SMPDL3b modulates insulin receptor signaling in diabetic kidney disease.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.

Adenosine A2 a receptor stimulation prevents proteinuria in diabetic rats by promoting an anti-inflammatory phenotype without affecting oxidative stress.

AIM: Diabetic patients are at increased risk for kidney disease. There is presently no clinical treatment available that effectively protects kidney function in diabetics. This study investigates whether chronic stimulation of the adenosine A2a receptor (A2a AR) protects kidney function in insulinopenic diabetic rats. METHODS: Streptozotocin-induced diabetic rats and corresponding controls were chronically treated with the adenosine A2a AR agonist CGS21680 throughout the four-week diabetes duration. Kidney function was thereafter investigated, and urine and plasma samples were collected for analysis of protein, oxidative stress and inflammatory markers. RESULTS: Glomerular filtration rate, renal blood flow, filtration fraction and diabetes-induced kidney hypoxia were all unaffected by chronic A2a AR stimulation. Furthermore, diabetic rats had increased oxidative stress, which was further increased by chronic A2a AR stimulation. However, the 10-fold increased urinary protein excretion observed in the diabetic rats was completely prevented by chronic A2a AR stimulation. These beneficial effects were accompanied by reduced levels of the pro-inflammatory TNF-α and increased levels of the anti-inflammatory IL-10 as well as decreased infiltration of macrophages, glomerular damage and basement membrane thickness. CONCLUSION: Chronic A2a AR stimulation prevents proteinuria and glomerular damage in experimental diabetes via an anti-inflammatory mechanism independent of oxidative stress and kidney hypoxia.

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression.

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Albumin induces endoplasmic reticulum stress and apoptosis in renal proximal tubular cells.

Chronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12.

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded vMIP-I and vMIP-II induce signal transduction and chemotaxis in monocytic cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)/ Human herpesvirus 8 encodes three chemokines, which are called viral macrophage inflammatory protein (vMIP)-I, -II, and -III. Here, we expressed the KSHV vMIP-I and vMIP-II proteins and analyzed their biological functions. Both vMIP-I and vMIP-II had an apparent molecular mass of 7.8 kDa and were localized to the cytoplasm in a body cavity-based lymphoma cell line BC-3, stimulated with phorbol ester. We next treated a human monocytic leukemia cell line, THP-1, with purified recombinant vMIP-I and vMIP-II, or vMIP-I and vMIP-II fused with alkaline phosphatase to study Ca(2+) signalling and in vitro chemotaxis in response to these proteins. Calcium mobilization was induced by both vMIP-I and vMIP-II. Furthermore, vMIP-I and vMIP-II induced Ca(2+) mobilization in K562 cells expressing the CC chemokine receptor 5 (CCR5), suggesting that both may be agonistic for CCR5. Additionally, vMIP-I induced Ca(2+) mobilization through the intermediary of CCR8. These viral MIPs were also capable of chemotactically activating the THP-1 cells. These results imply that vMIP-I and vMIP-II may play important roles in the propagation of KS and primary effusion lymphoma by inducing the chemotaxis of CCR5-expressing monocytes.

Glyoxalase I deficiency is associated with an unusual level of advanced glycation end products in a hemodialysis patient.

BACKGROUND: Advanced glycation of proteins and their attendant advanced glycation end products (AGEs) contribute to the complications associated with diabetes mellitus or uremia. Regulatory mechanisms of AGE formation in vivo remain an issue of particular interest. We investigated a role of the glyoxalase detoxification system of precursor reactive carbonyl compounds (RCOs) in the in vivo AGE formation. METHODS: Plasma levels of AGEs [pentosidine and Nepsilon-carboxymethyllysine (CML)], their RCO precursors, d-lactate (the final product resulting from the glyoxalase detoxification pathway), as well as of various compounds known to generate AGE precursors and surrogate markers for oxidative stress (antioxidant enzymes and glutathione), were measured in both hemodialysis (HD) patients and normal subjects. The activity and protein expression of glyoxalase I, an enzyme essential for the detoxification of alpha-oxoaldehydes, in red blood cells (RBC) were also examined. RESULTS: In one 69-year-old lady who had been on hemodialysis (HD) for three years and had suffered from recurrent cardiovascular complications despite the absence of significant risk factors, plasma levels of pentosidine (77.3 +/- 2.4 pmol/mg protein) and CML (330.8 +/- 8.2 pmol/mg protein) were markedly elevated as compared to other HD patients (N = 20: 26.6 +/- 11.8 pmol/mg protein for pentosidine and 224.4 +/- 51.7 pmol/mg protein for CML). The plasma level of RCO precursors for pentosidine and CML was also higher in this patient than in other HD patients. Further investigation disclosed a very low activity in RBC of glyoxalase I (1.5 +/- 0.4 mU/106 RBC), as compared to other HD patients (3.9 +/- 0.6 mU/106 RBC) or normal subjects (4.0 +/- 0.6 mU/106 RBC). The glyoxalase I protein level, assessed in RBC by immunoblot analysis with a specific antibody, was markedly lower than that observed in HD patients and normal subjects. The causes of this deficiency remain unknown. Nucleotide sequencing of the products of reverse transcription-polymerase chain reaction from the patient's mononuclear cells revealed no genetic mutation within the coding region of the glyoxalase I gene. Plasma d-lactate level was also in the lower range (0.18 +/- 0.03 mg/dL) of the values measured in the other HD patients (0.27 +/- 0.09 mg/dL) and normal subjects (0.35 +/- 0.12 mg/dL). The plasma levels of various compounds known to generate AGE precursors (glucose, lipids and ascorbic acid) were either normal or low. The surrogate markers for oxidative stress such as antioxidant enzymes (glutathione peroxidases and superoxide dismutase) and glutathione were all within the range observed in the other HD patients. CONCLUSION: The unusually high levels of AGEs in this patient implicate a deficient glyoxalase detoxification of RCO precursors. The present clinical observation implicates, to our knowledge for the first time, the glyoxalase detoxification system and, in particular, glyoxalase in the actual level of AGEs in a uremic patient.

Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy.

Mesangial cells play critical roles in maintaining a structure and function of the glomerulus. We previously cloned a novel mesangium-predominant gene, megsin, a new serine protease inhibitor. To clarify localization and roles of megsin protein, we raised polyclonal antibodies to megsin. By immunohistochemistry, megsin protein was specifically identified in the mesangial area. The amount of megsin protein was increased in glomeruli of patients with IgA nephropathy than in those of normal individuals and of patients with minimal change nephrotic syndrome or membranous nephropathy, suggesting a pathophysiological role of megsin as a functional modulator of mesangial functions in situ.

Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis.

BACKGROUND: We recently cloned a new human mesangium-predominant gene, megsin. Megsin is a novel member of the serine protease inhibitor (serpin) superfamily. To elucidate functional roles of this gene, we cloned megsin in rodents and investigated its role in a rat nephritis model. METHODS: Megsin homologues were cloned from cultured rat and mouse mesangial cDNAs utilizing polymerase chain reaction (PCR) with degenerative primers. Expression of megsin in three different types of resident glomerular cells was investigated by PCR. Levels of megsin mRNA expression at various time points in the anti-Thy1 rat nephritis model were studied by semiquantitative PCR and Northern blotting analysis. In order to investigate megsin protein expression in anti-Thy1 nephritis rats, we raised antibody against rat megsin-specific synthetic peptide, with which immunohistochemical studies were performed. RESULTS: Rat and mouse megsins were composed of 380 amino acids, which revealed 75.3 and 73.9% identity, respectively, with human megsin at the amino acid level. Characteristic features as an inhibitory serpin were conserved in both rat and megsin megsins. PCR analysis revealed expression of megsin in cultured mesangial cells but not in glomerular epithelial or endothelial cells. In anti-Thy1 nephritis rats, semiquantitative PCR and Northern blotting showed that expression of megsin mRNA was up-regulated in glomeruli at day 8. Immunohistochemical studies demonstrated the prominent accumulation of megsin in glomeruli at the same time point. Megsin was mainly localized in mesangial area. The megsin expression level returned to the basal level at day 28. CONCLUSION: Sequences of megsin were well conserved among different species. Rat megsin was also predominantly expressed in mesangial cells. Expression of megsin was up-regulated at the peak of hypercellularity and matrix accumulation in the mesangioproliferative glomerulonephritis model, suggesting that megsin may participate in the process of glomerulosclerosis by modulating extracellular matrix deposition or cell survival.

Glyoxal and methylglyoxal trigger distinct signals for map family kinases and caspase activation in human endothelial cells.

Carbonyl compounds with diverse carbon skeletons may be differentially related to the pathogenesis of vascular diseases. In this study, we compared intracellular signals delivered into cultured human umbilical vein endothelial cells (HUVECs) by glyoxal (GO) and methylglyoxal (MGO), which differ only by a methyl group. Depending on their concentrations, GO and MGO promoted phosphorylations of ERK1 and ERK2, which were blocked by the protein-tyrosine kinase (PTK) inhibitors herbimycin A and staurosporine, thereby being PTK-dependent. GO and MGO also induced phosphorylations of JNK, p38 MAPK, and c-Jun, either PTK-dependently (GO) or -independently (MGO). Next, we found that MGO, but not GO, induced degradation of poly(ADP-ribose) polymerase (PARP) as the intracellular substrate of caspase-3. Curcumin and SB203580, which inhibit JNK and p38 MAPK signaling pathways, but not herbimycin A/staurosporine, prevented the MGO-induced PARP degradation. We then found that MGO, but not GO, reduced the intracellular glutathione level, and that cysteine, but not cystine, inhibited the MGO-mediated activation of ERK, JNK, p38 MAPK, or c-Jun more extensively than did lysine or arginine. In addition, all the signals triggered by GO and MGO were blocked by amino guanidine (AG), which traps carbonyls. These results demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another for PTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.

Effect of dwell time on carbonyl stress using icodextrin and amino acid peritoneal dialysis fluids.

BACKGROUND: Deterioration of the peritoneal membrane limits the technical survival of peritoneal dialysis (PD). Advanced glycation of the membrane has been incriminated in this evolution. Advanced glycation end products (AGEs) develop under the influence of glucose and of its degradation products, mainly reactive carbonyl compounds (RCOs) such as glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG). The present study was undertaken to evaluate the impact of recently developed glucose-free PD fluids on AGE generation. METHODS: Recently developed glucose-free PD fluids containing either icodextrin or amino acids were investigated. GO, MGO, and 3-DG [high-performance liquid chromatography (HPLC)] and total RCOs (spectrophotometry) were measured in fresh solutions and in effluents after various dwell duration. The AGE formation potential of PD fluids and effluents was assessed by incubation at 37 degrees C, for one week, with bovine serum albumin and by the eventual measurement of pentosidine (HPLC) and Nepsilon-carboxymethyllysine (CML; gas chromatography/mass spectrometry). RESULTS: GO, MGO, and 3-DG (P < 0. 001) as well as total RCOs levels (P < 0.01) were significantly lower in icodextrin and amino acid PD fluid than in commercial, heat-sterilized, 1.36% glucose PD fluid. Pentosidine and CML generation were also significantly lower (P < 0.001) in icodextrin and amino acid PD fluid than in conventional 1.36% glucose PD fluid. The levels of total RCOs, however, increased in icodextrin and amino acid PD fluid effluents with dwell time. AGE formation potential rose accordingly, as demonstrated by a parallel increase in the generation of pentosidine and CML during incubation of PD effluents. CONCLUSION: The present data demonstrate lower RCO contents and AGE formation potential in fresh icodextrin and amino acid PD fluids than in fresh heat-sterilized glucose PD fluids. However, this difference decreases progressively during dwell time, mainly as a result of the influx of total RCOs.

Nitric oxide inhibits the formation of advanced glycation end products.

BACKGROUND: Advanced glycation end products (AGEs) are elevated in renal failure and have been implicated in the pathogenesis of several uremic complications. Their formation is closely associated with oxidative stress. The recent observation that nitric oxide (NO) has an antioxidant effect led us to examine the possible role of NO in the generation of AGEs. METHODS: We examined the effect of NO donors, 2, 2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the in vitro formation of pentosidine, which was used as a surrogate marker for AGEs. Bovine serum albumin was incubated under air at 37 degrees C in a medium containing either several AGE precursors or uremic plasma. To elucidate further the mechanism of the NO effect on AGE formation, we examined the generation of free radicals and carbonyls in pentose-driven pentosidine formation. RESULTS: NO donors significantly inhibit the formation of pentosidine in a dose-dependent manner. The effect is abolished by the addition of a NO scavenging agent, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). The inhibitory effect results from NO but not from the NO donor molecule. It is best explained by the ability of NO to scavenge carbon-centered radicals, hydroxyl radical, and carbonyl compounds. CONCLUSIONS: NO inhibits pentosidine formation by scavenging free radicals and by inhibiting carbonyl compound formation. NO might be implicated in the atherogenic and inflammatory effects of AGEs: Reduced NO production and increased oxidative stress associated with atherosclerotic lesions may accelerate AGE formation and, thus, exacerbate endothelial dysfunction and accelerate the development of atherosclerosis in uremia.

Human herpesvirus 8-encoded interleukin-6 homologue (viral IL-6) induces endogenous human IL-6 secretion.

We found that human herpesvirus 8-encoded IL-6 (vIL-6) induced endogenous human IL-6 (huIL-6) secretion from various cell lines (MT-4, THP-1, U937, Raji, and CESS) including patients with multicentric Castleman's disease. Especially, in MT-4 cells, huIL-6 was enhanced with vIL-6 by 30-fold compared with that of control. In addition, reverse transcriptase-polymerase chain reaction confirmed that vIL-6 induced huIL-6 expression in MT-4 cells. Our novel finding of the huIL-6 induction by vIL-6 indicates that vIL-6 may play a significant role in the pathogenesis of HHV-8 associated diseases.

Identification and analysis of the K5 gene of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes approximately 80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5' and 3' ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

Reactivation of human herpesvirus 6 by infection of human herpesvirus 7.

We have attempted to reactivate human herpesvirus 6 (HHV-6) by infection with HHV-7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV-6 and were infected by human herpesvirus 7 (HHV-7) in vitro. The antigen positive rate to HHV-6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV-6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock-infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV-7 is necessary to reactivate HHV-6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV-6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV-7 infection.

Expression of megsin mRNA, a novel mesangium-predominant gene, in the renal tissues of various glomerular diseases.

Mesangial cells play an important role in maintaining a structure and function of the glomerulus and in the pathogenesis of glomerular diseases. Recently, we discovered a new mesangium-predominant gene termed "megsin." Megsin is a novel protein that belongs to the serine protease inhibitor (serpin) superfamily. To elucidate the pathophysiologic role of megsin in the kidney, the expression and localization of megsin mRNA in renal tissues of patients with IgA nephropathy (IgA-N), diabetic nephropathy (DN), minimal change nephrotic syndrome (MCNS), membranous nephropathy (MN), and normal human kidney (NHK) was evaluated by in situ hybridization using digoxigenin-labeled oligonucleotide. Individual cells positive for megsin mRNA were observed only in glomeruli in all renal tissues. Their localization coincided with those of mesangial cells. The percentage of positive cells for megsin mRNA in total glomerular cells was significantly greater in IgA-N than in MCNS, MN, and NHK. It was also significantly greater in DN than in MCNS and NHK. In IgA-N, the percentage of megsin mRNA-positive cells was greater in tissues from those with mesangial cell proliferation and slightly mesangial matrix expansion (periodic acid-Schiff-positive area in the total glomerulus area, <30%; cell number in mesangial matrix area, >30; assessed in cross-sections through their vascular poles) than in tissues from those with severe mesangial matrix expansion (periodic acid-Schiff-positive area in total glomerulus area, >30%; cell number in mesangial matrix area, <30). In conclusion, megsin mRNA was predominantly expressed in glomerular mesangial cells in all renal tissues. The expression of megsin mRNA was upregulated in IgA-N and DN, both of which are diseases accompanied with mesangial cell proliferation and/or mesangial matrix expansion. These data suggest a link of megsin expression to the pathogenesis of IgA-N and DN, two major causes of end-stage renal failure.

Glucose degradation product methylglyoxal enhances the production of vascular endothelial growth factor in peritoneal cells: role in the functional and morphological alterations of peritoneal membranes in peritoneal dialysis.

Peritoneal membrane permeability deteriorates in peritoneal dialysis (PD) patients. We test whether glucose degradation products (GDPs) in PD fluids, glyoxal, methylglyoxal and 3-deoxyglucosone, stimulate the production of vascular endothelial growth factor (VEGF), a factor known to enhance vascular permeability and angiogenesis. VEGF increased in cultured rat mesothelial and human endothelial cells exposed to methylglyoxal, but not to glyoxal or 3-deoxyglucosone. VEGF also increased in peritoneal tissue of rats given intraperitoneally methylglyoxal. VEGF and carboxymethyllysine (CML) (formed from GDPs) co-localized immunohistochemically in mesothelial layer and vascular walls of the peritoneal membrane of patients given chronic PD. By contrast, in the peritoneum of non-uremic subjects, VEGF was identified only in vascular walls, in the absence of CML. VEGF production induced by GDPs may play a role in the progressive deterioration of the peritoneal membrane.

Oxidative protein damage with carbohydrates and lipids in uremia: 'Carbonyl stress'.

Chronic uremia appears to be in a state of an increased oxidative stress. Under oxidative stress, proteins are modified directly by reactive oxygen species with the eventual formation of oxidised amino acids. Proteins are also modified indirectly with reactive carbonyl compounds formed by the autoxidation of carbohydrates and lipids, with the eventual formation of the advanced glycation/lipoxidation end products (AGEs/ALEs). AGEs, pentosidine and carboxymethyllysine (CML), and ALE, malondialdehyde (MDA)-lysine, are elevated in plasma and matrix proteins of uremic patients several times above normal subjects. Precursor carbonyl compounds derived from carbohydrates and lipids are indeed elevated in uremic circulation. Uremia thus appears to be in a state of carbonyl overload with potentially damaging proteins (carbonyl stress). Carbonyl stress might be relevant to long-term complications associated with chronic renal failure and dialysis, such as dialysis-related amyloidosis and atherosclerosis. Immunohistochemical studies identified carbonyl stress in long-lived amyloid deposits and vascular lesions. Proteins modified under carbonyl stress exhibit several biological activities, which might, at least in part, account for the development of joint and vascular complications in uremia.

Increased carbonyl modification by lipids and carbohydrates in diabetic nephropathy.

BACKGROUND: In diabetic nephropathy (DN), possible mediators of untoward effects of hyperglycemia include the advanced glycation end products (AGEs). Indeed, an AGE, carboxymethyllysine (CML), accumulates in expanded mesangial matrix and nodular lesions. An advanced lipoxidation end product (ALE), malondialdehyde-lysine (MDA-lysine), generated on proteins during lipid peroxidation also accumulates in these lesions. As both ALEs and AGEs are formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of lipids and carbohydrates, their colocalization suggests an increased carbonyl modification of proteins. METHODS: To address this hypothesis, human diabetic renal tissues were examined to characterize carbonyl modification of proteins by lipids and carbohydrates: (a) ALEs, MDA-lysine and 4-hydroxynonenal (HNE) protein adduct, derived from lipids, and (b) AGEs, pentosidine and CML, derived from carbohydrates. Furthermore, to elucidate the biological effect of carbonyl modification on primary cultured human and rat mesangial cells, the intracellular protein phosphorylation was examined in the presence of various kinds of carbonyl compounds. RESULTS: The ALE and AGE adducts examined were identified in expanded mesangial matrix and nodular lesions. The exposure of cultured mesangial cells to carbonyl compounds resulted in phosphorylation of tyrosine residues of a number of intracellular proteins. CONCLUSIONS: These data suggest a broad derangement in nonenzymatic biochemistry involving both lipids and carbohydrates exists in diabetic glomerular lesions ("carbonyl stress").