Pathobiology of renal-specific oxidoreductase/myo-inositol oxygenase in diabetic nephropathy: its implications in tubulointerstitial fibrosis

Renal-specific oxido-reductase/ myoinositol oxygenase (RSOR/MIOX) is expressed in renal tubules. It catabolizes myo-inositol and its expression is increased in diabetic mice and in LLC-PK1 cells under high-glucose ambience. Aldose reductase (AR) is another aldo-keto reducase that is expressed in renal tubules. It regulates the polyol pathway and plays an important role in glucose metabolism, osmolyte regulation, and ECM pathobiology via the generation of advanced glycation end products, reactive oxygen species, and activation of transforming growth factor (TGF)-β. In view of the similarities between AR and RSOR/MIOX, the pathobiology of RSOR/MIOX and some of the cellular pathways affected by its overexpression were investigated. An increased expression of fibronectin was noted by transfection of LLC-PK1 cells with pcDNA3.1-RSOR/MIOX. Similar changes were observed in LLC-PK1 cells under high-glucose ambience, and they were notably lessened by RSOR/MIOX-small interfering (si) RNA treatment. The changes in tubulointerstitial fibronectin expression were also observed in the kidneys of db/db mice having high levels of RSOR. The pcDNA3.1-RSOR/MIOX transfectants had an increased NADH/NAD+ ratio, PKC and TGF-β activity, Raf1:Ras association, and p-ERK phosphorylation. These changes were significantly reduced by the inhibitors of PKC, aldose reductase, Ras farnesylation, and MEK1. Similar increases in various the above-noted parameters were observed under high-glucose ambience. Such changes were partially reversed with RSOR-siRNA treatment. Expression of E-cadherin and vimentin paralleled in cells overexpressing RSOR/MIOX or subjected to high-glucose ambience. These studies suggest that RSOR/MIOX modulates various downstream pathways affected by high-glucose ambience, and conceivably it plays a role in the pathobiology of tubulointerstitium in diabetic nephropathy.

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Alteration of urinary sorbitol excretion in WBN-kob diabetic rats - treatment with an aldose reductase inhibitor

Journal of Endocrinology ◽

10.1677/joe.0.1810429 ◽

2004 ◽

Vol 181 (3) ◽

pp. 429-435 ◽

Cited By ~ 7

Author(s):

T Tsugawa ◽

R Shinohara ◽

A Nagasaka ◽

I Nakano ◽

F Takeda ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Aldose Reductase ◽

Whole Blood ◽

Reductase Inhibitor ◽

Cell Function ◽

Polyol Pathway ◽

Diabetic Rats ◽

Aldose Reductase Inhibitor ◽

Tubular Damage ◽

Renal Tubules

An accelerated polyol pathway in diabetes contributes to the development of diabetic complications. To elucidate diabetic nephropathy involving also renal tubular damage, we measured urinary sorbitol concentration concomitantly with urinary N-acetyl-D-glucosaminidase (NAG) excretion in WBN-kob diabetic rats.Twenty-four-hour urinary sorbitol concentrations increased in the diabetic rats in parallel with whole blood sorbitol concentrations. An increase in 24-h urinary NAG excretion coincided with the elevated urinary sorbitol levels in the diabetic rats. The administration of epalrestat, an aldose reductase inhibitor, reduced the increased whole blood and urinary sorbitol concentrations and urinary NAG excretion concomitantly with renal aldose reductase inhibition in the diabetic rats.These results indicate that diabetic nephropathy involves distorted cell function of renal tubules, and that treatment with epalrestat may prevent at least the progress of the nephropathy.

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WJ-39, an Aldose Reductase Inhibitor, Ameliorates Renal Lesions in Diabetic Nephropathy by Activating Nrf2 Signaling

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/7950457 ◽

2020 ◽

Vol 2020 ◽

pp. 1-21

Author(s):

Xiaoyu Zhou ◽

Zheng Liu ◽

Ke Ying ◽

Huimin Wang ◽

Peng Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Diabetic Nephropathy ◽

Aldose Reductase ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Polyol Pathway ◽

Inflammatory Factors ◽

Related Factor ◽

Nuclear Factor ◽

Renal Lesions

Diabetic nephropathy (DN) is a chronic diabetic microvascular complication. Hyperactivity of the polyol pathway is involved in the pathogenesis of DN. Aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, is expected to be an effective target in the treatment of DN. WJ-39 is a novel inhibitor of AR. The present study aimed at exploring the effects of WJ-39 in DN. DN was induced in rats by injecting 30 mg/kg streptozotocin (STZ). After 14 weeks, WJ-39 (10, 20, and 40 mg/kg) was intragastrically administered to the rats for 12 weeks. Treatment with WJ-39 significantly inhibited AR activation and ameliorated renal dysfunction and fibrosis in DN rats. WJ-39 reduced oxidative stress in the kidneys of DN rats by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. WJ-39 suppressed the activation of the nuclear factor-kappa B (NF-κB) pathway and the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome to reduce the secretion of inflammatory factors. Rat mesangial cells (RMCs) were cultured under hyperglycemic conditions. WJ-39 abrogated the high glucose- (HG-) induced, excessive production of reactive oxygen species (ROS) and inflammatory factors. However, transfection with Nrf2 small interfering RNA abolished the effects of WJ-39. WJ-39 also blocked the transforming growth factor-β1/Smad pathway to reduce the production of glomerular extracellular matrix proteins, ultimately reducing fibrogenesis in DN. Our results show that WJ-39 ameliorated renal injury in DN rats, and its effects on oxidative stress and inflammation were associated with the activation of Nrf2 signaling. Thus, WJ-39 and its mechanism of amelioration of renal lesions in DN rats by reducing renal inflammation, oxidative stress, and fibrosis injury could be an effective strategy for the treatment of DN.

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Protective Pleiotropic Effect of Flavonoids on NAD+Levels in Endothelial Cells Exposed to High Glucose

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/894597 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Daniëlle M. P. H. J. Boesten ◽

Saskia N. I. von Ungern-Sternberg ◽

Gertjan J. M. den Hartog ◽

Aalt Bast

Keyword(s):

Endothelial Cells ◽

Enzyme Activity ◽

Aldose Reductase ◽

High Glucose ◽

Umbilical Vein ◽

Polyol Pathway ◽

Pleiotropic Effect ◽

Protective Role ◽

Parp Activation

NAD+is important for oxidative metabolism by serving as an electron transporter. Hyperglycemia decreases NAD+levels by activation of the polyol pathway and by overactivation of poly(ADP-ribose)-polymerase (PARP). We examined the protective role of three structurally related flavonoids (rutin, quercetin, and flavone) during high glucose conditions in anin vitromodel using human umbilical vein endothelial cells (HUVECs). Additionally we assessed the ability of these flavonoids to inhibit aldose reductase enzyme activity. We have previously shown that flavonoids can inhibit PARP activation. Extending these studies, we here provide evidence that flavonoids are also able to protect endothelial cells against a high glucose induced decrease in NAD+. In addition, we established that flavonoids are able to inhibit aldose reductase, the key enzyme in the polyol pathway. We conclude that this protective effect of flavonoids on NAD+levels is a combination of the flavonoids ability to inhibit both PARP activation and aldose reductase enzyme activity. This study shows that flavonoids, by a combination of effects, maintain the redox state of the cell during hyperglycemia. This mode of action enables flavonoids to ameliorate diabetic complications.

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Silencing of long noncoding RNA XIST protects against renal interstitial fibrosis in diabetic nephropathy via microRNA-93-5p-mediated inhibition of CDKN1A

AJP Renal Physiology ◽

10.1152/ajprenal.00254.2019 ◽

2019 ◽

Vol 317 (5) ◽

pp. F1350-F1358 ◽

Cited By ~ 6

Author(s):

Jindou Yang ◽

Yan Shen ◽

Xia Yang ◽

Yanjun Long ◽

Shuang Chen ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Molecular Mechanism ◽

High Glucose ◽

Noncoding Rna ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Kidney Tissue ◽

Luciferase Reporter ◽

Renal Interstitial Fibrosis ◽

Endogenous Expression

Long noncoding RNAs (lncRNAs) have been reported to play an important role in diabetic nephropathy (DN). However, the molecular mechanism involved in this process remains poorly understood. Thus, the present study aimed to explore the function and molecular mechanism of dysregulated lncRNA X-inactive specific transcript (XIST) in DN. DN mouse models were established by streptozotocin treatment, and human renal tubular epithelial HK-2 cells were exposed to high glucose to produce an in vitro model. XIST was highly expressed in renal tissues of patients with DN, mice with DN, and high glucose-exposed HK-2 cells. To identify the interaction among XIST, miR-93-5p, and cyclin-dependent kinase inhibitor 1A (CDKN1A) and to analyze the functional significance of their interaction in renal interstitial fibrosis, we altered endogenous expression of XIST and miR-93-5p and CDKN1A. Dual-luciferase reporter assay results suggested that XIST was highly expressed in the kidney tissue of DN mice and high glucose-exposed HK-2 cells. XIST was identified to be a lncRNA that could bind to miR-93-5p, and CDKN1A was a target of miR-93-5p. Downregulated expression of XIST led to an increase in miR-93-5p expression, thereby decreasing CDKN1A and suppressing renal interstitial fibrosis in DN. Consistently, XIST knockdown reduced the expression of fibrosis markers (fibronectin, collagen type IV, and transforming growth factor-β1). Restoration of CDKN1A or decreasing miR-93-5p yielded a reversed effect on renal interstitial fibrosis. In conclusion, our study demonstrated that silenced XIST inducing miR-93-5p-dependent CDKN1A inhibition was beneficial for preventing renal interstitial fibrosis in DN, which may provide a future strategy to prevent the progression of DN.

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Protective Effects of Luteolin on Diabetic Nephropathy in STZ-Induced Diabetic Rats

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2011/323171 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 33

Author(s):

Guo Guang Wang ◽

Xiao Hua Lu ◽

Wei Li ◽

Xue Zhao ◽

Cui Zhang

Keyword(s):

Diabetic Nephropathy ◽

Growth Factor ◽

High Glucose ◽

Transforming Growth Factor ◽

Antioxidant Properties ◽

Diabetic Rats ◽

Heme Oxygenase 1 ◽

Protective Effects ◽

Sod Activity ◽

Matrix Expression

Diabetic nephropathy is a long-term complication of diabetic mellitus. Many experimental evidences suggest that persistent hyperglycaemia generates intracellular reactive oxygen species (ROS) and upregulates transforming growth factor-b1 and extracellular matrix expression in mesangial and tubular epithelial cells, which is involved of free radicals in the pathogenesis of diabetes and more importantly in the development of diabetic complications. Antioxidants effectively inhibit high-glucose- and H2O2-induced transforming growth factor-b1 and fibronectin upregulation, thus providing evidence that ROS play an important role in high glucose-induced renal injury. The flavonoid luteolin has been shown to possess direct antioxidant activity, therefore we hypothesize that it may be useful in treatment of many chronic disease associated with oxidative stress, such as diabetic nephropathy via its antioxidant properties. Our results suggested that protection against development of diabetic nephropathy by luteolin treatment involved changes in superoxide dismutase (SOD) activity, the malondialdehyde (MDA) content and expression of Heme Oxygenase-1 (HO-1) protein.

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Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney

Cellular Physiology and Biochemistry ◽

10.1159/000488185 ◽

2018 ◽

Vol 45 (6) ◽

pp. 2369-2388 ◽

Cited By ~ 16

Author(s):

Claire Hills ◽

Gareth William Price ◽

Mark John Wall ◽

Timothy John Kaufmann ◽

Chi-Wai Tang ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Epithelial Cells ◽

Transforming Growth Factor ◽

Cell Communication ◽

Atp Release ◽

Immunoblot Analysis ◽

Biopsy Material ◽

Junctional Conductance ◽

Fibronectin Expression ◽

Tgf Β1

Background/Aims: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron. Methods: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFβ1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFβ1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-β1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-β1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression. Results: Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFβ1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFβ1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFβ1 and to 0.42±0.2nS after 7days TGFβ1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFβ1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06μM in control to 1.99±0.47μM after 48hr incubation with TGFβ1 (10ng/ml; P<0.05, n=3). TGF-β1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-β1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3). Conclusion: These data suggest that chronic exposure to glucose-evoked TGFβ1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy.

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DW1029M, a novel botanical drug candidate, inhibits advanced glycation end-product formation, rat lens aldose reductase activity, and TGF-β1 signaling

AJP Renal Physiology ◽

10.1152/ajprenal.00651.2013 ◽

2014 ◽

Vol 306 (10) ◽

pp. F1161-F1170 ◽

Cited By ~ 7

Author(s):

Joobyoung Yoon ◽

Hyunyong Lee ◽

Hwan Bong Chang ◽

Hyunsik Choi ◽

Yong Sung Kim ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Aldose Reductase ◽

Transforming Growth Factor ◽

Reductase Activity ◽

Product Formation ◽

Drug Candidate ◽

Advanced Glycation ◽

Active Constituents ◽

Rat Lens Aldose Reductase ◽

Botanical Extract

DW1029M is a botanical extract consisting of Morus bark and Puerariae radix, produced by Dong-Wha Pharmaceutical, for nephroprotective drug development; it has been in phase II clinical trials in Korea. In our mechanistic investigations, we found that DW1029M inhibits advanced glycation end products (AGEs), rat lens aldose reductase (RLAR), and transforming growth factor (TGF)-β1 signaling, all of which are implicated in diabetic complications such as diabetic nephropathy and diabetic retinopathy. DW1029M inhibits AGE formation via Fe2+ chelation. The extract contains 13 active constituents that inhibit AGE formation, 8 active constituents that inhibit RLAR activity, and 1 inhibitor of TGF-β1 signaling. Our results suggest DW1029M protects against diabetic nephropathy via blockade of AGE formation, RLAR activity, and TGF-β1 signaling.

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O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00108.2011 ◽

2011 ◽

Vol 301 (4) ◽

pp. E713-E726 ◽

Cited By ~ 50

Author(s):

Howard Goldberg ◽

Catharine Whiteside ◽

I. George Fantus

Keyword(s):

Diabetic Nephropathy ◽

P38 Mapk ◽

High Glucose ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Mesangial Cells ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Glomerular Mesangial Cells ◽

Matrix Accumulation

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β- N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins ( O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase ( O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr308 and Ser473 phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

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Transgenic overexpression of GLUT1 in mouse glomeruli produces renal disease resembling diabetic glomerulosclerosis

AJP Renal Physiology ◽

10.1152/ajprenal.00466.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. F99-F111 ◽

Cited By ~ 33

Author(s):

Youli Wang ◽

Kathleen Heilig ◽

Thomas Saunders ◽

Andrew Minto ◽

Dilip K. Deb ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Glomerular Disease ◽

Membrane Thickness ◽

Renal Tubules ◽

Glomerular Mesangial Cells ◽

Diabetic Glomerulosclerosis ◽

Basement Membrane Thickness ◽

Intracellular Glucose

Previous work identified an important role for hyperglycemia in diabetic nephropathy (The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977–986, 1993; UK Prospective Diabetes Study Group. Lancet 352: 837–853, 1998), and increased glomerular GLUT1 has been implicated. However, the roles of GLUT1 and intracellular glucose have not been determined. Here, we developed transgenic GLUT1-overexpressing mice (GT1S) to characterize the roles of GLUT1 and intracellular glucose in the development of glomerular disease without diabetes. GLUT1 was overexpressed in glomerular mesangial cells (MC) of C57BL6 mice, a line relatively resistant to diabetic nephropathy. Blood pressure, blood glucose, glomerular morphometry, matrix proteins, cell signaling, transcription factors, and selected growth factors were examined. Kidneys of GT1S mice overexpressed GLUT1 in glomerular MCs and small vessels, rather than renal tubules. GT1S mice were neither diabetic nor hypertensive. Glomerular GLUT1, glucose uptake, mean capillary diameter, and mean glomerular volume were all increased in the GT1S mice. Moderately severe glomerulosclerosis (GS) was established by 26 wk of age in GT1S mice, with increased glomerular type IV collagen and fibronectin. Modest increases in glomerular basement membrane thickness and albuminuria were detected with podocyte foot processes largely preserved, in the absence of podocyte GLUT1 overexpression. Activation of glomerular PKC, along with increased transforming growth factor-β1, VEGFR1, VEGFR2, and VEGF were all detected in glomeruli of GT1S mice, likely contributing to GS. The transcription factor NF-κB was also activated. Overexpression of glomerular GLUT1, mimicking the diabetic GLUT1 response, produced numerous features typical of diabetic glomerular disease, without diabetes or hypertension. This suggested GLUT1 may play an important role in the development of diabetic GS.

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