Diabetic Kidney Injury Research Articles

The Rab-Rabphilin system in injured human podocytes stressed by glucose overload and angiotensin II.

Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.

RETRACTED: Cathelicidin-BF attenuate kidney injury through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced diabetic rats

ObjectiveTo investigate protective efficacies and mechanisms of Cathelicidin-BF (BF-30) peptide on streptozotocin (STZ)-induced diabetic kidney injury. MethodsEffects of BF-30 on hydrogen peroxide induced oxidative damage in HK-2 renal cells were assessed by CCK-8 method. Forty STZ-induced diabetic rats with kidney injury were randomly divided into model control group, BF-30 group at different doses (0.1, 0.3 and 0.9 mg/kg). Blood biochemical and kidney related indexes as well adrenal morphological changes, inflammation related markers of diabetic rats were measured. ResultsCell viability of HK-2 cells with oxidative damage induced by hydrogen peroxide were significantly improved by BF-30 with 0.8 μg/mL for 56.5% and 1.6 μg/mL for 82.3% compared with control. Moreover, the decreased reactive oxygen species (ROS), and increased intracellular antioxidant enzymes GPX1, SOD2 and GSH were showed in BF-30 treated groups. In addition, co-incubation of BF-30 in HK-2 cells promoted the increase of p-AMPK and LC3, decreased activation of p-mTOR, BAX and Caspase 3. Chronic treatment of BF-30 improved the STZ-induced diabetic characteristics of diabetic kidney disease (DKD) model rats. Further renal histopathological examination revealed 12-week treatment of BF-30 effectively improved the morphology of nephropathy in DKD rats. Moreover, BF-30 also could ameliorate excessive oxidative stress, renal cell apoptosis and fibrosis, thereby protects renal tissues. ConclusionBF-30 exerted protective effects on STZ-induced kidney injury mainly through the inhibiting oxidative stress in kidney tissue, reducing renal fibrosis, increasing autophagy, and reducing the renal cell apoptosis related proteins to decrease the cell damage and protect nephrocytes.

P0993GYMNEMA SYLVESTRE EXTRACT ATTENUATE THE PATHOLOGICAL PROGESSION OF DIABETIC NEPHROPATHY IN RATS

Abstract Background and Aims Diabetic nephropathy is not only a common and severe microvascular complication of diabetes mellitus but also the leading cause of renal failure. Aqueous extract of leaves of Gymnema sylvestre (ALGS) has been reported to cause reversible increases in intracellular calcium, hypolipidemic activity and insulin secretion in mouse and human β cells with type 2 diabetes. The present study aimed to investigate the antidiabetic and renoprotective effects of ALGS in a rat model of type 2 diabetic mellitus. Method Male Sprague-Dawley rats with type 2 diabetes induced by a high-fat diet (HFD)/streptozotocin (STZ) were treated with ALGS at dosages of 0.5% and 1% (w/w) daily for 4 weeks. At the end of the experimental period, body weight, serum glucose levels, insulin levels, and kidney function were assessed. Furthermore, antioxidant enzyme and lipid peroxide levels were determined in the kidney with histopathological examination Molecular mechanism underlying the functioning of ALGS, mouse glomerular mesangial cells (MES-13) treated with high glucose (HG, 25 mM glucose) were chosen as a model for an examination of the signal transduction pathway of ALGS. Results The results revealed that ALGS improved diabetic kidney injury by reducing blood glucose, serum creatinine, and blood urea nitrogen levels and enhanced antioxidant enzyme activities in kidney tissue. Treatment with ALGS significantly reduced the malondialdehyde and 8-hydroxy-2-deoxyguanosine levels and increased serum insulin levels; expression of renal superoxide dismutase, catalase, and glutathione peroxidase activities; and glutathione content. Histological studies have also demonstrated that ALGS treatment inhibited the dilation of Bowman's capsule, which confirmed its renoprotective action in diabetes. In addition, treatment with ALGS attenuated 25 mM HG-induced suppressed nuclear factor erythroid 2-related factor 2 and antioxidant enzyme expression in MES-13 cells. Conclusion The results of present study reveal that ALGS extract could be useful intervention in the treatment of diabetes mellitus and, through antioxidative pathways.

P0704PROTECTIVE EFFECT OF DUAL ACTING LIGAND TARGETING A2A AND A3 ADENOSINE RECEPTOR ON OBSTRUCTION-INDUCED KIDNEY INJURY IN MICE

Abstract Background and Aims Chronic kidney disease (CKD) has been a worldwide public health problem, and the current therapeutic strategies against CKD are limited. Adenosine plays a significant role in protection against cellular damage in the regions with high metabolism. All 4 known subtypes of adenosine receptors such as A1AR, A2AAR, A2BAR, and A3AR are expressed in the kidney. While A2AAR agonists protect the kidney against inflammation, our previous study has demonstrated that an A3AR regulator effectively prevented diabetic kidney injury. The present study examined the preventive effect of LJ-4459, a newly synthesized dual acting ligand targeting A2AAR and A3AR, on unilateral ureteral obstruction (UUO)-induced kidney injury in mice. Method UUO surgery was performed in 6-week-old male C57BL/6 mice. LJ-4459 was administered by oral gavage at dose of either 1 or 10 mg/kg per day. The control group was administered an equal volume of vehicle (0.25% carboxymethyl cellulose). Results LJ-4459 effectively prevented tubular damage presented by significantly reduced NGAL expression and tubular necrosis in the obstructed kidney. LJ-4459 effectively inhibited elevation of a-SMA, collagen I, and collagen IV protein expression as well as picro sirius red staining in the obstructed kidney, suggesting antifibrotic effect of LJ-4459. LJ-4459 also inhibited inflammation as estimated by significantly reduced F4/80 positive-stained area and protein expression of p-NF-kB and ICAM1. Conclusion The results suggest that LJ-4459 may become a potential therapeutic agent against interstitial fibrosis, a common feature of CKD.

Phasor approach to autofluorescence lifetime imaging FLIM can be a quantitative biomarker of chronic renal parenchymal injury

Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, phasor autofluorescence lifetime imaging, which does not involve any staining, can be used to understand and evaluate the severity of kidney disease.

Hyperuricemia Predisposes to the Onset of Diabetes via Promoting Pancreatic β-Cell Death in Uricase-Deficient Male Mice.

Clinical studies have shown a link between hyperuricemia (HU) and diabetes, while the exact effect of soluble serum urate on glucose metabolism remains elusive. This study aims to characterize the glucose metabolic phenotypes and investigate the underlying molecular mechanisms using a novel spontaneous HU mouse model in which the uricase (Uox) gene is absent. In an attempt to study the role of HU in glycometabolism, we implemented external stimulation on Uox knockout (KO) and wild-type (WT) males with a high-fat diet (HFD) and/or injections of multiple low-dose streptozotocin (MLD-STZ) to provoke the potential role of urate. Notably, while Uox-KO mice developed glucose intolerance in the basal condition, no mice spontaneously developed diabetes, even with aging. HFD-fed Uox-KO mice manifested similar insulin sensitivity compared with WT controls. HU augmented the existing glycometabolism abnormality induced by MLD-STZ and eventually led to diabetes, as evidenced by the increased random glucose. Reduced β-cell masses and increased terminal deoxynucleotidyl TUNEL-positive β-cells suggested that HU-mediated diabetes was cell death dependent. However, urate-lowering therapy (ULT) cannot ameliorate the diabetes incidence or reverse β-cell apoptosis with significance. ULT displayed a significant therapeutic effect of HU-crystal–associated kidney injury and tubulointerstitial damage in diabetes. Moreover, we present transcriptomic analysis of isolated islets, using Uox-KO versus WT mice and streptozotocin-induced diabetic WT (STZ-WT) versus diabetic Uox-KO (STZ-KO) mice. Shared differentially expressed genes of HU primacy revealed Stk17β is a possible target gene in HU-related β-cell death. Together, this study suggests that HU accelerates but does not cause diabetes by inhibiting islet β-cell survival.

Attenuation of diabetic kidney injury in DPP4-deficient rats; role of GLP-1 on the suppression of AGE formation by inducing glyoxalase 1.

Dipeptidyl peptidase 4 (DPP4) inactivates incretin hormone glucagon-like peptide-1. DPP4 inhibitors may exert beneficial effects on diabetic nephropathy (DN) independently of glycemic control; however, the mechanisms underlying are not fully understood. Here, we investigated the mechanisms of the beneficial effects of DPP4 inhibition on DN using DPP4-deficient (DPP4-def) rats and rat mesangial cells.Blood glucose and HbA1c significantly increased by streptozotocin (STZ) and no differences were between WT-STZ and DPP4-def-STZ. The albumin level in urine decreased significantly and the albumin/creatinine ratio decreased slightly in DPP4-def-STZ. The glomerular volume in DPP4-def-STZ significantly decreased compared with that of WT-STZ. Advanced glycation end products formation, receptor for AGE (RAGE) protein expression, and its downstream inflammatory cytokines and fibrotic factors in kidney tissue, were significantly suppressed in the DPP4-def-STZ compared to the WT-STZ with increasing glyoxalase-1 (GLO-1) expression responsible for the detoxification of methylglyoxal (MGO). In vitro, exendin-4 suppressed MGO-induced AGEs production by enhancing the expression of GLO-1 and nuclear factor-erythroid 2 p45 subunit-related factor 2, resulting in decreasing pro-inflammatory cytokine levels. This effect was abolished by GLO-1 siRNA.Our data suggest that endogenously increased GLP-1 in DPP4-deficient rats contributes to the attenuation of DN partially by regulating AGEs formation via upregulation of GLO-1 expression.

Neutrophil Gelatinase-Associated Lipocalin as Early Sign of Diabetic Kidney Injury in Children

Neutrophil Gelatinase-Associated Lipocalin as Early Sign of Diabetic Kidney Injury in Children

Ursolic Acid Treatment Alleviates Diabetic Kidney Injury By Regulating The ARAP1/AT1R Signaling Pathway.

PurposeThis study aimed to investigate whether ursolic acid (UA) mitigates renal inflammation, oxidative stress and fibrosis by regulating the angiotensin II type 1 receptor-associated protein (ARAP1)/angiotensin II type 1 receptor (AT1R) signaling pathway and subsequently alleviating renal damage.Methodsdb/db mice were divided randomly into a diabetic nephropathy (DN) group and a UA treatment group. Light microscopy and electron microscopy were used to observe pathological changes in renal tissues. Immunohistochemistry (IHC) was employed to examine changes in the expression of ARAP1, AT1R, 8-hydroxydeoxyguanosine (8-OHdG), NADPH oxidase 2 (NOX2), the extracellular matrix protein fibronectin (FN), IL-1β and IL-18 in renal tissues. Western blotting and RT-qPCR were used to detect the respective changes in the protein and mRNA levels of ARAP1, AT1R, NOX4, NOX2, transforming growth factor-β1 (TGF-β1), FN, collagen IV, IL-1β and IL-18 in renal tissues and mesangial cells. In addition, immunofluorescence staining was employed to examine changes in FN and NOX2 expression in mesangial cells.ResultsUA treatment effectively reduced the body weights and blood glucose levels of db/db mice (p<0.05) as well as the urinary albumin/creatinine ratio (p<0.05). In addition, the renal tissue lesions and glomerulosclerosis index of the db/db mice were significantly improved after treatment (p<0.01). Histochemical analysis results showed significantly lower expression levels of ARAP1, AT1R, FN, NOX2, 8-OHdG, IL-1β and IL-18 in renal tissues in the UA treatment group than in the DN group. Western blotting and RT-qPCR data also revealed UA-induced decreases in the renal levels of the ARAP1, AT1, NOX4, NOX2, TGF-β1, FN, collagen IV, IL-1β and IL-18 proteins in vivo and/or in vitro (p<0.01). ARAP1 knockdown effectively reduced the expression of NOX2 and FN in vitro.ConclusionUA alleviated renal damage in type 2 diabetic db/db mice by downregulating proteins in the ARAP1/AT1R signaling pathway to inhibit extracellular matrix accumulation, renal inflammation, fibrosis and oxidative stress.

Chinese Herbal Medicine in Ameliorating Diabetic Kidney Disease via Activating Autophagy.

Diabetic kidney disease (DKD), a leading cause of end-stage renal disease (ESRD), has become a serious public health problem worldwide and lacks effective therapies due to its complex pathogenesis. Recent studies suggested defective autophagy involved in the pathogenesis and progression of DKD. Chinese herbal medicine, as an emerging option for the treatment of DKD, could improve diabetic kidney injury by activating autophagy. In this review, we briefly summarize underlying mechanisms of autophagy dysregulation in DKD, including AMP-activated protein kinase (AMPK), the mechanistic target of rapamycin (mTOR), and the sirtuin (Sirt) pathways, and we particularly concentrate on the current status of Chinese herbal medicine treating DKD by regulating autophagy. The advances in our understanding regarding the treatment of DKD via regulating autophagy with Chinese herbal medicine will enhance the clinical application of Chinese medicine as well as discovery of novel therapeutic agents for diabetic patients.

Deletion of Smad3 prevents renal fibrosis and inflammation in type 2 diabetic nephropathy

BackgroundTransforming growth factor (TGF)-β/Smad3 signaling is highly activated in kidneys of patients with type 2 diabetic nephropathy (T2DN), however, the precise role of Smad3 in the pathogenesis of diabetic nephropathy remains unclear. MethodsSmad3 knockout (KO)-db/db mice were generated by intercrossing of male and female double-heterozygous Smad3+/− db/m mice. Renal functions including urinary albumin excretion and serum creatinine were determined. Renal histological injury including renal fibrosis and inflammation were examined by periodic acid Schiff (PAS), periodic acid-silver methenamine (PASM), and immunohistochemistry (IHC) staining. ResultsSmad3 knockout (KO)-db/db mice were protected from the development of diabetic kidney injury, characterized by the normal levels of urinary albumin excretion and serum creatinine without any evidence for renal fibrosis and inflammation. In contrast, Smad3 wild-type (WT) db/db and Smad3+/− db/db mice developed progressively decline in renal function over the 12 to 32-week time course, including increased microalbuminuria and elevated levels of serum creatinine. Pathologically, Smad3 WT db/db and Smad3+/− db/db mice exhibited a marked deposition of collagen-I (colI), collagen-IV(col-IV), and an increased infiltration of F4/80+ macrophages in kidney. Mechanistically, Smad3 deficiency decreased the lncRNA Erbb4-IR transcription, while increased miR-29b transcription and therefore protected the kidney from progressive renal injury in db/db mice. ConclusionResults from this study imply that Smad3 may represent as a novel and effective therapeutic target for T2DN.

Prostaglandin E1 attenuates high glucose-induced apoptosis in proximal renal tubular cells by inhibiting the JNK/Bim pathway.

Proximal renal tubular damage is a critical process underlying diabetic kidney disease (DKD). Our previous study shows that prostaglandin E1 (PGE1) reduces the apoptosis of renal tubular cells in DKD rats. But its underlying mechanisms remain unclear. In this study we investigated the protective effects of PGE1 in DKD rats and high glucose (HG, 30 mM)-treated HK-2 proximal tubular cells. Four weeks after uninephrectomized streptozotocin-induced diabetic rats were established, the DKD rats were administered PGE1 (10 µg· kg−1· d−1, iv.) for 10 consecutive days. We showed that PGE1 administration did not change blood glucose levels, but alleviated diabetic kidney injury in the DKD rats, evidenced by markedly reduced proteinuria and renal tubular apoptosis. In the in vitro experiments, PGE1 (0.1–100 µM) significantly enhanced HG-reduced HK-2 cell viability. In HG-treated HK-2 cells, PGE1 (10 µM) significantly suppressed the c-Jun N-terminal kinase (JNK) and the mitochondrial apoptosis-related protein expressions such as Bim, Bax, caspase-3 and cleaved caspase-3; similar changes were also observed in the kidney of PGE1-treated DKD rats. By using two pharmacological tools-JNK activator anisomycin (AM) and JNK inhibitor SP600125, we revealed that PGE1 blocked HG-triggered activation of JNK/Bim pathway in HK-2 cells; JNK was an upstream regulator of Bim. In conclusion, our results demonstrate that the nephroprotective effects of PGE1 against apoptosis of proximal renal tubule in DKD rats via suppressing JNK-related Bim signaling pathway.

Cigarette smoke inhalation aggravates diabetic kidney injury in rats.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Epidemiological studies have demonstrated that cigarette smoke or nicotine is a risk factor for the progression of chronic kidney injury. The present study analyzed the kidney toxicity of cigarette smoke in experimental rats with DKD. Experimental diabetes was induced in 7-week-old Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin (60 mg kg-1). Four weeks after the induction of diabetes, rats were exposed to cigarette smoke (200 μg L-1), 4 h daily, and 5 days per week for 4 weeks. Cigarette smoke did not affect the levels of plasma glucose, hemoglobin A1c, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol or non-esterified fatty acids in both control and diabetic rats under the experimental conditions. Cigarette smoke, however, significantly increased diabetes-induced glomerular hypertrophy and urinary kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion, suggesting exacerbation of diabetic kidney injury. Cigarette smoke promoted macrophage infiltration and fibrosis in the diabetic kidney. As expected, cigarette smoke increased oxidative stress in both control and diabetic rats. These data demonstrated that four weeks of exposure to cigarette smoke aggravated the progression of DKD in rats.

Glycogen synthase kinase 3β hyperactivity in urinary exfoliated cells predicts progression of diabetic kidney disease

Burgeoning evidence points to glycogen synthase kinase (GSK)3β as a key player in diverse kidney diseases. However, as a pivotal transducer of the insulin signaling pathway, the role of GSK3β in diabetic kidney disease remains uncertain. In db/db mice, renal expression of total and activated GSK3β was increasingly elevated. This preceded the development of diabetic kidney disease, and correlated with the progression of signs of diabetic kidney injury, including albuminuria and extracellular matrix accumulation in glomeruli and tubulointerstitia. Invitro, exposure of glomerular podocytes, mesangial cells, and renal tubular cells to a diabetic milieu induced GSK3β overexpression and hyperactivity, which seem essential and sufficient for eliciting diabetic cellular damages in kidney cells, because the cytopathic effect of the diabetic milieu was mitigated by GSK3β knockdown, but was mimicked by ectopic expression of constitutively active GSK3β even in the normal milieu. In consistency, kidney biopsy specimens procured from patients with varying stages of diabetic nephropathy revealed an amplified expression of total and activated GSK3β in glomeruli and renal tubules, associated with the severity of diabetic nephropathy. Moreover, in retrospective cohorts of type 2 diabetic patients that were followed for over five years, the relative activity of GSK3β in banked urinary exfoliated cells represented an independent risk factor for development or progression of renal impairment. Furthermore, receiver operating characteristic curve analysis demonstrated that GSK3β activity in urinary exfoliated cells provided much better power than albuminuria in discriminating diabetic patients with progressive renal impairment from those with stable kidney function. Thus, renal expression and activity of GSK3β are amplified in experimental and clinical diabetic nephropathy. Hence, GSK3β in urinary exfoliated cells may serve as a novel biomarker for predicting diabetic kidney disease progression.

Bupleurum polysaccharides ameliorated renal injury in diabetic mice associated with suppression of HMGB1-TLR4 signaling.

Bupleurum polysaccharides (BPs) is isolated from Bupleurum smithii var. parvifolium, a key traditional Chinese medicine. The study was to investigate the effects of BPs on diabetic kidney injury. After two intraperitoneal injections of streptozotozin (STZ) 100 mg·kg-1, renal injury in diabetic mice was induced and BPs was orally administrated at dosages of 30 and 60 mg·kg-1·d-1. The STZ injected mice developed renal function damage, renal inflammation and fibrosis known as diabetic kidney disease (DKD). BPs significantly reduced serum creatinine level and urinary albumin excretion rate, with the attenuated swelling of kidneys. BPs treatment obviously alleviated the pathological damage of renal tissue. The progression of renal injury in BPs treated mice was inhibited with less expression of type IV collagen (Col IV), fibronectin (FN) and α-smooth muscle actin (α-SMA). The inhibition of inflammation in kidney was associated with the reduced level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). BPs administration suppressed the over-expression of toll like receptor 4 (TLR4) and high-mobility group box 1 (HMGB1) with lowered activity of nuclear factor kappa B (NF-κB) in renal tissue of diabetic mice. Oral administration of BPs effectively prevented the development ofrenal injury in diabetic mice. This study suggested that the protection provided by BPs might affect through the interruption of HMGB1-TLR4 pathway, leading to the inhibition of renal inflammation and fibrotic process.

Establishing equivalent diabetes in male and female Nos3-deficient mice results in a comparable onset of diabetic kidney injury.

Clinical studies indicate that sex differences exist in susceptibility for developing diabetic kidney disease (DKD), supporting the need to examine both sexes in animal studies of DKD. Streptozotocin (STZ) is commonly used in male mice to induce diabetes and DKD. However, females are not normally included because their sex hormones partially protect them from STZ‐induced islet injury and consequent diabetes. To address this issue, we identified a strategy to induce comparable diabetes in male and female mice using STZ and determined whether both sexes develop equivalent renal injury. Male and female mice lacking the gene for endothelial nitric oxide synthase (Nos3‐/‐) were made diabetic with five or six low‐dose STZ injections, respectively. Groups of male and female mice with equivalent hyperglycemia at week 3 after STZ were assessed for DKD at week 8. STZ‐treated male and female Nos3‐/‐ mice maintained comparable hyperglycemia between weeks 3 and 8 had an equivalent increase in HbA1c levels and comparable hypertension. Urine albumin/creatinine levels were elevated eightfold in mice of both sexes at week 8, accompanied by an equivalent loss of podocytes. In diabetic males and females, plasma cystatin C levels and glomerular collagen deposition were similarly increased. Kidney mRNA levels of proinflammatory and profibrotic markers and kidney injury molecule‐1 (KIM‐1) were equally elevated in males and females, indicating comparable kidney injury. This study shows that equivalent diabetes induces a comparable onset of DKD in male and female Nos3‐/‐ mice, demonstrating that it is possible to include males and females together in studies of DKD.

LC-MS-based metabolomics analysis to identify meprin-β-associated changes in kidney tissue from mice with STZ-induced type 1 diabetes and diabetic kidney injury.

Meprin metalloproteases have been implicated in the pathophysiology of diabetic kidney disease (DKD). Single-nucleotide polymorphisms in the meprin-β gene have been associated with DKD in Pima Indians, a Native American ethnic group with an extremely high prevalence of DKD. In African American men with diabetes, urinary meprin excretion positively correlated with the severity of kidney injury. In mice, meprin activity decreased at the onset of diabetic kidney injury. Several studies have identified meprin targets in the kidney. However, it is not known how proteolytic processing of the targets by meprins impacts the metabolite milieu in kidneys. In the present study, global metabolomics analysis identified differentiating metabolites in kidney tissues from wild-type and meprin-β knockout mice with streptozotocin (STZ)-induced type 1 diabetes. Kidney tissues were harvested at 8 wk post-STZ and analyzed by hydrophilic interaction liquid chromatography ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Principal component analysis identified >200 peaks associated with diabetes. Meprin expression-associated metabolites with strong variable importance of projection scores were indoxyl sulfate, N-γ-l-glutamyl-l-aspartic acid, N-methyl-4-pyridone-3-carboxamide, inosine, and cis-5-decenedioic acid. N-methyl-4-pyridone-3-carboxamide has been previously implicated in kidney injury, and its isomers, 4-PY and 2-PY, are markers of peroxisome proliferation and inflammation that correlate with creatinine clearance and glucose tolerance. Meprin deficiency-associated differentiating metabolites with high variable importance of projection scores were cortisol, hydroxymethoxyphenylcarboxylic acid-O-sulfate, and isovaleryalanine. The data suggest that meprin-β activity enhances diabetic kidney injury in part by altering the metabolite balance in kidneys, favoring high levels of uremic toxins such as indoxyl sulfate and N-methyl-pyridone-carboxamide.

TLR7 mediates increased vulnerability to ischemic acute kidney injury in diabetes.

Diabetes is a risk factor for acute kidney injury (AKI). However, its mechanism of pathogenesis has not been elucidated. The aim of the study was to investigate the role of inflammation and the toll-like receptor 7 (TLR7) in ischemic AKI for diabetes. A high glucose hypoxia-reoxygenation model of human renal tubular epithelial (HK-2) cells was used to generate AKI induced by ischemia-reperfusion in diabetes. The activity of cells was measured by CCK-8 assay and LDH activity. Inflammatory cytokines were assessed by ELISA. TLR7, MyD88, and NF-κB expressions were examined by western blotting. Apoptosis was evaluated by flow cytometry. The high glucose group and low glucose group were subjected to hypoxia-reoxygenation. The low glucose group developed only mild cell damage, apoptosis, and inflammatory response. In contrast, an equivalent hypoxia-reoxygenation injury provoked severe cell damage, apoptosis, and inflammatory response in the high glucose group. Expression of TLR7 and its related proteins were measured in the high glucose group before and after hypoxia-reoxygenation. The high glucose group exhibited more significant increases in TLR7 expression following hypoxia-reoxygenation than the low glucose group. In addition, the expression of TLR7 and its related proteins after hypoxia-reoxygenation were higher in the high glucose group than in the low glucose group. Inhibition of TLR7 provides significant protection against ischemic injury in diabetes. Our results suggest that diabetes increases the vulnerability to ischemia-induced renal injury. This increased vulnerability originates from a heightened inflammatory response involving the TLR7 signal transduction pathway.

&lt;p&gt;A small molecule inhibitor MCC950 ameliorates kidney injury in diabetic nephropathy by inhibiting NLRP3 inflammasome activation&lt;/p&gt;

BackgroundDiabetic nephropathy (DN) is a lethal diabetic microvascular complication characterized by chronic low-grade inflammation. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is implicated in the progression of DN. MCC950 is a selective and potent inhibitor of NLRP3; however, its efficacy in DN requires further investigation.MethodsTo investigate the efficacy of MCC950 in DN, eight-week-old type 2 diabetic db/db mice received injections of MCC950 intraperitoneally (10 mg/kg) twice per week for 12 weeks. Urinary albumin-to-creatinine ratio (ACR) and neutrophil gelatinase-associated lipocalin (NGAL), renal function, pathological changes, markers of podocyte and fibrosis and NLPR3/caspase-1/IL-1β expression in the renal cortices of db/db mice were evaluated. High-glucose (HG)-treated rat glomerular mesangial cells were treated with various concentrations of MCC950 for 48 hrs. Markers of fibrosis and NLPR3/caspase-1/IL-1β expression in the glomerular mesangial cells were measured.ResultsThe NLRP3 inflammasome was activated in db/db mice and HG-induced mesangial cells by upregulating NLRP3/caspase-1/IL-1β pathway. Inhibition of the NLRP3 inflammasome with MCC950 reduced the production of active caspase-1 and active IL-1β in db/db mice and HG-induced mesangial cells. MCC950 reduced serum creatinine, urinary ACR and NGAL, attenuated mesangial expansion with increased matrix and tubular dilatation, alleviated thickened glomerular basement membrane (GBM) and foot process fusion without affecting body weight and blood glucose levels in db/db mice. MCC950 increased the expression of podocin in db/db mice, and decreased the expression of TGF-β1, fibronectin, collagen I and α-smooth muscle actin (α-SMA) in renal cortices of db/db mice and HG-induced mesangial cells.ConclusionMCC950 ameliorated renal function, thickened GBM, podocyte injury and renal fibrosis in db/db mice, and decreased the production of fibrosis markers in HG-induced mesangial cells. MCC950 effectively ameliorated diabetic kidney injury by inhibiting NLRP3/caspase-1/IL-1β pathway, which may be a potential therapeutic strategy to prevent the progression of DN.

SUN-302 The Relative Roles of Pro-oxidant Ezymes Nox4 versus Nox5 in Diabetic Kidney Disease

Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional mice/rats models of DKD. We examined the role of Nox5 in human diabetic nephropathy, in human renal cell populations as well as in a high fat fed rabbit model of kidney disease. In addition, in vivo, we also examined the contribution of Nox5 in the pathogenesis of DN in the absence of Nox4 expression. Protein expression of Nox4 and Nox5 and their localization in glomerular (podocytes and mesangial cells) and tubular cells were examined by immunostaining in human kidney biopsies obtained from non-diabetic and diabetic individuals. In vitro, human mesangial cells, podocytes and proximal tubules were exposed to high glucose, TGF-β and AngII and Nox4 and Nox5 were knockdown in these renal cells. Cell morphology, gene and protein expression of markers of fibrosis and inflammation as well as putative signalling pathways and the level of ROS were assessed in these human renal cells. In addition, we have developed a novel mode of diabetic complications which is more similar to the human context, the New Zealand White rabbit which expresses Nox5 and the other Nox isoforms as in humans. We have exposed the rabbits to high fat feeding (as a model of type 2 diabetes) as well as inducing insulin deficient diabetes (type 1 diabetes) using alloxan. As a world first, we have generated a Nox5 knockout rabbit using CRISP/Cas9.We have also generated a unique humanised Nox5 transgenic mouse with concomitant Nox4 KO with the aim to definitively delineate the relative effects of Nox4 versus Nox5 in diabetic complications. Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals when compared to non-diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox5 and that Nox5 inhibition also downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits develop increased mesangial area, increased Nox5 expression in renal cortex and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated rabbit models of diabetes (type 1 and type 2) as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy.