Hsp74/14‐3‐3σ Complex Mediates Centrosome Amplification by High Glucose,Insulin, and Palmitic Acid

It has been reported recently that type 2 diabetes promotes centrosome amplification via 14‐3‐3σ/ROCK1 complex. In the present study, 14‐3‐3σ interacting proteins are characterized and their roles in the centrosome amplification by high glucose, insulin, and palmitic acid are investigated. Co‐immunoprecipitation in combination with MS analysis identified 134 proteins that interact with 14‐3‐3σ, which include heat shock 70 kDa protein 4 (Hsp74). Gene ontology analyses reveal that many of them are enriched in binding activity. Kyoto Encyclopedia of Genes and Genomes analysis shows that the top three enriched pathways are ribosome, carbon metabolism, and biosynthesis of amino acids. Molecular and functional investigations show that the high glucose, insulin, and palmitic acid increase the expression and binding of 14‐3‐3σ and Hsp74 as well as centrosome amplification, all of which are inhibited by knockdown of 14‐3‐3σ or Hsp74. Moreover, molecular docking analysis shows that the interaction between the 14‐3‐3σ and the Hsp74 is mainly through hydrophobic contacts and a lesser degree ionic interactions and hydrogen bond by different amino acids residues. In conclusion, the results suggest that the experimental treatment triggers centrosome amplification via upregulations of expression and binding of 14‐3‐3σ and Hsp74.     

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF‐β1 and HIF‐1α in Ang II‐mediated fibrosis after myocardial infarction

Transforming growth factor (TGF)‐β1 is a known factor in angiotensin II (Ang II)‐mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor‐1 (Hif‐1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif‐1α contributed to the Ang II‐mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif‐1α and TGF‐β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague–Dawley rats with MI daily for 1 week; saline and hydralazine (another anti‐hypertensive agent like valsartan) was used as control. The fibrosis‐related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up‐regulation of Ang II, TGF‐β/Smad and Hif‐1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up‐regulation of TGF‐β/Smad and Hif‐1α was through the Ang II‐mediated pathway. By administering TGF‐β or dimethyloxalylglycine, we determined that both TGF‐β/Smad and Hif‐1α contributed to Ang II‐mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF‐β/Smad, Hif‐1α and fibrosis‐related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II‐induced cardiac fibrosis as well as into the cardiac protection of valsartan.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Isoform‐specific cleavage of 14‐3‐3 proteins in apoptotic JURL‐MK1 cells

The proteins of 14‐3‐3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14‐3‐3 isoforms (β, γ, ε, τ, and ζ) during the apoptosis of JURL‐MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C‐terminal amino acids. The observed 14‐3‐3 modifications were partially blocked by caspase‐3 inhibition. In addition to caspases, a non‐caspase protease is likely to contribute to 14‐3‐3's cleavage in an isoform‐specific manner. While 14‐3‐3 γ seems to be cleaved mainly by caspase‐3, the alternative mechanism is essentially involved in the case of 14‐3‐3 τ, and a combined effect was observed for the isoforms ε, β, and ζ. We suggest that the processing of 14‐3‐3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. J. Cell. Biochem. 106: 673–681, 2009. © 2009 Wiley‐Liss, Inc.     

Petchiether A attenuates obstructive nephropathy by suppressing TGF‐β/Smad3 and NF‐κB signalling

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.     

14‐3‐3ζ binds to and stabilizes phospho‐beclin 1S295 and induces autophagy in hepatocellular carcinoma cells

Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14‐3‐3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up‐regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14‐3‐3ζ’s role in regulating autophagy in HCC cells, with a focus on beclin 1. The co‐localization of 14‐3‐3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT‐2 and HCC‐LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14‐3‐3ζ and phospho‐beclin 1^S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT‐2 and HCC‐LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS²⁹⁵VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14‐3‐3ζ binding motif. CO‐IP results confirmed that 14‐3‐3ζ bound to beclin 1, and this connection was markedly weakened when S²⁹⁵ was mutated into A²⁹⁵ (alanine). Further, 14‐3‐3ζ overexpression prevented phospho‐beclin 1^S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14‐3‐3ζ enhanced the chemoresistance of HCC cells to cis‐diammined dichloridoplatium by activating autophagy. Our work reveals that 14‐3‐3ζ binds to and stabilizes phospho‐beclin 1^S295 and induces autophagy in HCC cells to resist chemotherapy.     

Endogenous hormone 2‐methoxyestradiol suppresses venous hypertension‐induced angiogenesis through up‐ and down‐regulating p53 and id‐1

Brain arteriovenous malformations (AVMs) which associate with angiogenesis due to local hypertension, chronic cerebral ischaemia and tissue hypoxia usually lead to haemorrhage, however, the therapeutic medicine for the disease is still lacking. 2‐methoxyestradiol (2‐ME) has been shown effective in the anti‐angiogenic treatment. This study was conducted to examine whether and how 2‐ME could improve the vascular malformations. Intracranial venous hypertension (VH) model produced in adult male Sprague‐Dawley rats and culture of human umbilical vein endothelial cells (HUVECs) at the anoxia condition were used to induce in vivo and in vitro angiogenesis, respectively. Lentiviral vectors of ID‐1 and p53 genes and of their siRNA were intracranially injected into rats and transfected into HUVECs to overexpress and down‐regulate these molecules. 2‐ME treatment not only reduced the in vivo progression of brain tissue angiogenesis in the intracranial VH rats and the in vitro increases in microvasculature formation, cellular migration and HIF‐1α expression induced by anoxia in HUVECs but also reversed the up‐regulation of ID‐1 and down‐regulation of p53 in both the in vivo and in vitro angiogenesis models. All of the anti‐angiogenesis effects of 2‐ME observed in VH rats and anoxic HUVECs were abrogated by ID‐1 overexpression and p53 knockdown. Our data collectively suggest that 2‐ME treatment inhibits hypoxia/anoxia‐induced angiogenesis dependently on ID‐1 down‐regulation and p53 up‐regulation, providing a potential alternative medical treatment for un‐ruptured AVM patients.     

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14‐3‐3 proteins

Alkaline/neutral invertases (A/N‐Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At‐A/N‐InvG or AT1G35580). We demonstrate that Ser547 at the extreme C‐terminus of the AtCINV1 protein is a substrate of calcium‐dependent kinases (CPK3 and 21) and that phosphorylation creates a high‐affinity binding site for 14‐3‐3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14‐3‐3 proteins enhances its activity. The analysis of three quadruple 14‐3‐3 mutants generated from six T‐DNA insertion mutants of the non‐epsilon family shows both specificity as well as redundancy for this function of 14‐3‐3 proteins. The strong reduction in hexose levels in the roots of one 14‐3‐3 quadruple mutant plant is in line with the activating function of 14‐3‐3 proteins. The physiological relevance of this mechanism that affects A/N‐invertase activity is underscored by the light‐induced activation and is another example of the central role of 14‐3‐3 proteins in mediating dark/light signaling. The nature of the light‐induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca⁺⁺ changes that activate calcium‐dependent kinases, await further study.     

MiR‐802 causes nephropathy by suppressing NF‐κB‐repressing factor in obese mice and human

Obesity is associated with significant microvascular complications including renal injuries and may induce end‐stage renal disease. Emerging studies have demonstrated microRNAs (miRNAs) are potential mediators in the pathophysiological process of nephropathy. The present study aimed to investigate the role of miR‐802 in obesity‐related nephropathy and potential molecular mechanisms. Through utilizing obese mouse model and human subjects, we explored the therapeutic benefits and clinical application of miR‐802 in protecting against nephropathy. Renal miR‐802 level was positively correlated with functional parameters, including blood urea nitrogen and creatinine in obese mice. Specific silencing of renal miR‐802 improved high fat diet (HFD)‐induced renal dysfunction, structural disorders and fibrosis. The up‐regulated inflammatory response and infiltrated macrophages were also significantly decreased in miR‐802 inhibitor‐treated obese mice. Mechanistically, miR‐802 directly bond to 3?‐UTR of NF‐κB‐repressing factor (NRF) and suppressed its expression. In clinical study, the circulating miR‐802 level was significantly increased in obese subjects, and positively correlated with plasma creatinine level but negatively correlated with creatinine clearance. Taken together, our findings provided evidence that miR‐802/NRF signalling was an important pathway in mediating obesity‐related nephropathy. It is a possible useful clinical approach of treating miR‐802 inhibitor to combat nephropathy.     

Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β‐catenin activity

Salidroside is a major phenylethanoid glycoside in Rhodiola rosea L., a traditional Chinese medicine, with multiple biological activities. It has been shown that salidroside possesses protective effects for alleviating diabetic renal dysfunction, contrast‐induced‐nephropathy and other kidney diseases. However, the involved molecular mechanism was still not understood well. Herein, we examined the protective effects of salidroside in mice with Adriamycin (ADR)‐induced nephropathy and the underlying molecular mechanism. The results showed that salidroside treatment ameliorates proteinuria; improves expressions of nephrin and podocin; and reduces kidney fibrosis and glomerulosclerosis induced by ADR. Mechanistically, ADR induces a robust accumulation of β‐catenin in the nucleus and stimulates its downstream target gene expression. The application of salidroside largely abolishes the nuclear translocation of β‐catenin and thus inhibits its activity. Furthermore, the activation of β‐catenin almost completely counteracts the protective roles of salidroside in ADR‐injured podocytes. Taken together, our data indicate that salidroside ameliorates proteinuria, renal fibrosis and podocyte injury in ADR nephropathy, which may rely on inhibition of β‐catenin signalling pathway.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Calycosin alleviates allergic contact dermatitis by repairing epithelial tight junctions via down‐regulating HIF‐1α

Calycosin, a bioactive component derived from Astragali Radix (AR; Huang Qi), has been shown to have an effect of anti‐allergic dermatitis with unknown mechanism. This study aims to investigate the mechanism of calycosin related to tight junctions (TJs) and HIF‐1α both in FITC‐induced mice allergic contact dermatitis and in IL‐1β stimulated HaCaT keratinocytes. Th2 cytokines (IL‐4, IL‐5 and IL‐13) were detected by ELISA. The epithelial TJ proteins (occludin, CLDN1 and ZO‐1), initiative key cytokines (TSLP and IL‐33) and HIF‐1α were assessed by Western blot, real‐time PCR, immunohistochemistry or immunofluorescence. Herein, we have demonstrated that allergic inflammation and the Th2 cytokines in ACD mice were reduced significantly by calycosin treatment. Meanwhile, calycosin obviously decreased the expression of HIF‐1α and repaired TJs both in vivo and in vitro. In HaCaT keratinocytes, we noted that IL‐1β induced the deterioration of TJs, as well as the increased levels of TSLP and IL‐33, which could be reversed by silencing HIF‐1α. In addition, administration of 2‐methoxyestradiolin (2‐ME), a HIF‐1α inhibitor,significantly repaired the TJs and alleviated the allergic inflammation in vivo. Furthermore, TJs were destroyed by DMOG or by overexpressing HIF‐1α in HaCaT keratinocytes, and simultaneously, calycosin down‐regulated the expression of HIF‐1α and repaired the TJs in this process. These results revealed that calycosin may act as a potential anti‐allergy and barrier‐repair agent via regulating HIF‐1α in AD and suggested that HIF‐1α and TJs might be possible therapy targets for allergic dermatitis.     

Recognition of an intra‐chain tandem 14‐3‐3 binding site within PKCε

The phosphoserine/threonine binding protein 14‐3‐3 stimulates the catalytic activity of protein kinase C‐ε (PKCε) by engaging two tandem phosphoserine‐containing motifs located between the PKCε regulatory and catalytic domains (V3 region). Interaction between 14‐3‐3 and this region of PKCε is essential for the completion of cytokinesis. Here, we report the crystal structure of 14‐3‐3ζ bound to a synthetic diphosphorylated PKCε V3 region revealing how a consensus 14‐3‐3 site and a divergent 14‐3‐3 site cooperate to bind to 14‐3‐3 and so activate PKCε. Thermodynamic data show a markedly enhanced binding affinity for two‐site phosphopeptides over single‐site 14‐3‐3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14‐3‐3 ligand. This dual‐site intra‐chain recognition has implications for other 14‐3‐3 targets, which seem to have only a single 14‐3‐3 motif, as other lower affinity and cryptic 14‐3‐3 gatekeeper sites might exist.     

IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14‐3‐3 PROTEINS IN THE MOSQUITO Aedes aegypti,AN IMPORTANT ARBOVIRUSES VECTOR

The 14‐3‐3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14‐3‐3 proteins are scaffold molecules that form homo‐ or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation‐dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14‐3‐3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix‐forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to ? and ζ isoforms (Aeae14‐3‐3ε and Aeae14‐3‐3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14‐3‐3ζ except in the midgut and ovaries of adult females. The 14‐3‐3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.     

New Alkaloids and α‐Glucosidase Inhibitory Flavonoids from Ficus hispida

Two new pyrrolidine alkaloids, ficushispimines A ( 1 ) and B ( 2 ), a new ω‐(dimethylamino)caprophenone alkaloid, ficushispimine C ( 3 ), and a new indolizidine alkaloid, ficushispidine ( 4 ), together with the known alkaloid 5 and 11 known isoprenylated flavonoids 6  –  16 , were isolated from the twigs of Ficus hispida. Their structures were elucidated by spectroscopic methods. Isoderrone ( 8 ), 3′‐(3‐methylbut‐2‐en‐1‐yl)biochanin A ( 11 ), myrsininone A ( 12 ), ficusin A ( 13 ), and 4′,5,7‐trihydroxy‐6‐[(1R*,6R*)‐3‐methyl‐6‐(1‐methylethenyl)cyclohex‐2‐en‐1‐yl]isoflavone ( 14 ) showed inhibitory effects on α‐glucosidase in vitro.     

Circ‐AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR‐296‐3p/E‐cadherin signals

Diabetic nephropathy is a leading cause of end‐stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ‐AKT3/miR‐296‐3p/E‐cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ‐AKT3 and fibrosis‐associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT‐PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40‐MES13 cells. Luciferase reporter assays were used to investigate interactions among E‐cadherin, circ‐AKT3 and miR‐296‐3p in mouse mesangial SV40‐MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ‐AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40‐MES13 cells treated with high‐concentration (25 mmol/L) glucose. In addition, circ‐AKT3 overexpression inhibited the level of fibrosis‐associated protein, such as fibronectin, collagen type I and collagen type IV. Circ‐AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40‐MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ‐AKT3 could act as a sponge of miR‐296‐3p and E‐cadherin was the miR‐296‐3p direct target. Moreover, circ‐AKT3/miR‐296‐3p/E‐cadherin modulated the extracellular matrix of mouse mesangial cells in high‐concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ‐AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR‐296‐3p/E‐cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.