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Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN., Graphical abstract Diabetic nephropathy is one of the most severe complications of diabetes mellitus. Chinese medicines have been intensively studied in treating diabetic nephropathy. This review comprehensively summarizes and discusses the clinical efficacies and underlying mechanisms of Chinese medicines for diabetic nephropathy, providing deep insights and profound perspectives into this field.Image 1

Diabetic nephropathy (DN) is considered the primary causes of end-stage renal disease (ESRD) and is related to abnormal glycolipid metabolism, hemodynamic abnormalities, oxidative stress and chronic inflammation. Antagonism of vascular endothelial growth factor B (VEGF-B) could efficiently ameliorate DN by reducing renal lipotoxicity. However, this pharmacological strategy is far from satisfactory, as it ignores numerous pathogenic factors, including anomalous reactive oxygen species (ROS) generation and inflammatory responses. We found that the upregulation of VEGF-B and downregulation of interleukin-22 (IL-22) among DN patients were significantly associated with the progression of DN. Thus, we hypothesized that a combination of a VEGF-B antibody and IL-22 could protect against DN not only by regulating glycolipid metabolism but also by reducing the accumulation of inflammation and ROS. To meet these challenges, a novel anti-VEGFB/IL22 fusion protein was developed, and its therapeutic effects on DN were further studied. We found that the anti-VEGFB/IL22 fusion protein reduced renal lipid accumulation by inhibiting the expression of fatty acid transport proteins and ameliorated inflammatory responses via the inhibition of renal oxidative stress and mitochondrial dysfunction. Moreover, the fusion protein could also improve diabetic kidney disease by increasing insulin sensitivity. Collectively, our findings indicate that the bifunctional VEGF-B antibody and IL-22 fusion protein could improve the progression of DN, which highlighted a novel therapeutic approach to DN., Graphical abstract The expression of VEGF-B was increased and that of IL-22 was decreased in diabetic nephropathy (DN) patients. Anti-VEGFB/IL22 fusion protein combining VEGF-B antibody and IL22 ameliorated DN by alleviating ectopic lipid accumulation, inflammatory responses and insulin resistance.Image 1

Type 2 diabetes mellitus (T2D) is a metabolic disorder characterized by inappropriate insulin function. Despite wide progress in genome studies, defects in gene expression for diabetes prognosis still incompletely identified. Prolonged hyperglycemia activates NF-κB, which is a main player in vascular dysfunctions of diabetes. Activated NF-κB, triggers expression of various genes that promote inflammation and cell adhesion process. Alteration of pro-inflammatory and profibrotic gene expression contribute to the irreversible functional and structural changes in the kidney resulting in diabetic nephropathy (DN). To identify the effect of some important NF-κB related genes on mediation of DN progression, we divided our candidate genes on the basis of their function exerted in bloodstream into three categories (Proinflammatory; NF-κB, IL-1B, IL-6, TNF-α and VEGF); (Profibrotic; FN, ICAM-1, VCAM-1) and (Proliferative; MAPK-1 and EGF). We analyzed their expression profile in leukocytes of patients and explored their correlation to diabetic kidney injury features. Our data revealed the overexpression of both proinflammatory and profibrotic genes in DN group when compared to T2D group and were associated positively with each other in DN group indicating their possible role in DN progression. In DN patients, increased expression of proinflammatory genes correlated positively with glycemic control and inflammatory markers indicating their role in DN progression. Our data revealed that the persistent activation NF-κB and its related genes observed in hyperglycemia might contribute to DN progression and might be a good diagnostic and therapeutic target for DN progression. Large-scale studies are needed to evaluate the potential of these molecules to serve as disease biomarkers.

Background: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves’ disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. Methods: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. Findings: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of DIO3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvβ3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. Interpretation: The study provided a mechanistic insight into how D3-αvβ3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves’ disease, explained a plausible link between thyroid disorder and NS. Funding: Funding support from American Thyroid Association (ATA) to NJT (ATA-2018-050.R1). Declaration of Interest: Jochen Reiser has patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is the co-founder of Walden Biosciences (Cambridge, MA, USA), a biotechnology company in which he has financial interest, including stock. Other authors have nothing to disclose and there are no competing or conflicting interests. Ethical Approval: All animal experiments were carried out according to the NIH’s Guide for the Care and Use of Experimental Animals (National Academies Press, 2011), and approved by the Institutional Animal Care and Use Committee (IACUC) at Rush University (Chicago, Illinois, USA). Human biopsy kidney sections from healthy donors and patients with FSGS, DN and MCD were procured in accordance with guidelines on human research and with approval of the Institutional Review Board of Rush University Medical Center (Chicago, Illinois, USA).

We identified a genetic variant, an 8-residue appendage, of the α345 hexamer of collagen IV present in patients with glomerular basement membrane (GBM) disease, Goodpasture's disease (GP) and Alport syndrome (AS) (see Companion Paper I), and determined the long-awaited crystal structure of hexamer (see Companion Paper II). We sought to elucidate how variants cause GBM disease by exploring the mechanism of hexamer assembly. Chloride ions induced in vitro hexamer assembly in a composition-specific manner in the presence of equimolar concentrations of α3, α4 and α5 NC1 monomers. Chloride ions, together with sulfilimine crosslinks, stabilized the assembled hexamer. Furthermore, the chloride ion-dependent assembly revealed conformational plasticity of the LCL bioactive sites, a critical property underlying bioactivity and pathogenesis. We explored the native mechanism by expressing recombinant α345 mini-protomers in cell culture and characterizing the expressed proteins. Our findings revealed: NC1-directed trimerization, forming protomers inside the cell, and hexamerization forming scaffolds outside the cell; and a Cl gradient signaled hexamerization. This assembly detail, along with a crystal structure, provides a framework for understanding hexamer dysfunction. Restoration of the native conformation of bioactive sites or α345 hexamer replacement are prospective approaches to therapeutic intervention.

NLRP3/IL-1β activation via thioredoxin (TRX)/thioredoxin-interacting protein (TXNIP) following mitochondria ROS (mtROS) overproduction plays a key role in inflammation. However, the involvement of this process in tubular damage in the kidneys of patients with diabetic nephropathy (DN) is unclear. Here, we demonstrated that mtROS overproduction is accompanied by decreases in TRX expression and TXNIP up-regulation. In addition, we discovered that mtROS overproduction is also associated with increases in NLRP3/IL-1β and TGF-β expression in the kidneys of patients with DN and db/db mice. We reversed these changes in db/db mice by administering a peritoneal injection of MitoQ, an antioxidant targeting mtROS. Similar results were observed in human tubular HK-2 cells subjected to high-glucose (HG) conditions and treated with MitoQ. Treating HK-2 cells with MitoQ suppressed the dissociation of TRX from TXNIP and subsequently blocked the interaction between TXNIP and NLRP3, leading to the inhibition of NLRP3 inflammasome activation and IL-1β maturation. The effects of MitoQ were enhanced by pretreatment with TXNIP siRNA and abolished by pretreatment with monosodium urate (MSU) and TRX siRNA in vitro. These results suggest that mitochondrial ROS-TXNIP/NLRP3/IL-1β axis activation is responsible for tubular oxidative injury, which can be ameliorated by MitoQ via the inhibition of mtROS overproduction., Graphical abstract fx1, Highlights • Reactive oxygen species promotes renal damage in diabetic nephropathy. • Mitochondrial ROS- TXNIP-NLRP3 biological axis involved in tubular injury of DN. • Inhibition of mitochondrial ROS by MitoQ ameliorated the renal tubular injury.

Aims Probucol is an anti-hyperlipidemic agent and a potent antioxidant drug that can delay progression of diabetic nephropathy (DN) and reverses renal oxidative stress in diabetic animal models; however, the mechanisms underlying these effects remain unclear. p66Shc is a newly recognized mediator of mitochondrial ROS production in renal cells under high-glucose (HG) ambience. We previously showed that p66Shc can serve as a biomarker for renal oxidative injury in DN patients and that p66Shc up-regulation is correlated with renal damage in vivo and in vitro. Here, we determined whether probucol ameliorates renal injury in DN by inhibiting p66Shc expression. Results We found that the expression of SIRT1, Ac-H3 and p66Shc in kidneys of DN patients was altered. Also, probucol reduced the levels of serum creatinine, urine protein and LDL-c and attenuated renal oxidative injury and fibrosis in STZ induced diabetic mice. In addition, probucol reversed p-AMPK, SIRT1, Ac-H3 and p66Shc expression. Correlation analyses showed that p66Shc expression was correlated with p-AMPK and Sirt1 expression and severity of renal injury. In vitro pretreatment of HK-2 cells with p-AMPK and SIRT1 siRNA negated the beneficial effects of probucol. Furthermore, we noted that probucol activates p-AMPK and Sirt1 and inhibits p66shc mRNA transcription by facilitating the binding of Sirt1 to the p66Shc promoter and modulation of Ac-H3 expression in HK-2 cells under HG ambience. Innovation and conclusion Our results suggest for the first time that probucol ameliorates renal damage in DN by epigenetically suppressing p66Shc expression via the AMPK-SIRT1-AcH3 pathway.

Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer-trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture’s disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

Diabetic nephropathy is a major complication of diabetes mellitus, and thus novel biomarkers are desired to evaluate the presence and progression of diabetic nephropathy. In this study, we sought to identify possible metabolites related to diabetic nephropathy among urinary eicosanoids and related mediators. Using liquid chromatogram-tandem mass spectrometry, we optimized the lipid extraction from urine using the Monospin C18 as a solid-phase extraction cartridge, and measured the urinary lipid mediators in 111 subjects with type 2 diabetes mellitus as well as 33 heathy subjects. We observed that 14 metabolites differed significantly among the clinical stages of nephropathy. Among them, levels of tetranor-prostaglandin E metabolite (tetranor-PGEM), an arachidonic acid metabolite, were significantly higher in subjects with stage 1 nephropathy than in healthy subjects, and increased with the progression of nephropathy. We also observed that levels of maresin-1, a docosahexaenoic acid metabolite, and leukotriene B4-ethanolamide, an arachidonoyl ethanolamide metabolite, were significantly lower in subjects with stage 3-4 nephropathy than in healthy subjects and those with stage 1-2 nephropathy. Finally, using a comprehensive analysis of urinary eicosanoids and related mediators, we concluded that tetranor-PGEM was capable of discriminating clinical stages of nephropathy, and thus useful as a novel biomarker for diabetic nephropathy.

Diabetes mellitus (DM), an endocrine disorder, will be one of the leading causes of death world-wide in about two decades. Cellular injuries and disorders of energy metabolism are two key factors in the pathogenesis of diabetes, which also become the important causes for the process of diabetic complications. AMPK is a key enzyme in maintaining metabolic homeostasis and has been implicated in the activation of autophagy in distinct tissues. An increasing number of researchers have confirmed that autophagy is a potential factor to affect or induce diabetes and its complications nowadays, which could remove cytotoxic proteins and dysfunctional organelles. This review will summarize the regulation of autophagy and AMPK in diabetes and its complications, and explore how AMPK stimulates autophagy in different diabetic syndromes. A deeper understanding of the regulation and activity of AMPK in autophagy would enhance its development as a promising therapeutic target for diabetes treatment., Graphical abstract This review summarizes the regulation of autophagy and AMPK in diabetes and its complications, and explores how AMPK stimulates autophagy in different diabetic syndromes. A deeper understanding of the regulation and activity of AMPK in autophagy would enhance its development as a promising therapeutic target for diabetes treatment.

Both acute kidney injury (AKI) and chronic kidney disease (CKD) that lead to diminished kidney function are interdependent risk factors for increased mortality. If untreated over time, end stage renal disease (ESRD) is an inevitable outcome. Acute and chronic kidney diseases occur partly due to imbalance between the molecular mechanisms that govern oxidative stress, inflammation, autophagy and cell death. Oxidative stress refers to the cumulative effects of highly reactive oxidizing molecules that cause cellular damage. Autophagy removes damaged organelles, protein aggregates and pathogens by recruiting these substrates into double membrane vesicles called autophagosomes which subsequently fuse with lysosomes. Mounting evidence suggests that both oxidative stress and autophagy are significantly involved in kidney health and disease. However, very little is known about the signaling processes that link them. This review is focused on understanding the role of oxidative stress and autophagy in kidney diseases. In this review, we also discuss the potential relationships between oxidative stress and autophagy that may enable the development of better therapeutic intervention to halt the progression of kidney disease and promote its repair and resolution., Graphical abstract, Highlights • The molecular mechanisms underlying the regulation of oxidative stress responses and autophagy may exhibit considerable cross-talk. • The autophagy pathway may be regulated in the context of kidney diseases. Failure or disruption of the autophagy pathway may contribute to the pathogenesis of kidney diseases. • Targeting the autophagy pathway may show considerable therapeutic potential in the treatment and management of kidney disorders.

Many studies have accessed the association between eNOS-4b/a polymorphism and the risk of diabetic nephropathy (DN) among type 2 diabetic subjects. However, the results are conflicting and inconclusive. The aim of current meta-analysis was to more precisely estimate the relationship. Pubmed, Embase, the China National Knowledge Infrastructure and the Wanfang Database were searched for articles published up to May 26th, 2013 that addressed eNOS-4b/a polymorphism and the risk of DN among type 2 diabetic subjects. 18 studies were included in this meta-analysis. eNOS-4b/a polymorphisms were associated with an overall significantly increased risk of DN (allele model: OR = 1.44, 95% CI = 1.14-1.82; additive model: OR = 2.03, 95% CI = 1.14-3.62; dominant model: OR = 1.34, 95% CI = 1.07-1.68; recessive model: OR = 2.01, 95% CI = 1.12-3.61). Subgroup analysis revealed a significant association between the eNOS-4b/a polymorphism and DN in Asian population, especially in Chinese population, but not in non Asian populations. Our meta-analysis supported an association between the 4b/a polymorphism of eNOS gene and increased risk of DN in type 2 diabetes among Asians, especially in Chinese population.

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane., Graphical abstract fx1, Highlights • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.

Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O2− (superoxide anion) and NOx (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O2− and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NOx production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

The accumulation of advanced glycation end products (AGE) plays significant role in developing tubular hypertrophy during diabetic nephropathy (DN). Reactive oxygen species and nitric oxide (NO) are directly involved in the progression of DN. We have studied the effect of standardized Gymnemasylvestre organic extract (GE) on AGE induced cellular hypertrophy using rat renal tubular epithelial cells (NRK 52E). AGE (400 μg/ml) induced cytotoxicity to NRK 52E cells as determined by MTT assay at 0–72 h. We report cellular hypertrophy mediated cytotoxicity by AGE which was the result of significant reduction in the cellular nitric oxide and cGMP levels associated with increased lipid peroxidation and antioxidant depletion (P < 0.05). Upon treatment with GE the cell viability was increased with reduced cellular hypertrophy by 1.7 folds when compared to AGE treated group. GE could significantly increase NO by 1.9 folds and cGMP by 2.8 folds and inhibited GSH depletion by 50% during AGE induced toxicity. The antioxidant enzyme activity of catalase was increased by 50% while, glutathione peroxidase and superoxide dismutase enzyme activities were significantly increased by 42% and 67% with decreased lipid peroxidation (49%) upon GE treatment. Thus, GE attenuates AGE induced hypertrophic growth by inhibiting GSH depletion and partly through increased NO/cGMP signaling.