseases, diabetes, cardiovascular disease, and other age-related diseases. In the kidneys, SIRT1 may inhibit renal cell apoptosis, inflammation, and fibrosis. Therefore its activation may also become a new therapeutic target in the patients with chronic kidney disease including diabetic nephropathy. In this paper, we would like to review the protective functions of sirtuins and the role of SIRT1 in the onset of kidney disease based on previous studies, including diabetic nephropathy, acute renal injury, chronic kidney disease as well as lupus nephritis.

Role of 12-lipoxygenase in decreasing P-cadherin and increasing angiotensin II type 1 receptor expression according to glomerular size in type 2 diabetic rats.

12-lipoxygenase (12-LO) was implicated in the development of diabetic nephropathy (DN), in which the proteinuria was thought to be associated with a decreased expression of glomerular P-cadherin. Therefore, we investigated the role of 12-LO in the glomerular P-cadherin expression in type 2 diabetic rats according to the glomerular sizes. Rats fed with high-fat diet for 6 wk were treated with low-dose streptozotocin. Once diabetes onset, diabetic rats were treated with 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) for 8 wk. Then glomeruli were isolated from diabetic and control rats with a sieving method. RT-PCR, Western blotting, and immunofluorescent staining were used for mRNA and protein expressions of P-cadherin and angiotensin II (Ang II) type 1 receptor (AT1). We found that CDC did not affect the glucose levels but completely attenuated diabetic increases in glomerular volume and proteinuria. Diabetes significantly decreased the P-cadherin mRNA and protein expressions and increased the AT1 mRNA and protein expressions in the glomeruli. These changes were significantly prevented by CDC and recaptured by direct infusion of 12-LO product [12(S)-HETE] to normal rats for 7 days. The decreased P-cadherin expression was similar between large and small glomeruli, but the increased AT1 expression was significantly higher in the large than in the small glomeruli from diabetic and 12(S)-HETE-treated rats. Direct infusion of normal rats with Ang II for 14 days also significantly decreased the glomerular P-cadherin expression. These results suggest that diabetic proteinuria is mediated by the activation of 12-LO pathway that is partially attributed to the decreased glomerular P-cadherin expression.

Angiotensin II type 1 receptor expression is increased via 12-lipoxygenase in high glucose-stimulated glomerular cells and type 2 diabetic glomeruli.

BACKGROUND: Angiotensin II type 1 receptor (AT1) plays an important role in the development of diabetic nephropathy (DN). However, the roles played by 12-lipoxygenase (12-LO) in the AT1 expression in glomerular cells exposed to high glucose (HG) and diabetic glomeruli remain unclear. Our objective in the present study was to investigate the role of 12-LO in the AT1 expression in glomerular cells and glomeruli under diabetic conditions. METHODS: Mesangial cells (MCs), podocytes and glomeruli isolated from rats were used in this study. The rats fed a high fat diet received low-dose streptozotocin to make type 2 diabetes. The 12-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] was infused to rats by osmotic mini-pump. Morphometric measurement for glomerular volume, competitive reverse transcription polymerase chain reaction for mRNA expression, western blot and immunohistochemistry for protein expression were performed, respectively. RESULTS: Both the 12(S)-HETE and HG increased AT1 protein expression in MCs and podocytes. Furthermore, the levels of the AT1 were significantly higher in glomeruli derived from 12(S)-HETE-treated rats compared with control rats. In addition, HG-induced AT1 expression was significantly reduced by the 12-LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cynanocinnamate (CDC). Compared with the non-diabetic controls, DN rats showed significant glomerular hypertrophy and albuminuria. This was associated with significant increases in AT1 protein expression. These abnormalities were prevented by treatment of the CDC. CONCLUSIONS: These results indicate that AT1 expression is enhanced, at least in part, by 12-LO in the type 2 diabetic glomeruli, and 12-LO inhibition can ameliorate DN progression through downregulation of AT1 expression.

Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes.

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-beta, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN.

Products of 12/15-lipoxygenase upregulate the angiotensin II receptor.

Angiotensin II and its type 1 receptor (AT1R) play important roles in the pathogenesis of renal disease and diabetic nephropathy. The 12/15-lipoxygenase pathway of arachidonate metabolism and its lipid products have also been implicated in diabetic nephropathy. However, it is unclear whether 12/15-lipoxygenase regulates expression of AT1R. In cultured rat mesangial cells, we found that the 12/15-lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) increased AT1R mRNA and protein expression, primarily by stabilizing AT1R mRNA. Pretreatment with 12(S)-HETE also amplified the signaling effects of angiotensin II, likely due to the increased AT1R expression. Levels of AT1R protein expression decreased when 12/15-lipoxygenase was knocked down with specific short hairpin RNA (shRNA) compared with control cells. Similarly, levels of the AT1 receptor, but not the AT2 receptor, were significantly lower in mesangial cells and glomeruli derived from 12/15-lipoxygenase knockout mice compared with control mice. Reciprocally, stable overexpression of 12/15-lipoxygenase increased AT1R expression in cultured mesangial cells. In vivo, modified siRNA targeting 12/15-lipoxygenase reduced glomerular AT1R expression in a diabetic mouse model. Interestingly, angiotensin II induced greater levels of 12/15-lipoxygenase, TGF-beta1, and fibronectin (FN) in AT1R-overexpressing mesangial cells compared with control cells. Therefore, oxidized lipids generated by the 12/15-lipoxygenase-mediated metabolism of arachidonic acid can enhance AT1R expression in mesangial cells and augment the profibrotic effects of angiotensin II.

Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes.

Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor-beta (TGF-beta) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB-beta could be problematic. Several downstream effects of TGF-beta are mediated by connective tissue growth factor (CTGF), which is up-regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozotocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleotide, -treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and proteinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI-1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes-induced phosphorylation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.