miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: As microRNA-21 (miR-21) plays a pathological role in fibrosis, we hypothesised that it may be a therapeutic target for diabetic nephropathy. METHODS: Abundance of miR-21 was examined in diabetic kidneys from db/db mice. The therapeutic potential of miR-21 in diabetic kidney injury was examined in db/db mice by an ultrasound-microbubble-mediated miR-21 small hairpin RNA transfer. In addition, the role and mechanisms of miR-21 in diabetic renal injury were examined in vitro under diabetic conditions in rat mesangial and tubular epithelial cell lines by overexpressing or downregulating miR-21. RESULTS: In db/db mice, a mouse model of type 2 diabetes, renal miR-21 at age 20 weeks was increased twofold compared with db/m (+) mice at the same age, and this increase was associated with the development of microalbuminuria and renal fibrosis and inflammation. More importantly, gene transfer of miR-21 knockdown plasmids into the diabetic kidneys of db/db mice at age 10 weeks significantly ameliorated microalbuminuria and renal fibrosis and inflammation at age 20 weeks, revealing a therapeutic potential for diabetic nephropathy by targeting miR-21. Overexpression of miR-21 in kidney cells enhanced, but knockdown of miR-21 suppressed, high-glucose-induced production of fibrotic and inflammatory markers. Targeting Smad7 may be a mechanism by which miR-21 regulates renal injury because knockdown of renal miR-21 restored Smad7 levels and suppressed activation of the TGF-ß and NF-κB signalling pathways. CONCLUSIONS/INTERPRETATION: Inhibition of miR-21 might be an effective therapy for diabetic nephropathy.

Successful treatment of massive ascites with intraperitoneal administration of a steroid in a case of systemic lupus erythematosus.

Massive ascites is a rare manifestation of systemic lupus erythematosus (SLE) and has a poor response to glucocorticoid therapy, probably because of impaired vascular circulation due to persistent peritoneal inflammation. We describe a young woman presenting with massive painless ascites as the predominant manifestation of SLE. Peritoneal effusion was resistant to the oral administration of steroids and the conventional therapies for ascites. Intraperitoneal injection of triamcinolone, an insoluble glucocorticoid, induced dramatic remission of massive ascites, with no adverse event or recurrence.

Natural history and impact on outcomes of acute kidney injury in patients with road traffic injury.

INTRODUCTION: The incidence and outcomes of posttraumatic acute kidney injury (AKI) have not been well-established because of the alterations in the definition used to characterize renal dysfunction. The natural history of AKI after road traffic injury (RTI) has not been studied. MATERIALS AND METHODS: We conducted a retrospective analysis of a tertiary care medical center database, on 3,945 RTI patients admitted between 2002 and 2006. RESULTS: AKI as defined by RIFLE criteria developed in 423 (10.7%) RTI patients, with maximum RIFLE class risk, injury and failure in 43.0%, 28.6%, and 28.4% respectively. A total of 59 patients (13.9% of AKI cohort) required renal replacement therapy and 77.5% of patients surviving AKI had complete renal recovery before discharge. Infusing vasopressors >= 4 h, using high-dose diuretics, and delayed transport time were identified as the independent risk factors for occurrence of AKI. Patients with maximum RIFLE class risk, injury and failure had hospital mortality rates of 37.4, 52.9 and 79.2%, respectively, compared with 7.1% for patients without AKI. RIFLE classification was also associated with the probability of making a complete renal recovery. CONCLUSIONS: Development of AKI in RTI patients represents a substantial risk for mortality in this population. Shortening the transport time and appropriate early intervention may reduce the risk of AKI. RIFLE provides a well-balanced classification system for determining AKI and predicting its outcome in this population.

Induction treatment of proliferative lupus nephritis with leflunomide combined with prednisone: a prospective multi-centre observational study.

To evaluate the efficacy and safety of leflunomide in the treatment of proliferative lupus nephritis, a prospective multi-centre observational study was conducted. Patients with biopsy proven proliferative lupus nephritis were assigned to receive either leflunomide or cyclophosphamide with concomitant prednisone. Leflunomide was given orally with a loading dose of 1 mg/kg/day for 3 days followed by 30 mg/day. Intravenous cyclophosphamide was administered monthly at a dose of 0.5 g/m2 of body-surface area. A total of 110 patients were enrolled, 70 in the leflunomide group and 40 in the cyclophosphamide group. The complete remission rate in the leflunomide group was 21% and partial remission rate 52%, as compared with 18% and 55%, respectively, in the cyclophosphamide group. Renal parameters and systemic lupus erythematosus disease activity index improved significantly and similarly in both groups. Serum creatinine decreased or stabilized in both treatment groups. No significant difference was noted with respect to clinical outcome between groups. Repeat biopsy also showed a significant reduction of active lesions in kidney pathology after 6 months of leflunomide treatment. Major adverse events, similar in both treatment groups, included infection, alopecia and hypertension. Leflunomide, compared with cyclophosphamide, in combination with prednisone was effective in the induction therapy of proliferative lupus nephritis and was generally well-tolerated.

Advanced glycation end products upregulate C-reactive protein synthesis by human hepatocytes through stimulation of monocyte IL-6 and IL-1 beta production.

Patients with chronic renal failure are characterized by increased plasma levels of C-reactive protein (CRP) and advanced glycation end products (AGE). AGE have been identified as a class of proinflammator mediators. To investigate whether AGE can stimulate hepatocytes to produce CRP, primary human fetal hepatocytes (HFH) were incubated with AGE-modified human serum albumin (AGE-HSA) or conditioned medium from AGE-HSA-stimulated monocytes (AGE-MCM). CRP concentrations in the supernatants were determined by an ELISA and CRP mRNA levels were determined by a quantitative RT-PCR. Exposure of HFH with AGE-HSA for 12-72 h did not change CRP concentrations in the supernatants. CRP protein and mRNA expression were significantly upregulated in a time- and dose-dependent manner when HFH were incubated with AGE-MCM. This stimulating effect was partially inhibited when AGE-MCM were preincubated with antibodies against interleukin-6 (anti-IL-6), interleukin-1 beta (anti-IL-1 beta), or soluble IL-1 receptor and was completely inhibited when AGE-MCM were preincubated with anti-IL-6 and anti-IL-1 beta simultaneously. The inhibiting effect did not occur when AGE-MCM was preincubated with antibody of tumour necrosis factor-alpha (anti-TNF-alpha) and soluble TNF receptor. Exposure of HFH with exogenous IL-6 and IL-1 beta, at the same concentrations as contained in AGE-MCM, also increased CRP production, but exogenous TNF-alpha had no effect. These results suggest that AGE cannot directly stimulate hepatocytes to produce CRP, but rather indirectly enhance CRP expression via stimulation of IL-6 and IL-1 beta production by human monocytes.

Restricted intake of dietary advanced glycation end products retards renal progression in the remnant kidney model.

Diet-derived advanced glycation end products (AGEs) contribute significantly to accumulation of AGEs in renal insufficiency. To test whether modulation of dietary AGEs would impact on progression of chronic renal disease, 5/6 nephrectomy rats were randomly placed on three diets that differed only in AGEs content (low AGEs diet (LAD), high AGEs diet (HAD), and standard rodent diet (SRD)) for 5-13 weeks. Compared with SRD- or HAD-fed rats, LAD-treated animals showed decreased proteinuria and retarded decline of creatinine clearance without alteration of blood pressure. Glomerular volume was reduced by 23% compared with HAD-fed rats at week 13 (P<0.001). Renal fibrosis progressed with time in the remnant kidneys from HAD-fed rats. However, LAD-fed animals presented a better-preserved structure of the kidneys. LAD-fed rats demonstrated significantly decreased serum and renal AGEs concentration (P<0.01 and P<0.01). This was associated with marked decrease of intrarenal advanced oxidation protein products and thiobarbituric acid reactive substances, as well as increase of glutathione peroxidase activity. LAD treatment also downregulated expression of monocyte chemoattractant protein-1 and transforming growth factor-1 and ameliorated macrophage infiltration in the remnant kidney. These results demonstrated that restriction of dietary AGEs intake retards progression of renal fibrosis and dysfunction in the remnant kidney model.

Pathogenesis of beta(2)-microglobulin amyloidosis: role of monocytes/macrophages.

beta(2)-microglobulin (beta(2)M) amyloidosis (A beta(2)M) is a serious, often incapacitating complication for patients undergoing long-term hemodialysis. Amyloid deposits composed of beta(2)M fibrils as the major constituent protein are mainly localized in joints and periarticular bone and lead to chronic arthralgias, carpal tunnel syndrome, and eventually destructive arthropathy. Although recent histologic studies have shown the accumulation of monocytes/macrophages around amyloid deposits, the factor(s) causing their infiltration and pathologic involvement have yet to be fully elucidated. Immunohistochemical staining reveals that macrophages in tenosynovial tissues express CD13, CD14, CD33, HLA-DR, and CD68 antigens on their surfaces and express interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6. Many of these cells also express LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and VLA-4 (CD49d/CD29) on their surfaces. AGE-modified beta(2)M enhances chemotaxis of monocytes and stimulates macrophages to release bone-resorbing cytokines, such as IL-1 beta, TNF-alpha and IL-6. Via a RAGE-mediated pathway, AGE-modified, but not unmodified beta(2)M, significantly delays constitutive apoptosis of human peripheral blood monocytes. Monocytes survival in an advanced glycation end product (AGE) beta(2)M-containing microenvironment is associated with their phenotypic alteration into macrophage-like cells that generate more reactive oxygen species and elaborate greater quantities of IL-1 beta and TNF-alpha. Thus through regulation of their survival and differentiation, AGE beta(2)M in amyloid deposits may be able to influence the presence and quantity of infiltrated monocytes, and hence their biologic effects.

beta(2)-Microglobulin modified with advanced glycation end products delays monocyte apoptosis.

BACKGROUND: A local inflammatory reaction to beta(2)-microglobulin (beta(2)m) amyloid deposits by monocytes/macrophages is a characteristic histologic feature of dialysis-related amyloidosis (DRA). Since beta(2)m modified with advanced glycation end products (AGE-beta(2)m) is a major constituent of amyloid in DRA, we tested the hypothesis that AGE-beta(2)m affects apoptosis and phenotype of human monocytes. METHODS: Human peripheral blood monocytes were incubated with or without in vitro-derived AGE-beta(2)m, and their viability, extent of apoptosis, morphology, and function examined over the subsequent four days. RESULTS: AGE-modified but not unmodified beta(2)m significantly delayed spontaneous apoptosis of human peripheral blood monocytes in adherent and nonadherent cultures. The effect of AGE-beta(2)m on monocytes apoptosis was time- and dose-dependent and was attenuated by a blocking antibody directed against the human AGE receptor (RAGE). There was no difference in effect between AGE-beta(2)m and that of AGE-modified human serum albumin. Culture of monocytes with AGE-beta(2)m did not alter membrane expression of Fas or Fas ligand. Monocytes cultured with AGE-beta(2)m underwent substantial changes in morphology similar to those observed when monocytes differentiate into macrophages. The cultured cells increased in size and vacuolization, and their content of beta-glucuronidase and acid phosphatase increased by 5- to 10-fold at day 4. Expression of the monocyte--macrophage membrane antigens HLA-DR, CD11b, and CD11c also increased at day 4. Although exhibiting phenotypic characteristics of macrophages, monocytes cultured with AGE-beta(2)m functioned differently than macrophages cultured with serum. Superoxide production in response to phorbol myristic acetate was maintained in monocytes cultured with AGE-beta(2)m, but declined with time in cells cultured with serum. Constitutive synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2) increased in monocytes cultured for four to six days with AGE-beta(2)m. CONCLUSIONS: These findings support a novel role for AGE-modified proteins such as AGE-beta(2)m that may contribute to the development of a local inflammatory response, with predominant accumulation of monocytes/macrophages, in DRA.

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures.

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.

Beta 2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts.

Beta 2-microglobulin amyloidosis (A beta 2m) is a serious complication for patients undergoing long-term dialysis. beta 2-microglobulin modified with advanced glycation end products (beta 2m-AGE) is a major component of the amyloid in A beta 2m. It is not completely understood whether beta 2m-AGE plays an active role in the pathogenesis of A beta 2m, or if its presence is a secondary event of the disease. beta 2-microglobulin amyloid is mainly located in tendon and osteo-articular structures that are rich in collagen, and local fibroblasts constitute the principal cell population in the synthesis and metabolism of collagen. Recent identification of AGE binding proteins on human fibroblasts lead to the hypothesis that the fibroblast may be a target for the biological action of beta 2m-AGE. The present study demonstrated that two human fibroblast cell lines exhibited a decrease in procollagen type I mRNA and type I collagen synthesis after exposure to beta 2m-AGE for 72 hours. Similar results were observed using AGE-modified albumin. Antibody against the RAGE, the receptor for AGE, attenuated this decrease in synthesis, indicating that the response was partially mediated by RAGE. In addition, antibody against epidermal growth factor (EGF) attenuated the decrease in type I procollagen mRNA and type I collagen induced by beta 2m-AGE, suggesting that EGF acts as an intermediate factor. These findings support the hypothesis that beta 2m-AGE actively participates in connective tissue and bone remodeling via a pathway involving fibroblast RAGE, and at least one interposed mediator, the growth factor EGF.

Aminoguanidine inhibits advanced glycation end products formation on beta2-microglobulin.

Because advanced glycation end products (AGE)-modified beta2-microglobulin (AGE-beta2M) is a dominant constituent of amyloid in dialysis-related amyloidosis (DRA), AGE-beta2M may be directly involved in the pathobiology of DRA. In experimental diabetes mellitus, blocking the formation of AGE prevents AGE-mediated tissue damage. In this study, it is postulated that similar pharmacologic intervention may be beneficial in DRA. Aminoguanidine, a nucleophilic hydrazine compound that prevents AGE formation on collagen, may have a similar effect on the advanced glycation of beta2M. To test this hypothesis, beta2M was incubated in vitro with 50 or 100 mM D-glucose for 3 wk in the presence and absence of incremental concentrations of aminoguanidine. On the basis of enzyme-linked immunosorbent assay and immunoblots using anti-AGE-keyhole limpet hemocyanin antibody, aminoguanidine inhibited glucose-induced N(epsilon)-(carboxymethyl)lysine formation on beta2M. At aminoguanidine-glucose molar ratios of 1:8 to 1:1, 26 to 53% inhibition occurred. Fluorospectrometry examination showed that aminoguanidine also inhibited the formation of fluorescent AGE on beta2M in a dose-dependent manner. At aminoguanidine-glucose molar ratios of 1:8 to 1:1, fluorescent product generation was inhibited by 30 to 70%. Furthermore, aminoguanidine suppressed the AGE formation on beta2M bound to AGE-modified collagen. If aminoguanidine is similarly active in vivo, this compound may be of clinical utility for treating DRA in patients on maintenance dialysis.

Interaction between beta 2-microglobulin and advanced glycation end products in the development of dialysis related-amyloidosis.

Dialysis related amyloidosis (DRA) is a progressive debilitating complication of long-term dialysis. beta 2-microglobulin (beta 2m) amyloid deposition occurs preferentially in older patients and initially is located in collagen-rich osteo-articular tissues. Since an age-dependent increase in the formation of advanced glycation end products (AGE) has been observed in collagen-containing structures, we hypothesized that AGE-modified beta 2m in the amyloid of DRA may be formed locally in osteo-articular structures as a subsequent event of its binding to collagen-AGE. Based on this hypothesis, we investigated the binding between beta 2m and AGE-modified collagen (collagen-AGE) in vitro. Significantly larger amounts of human beta 2m were bound to types I to IV of immobilized collagen-AGE than to unmodified collagens (P < 0.0001). The quantity of beta 2m bound to collagen-AGE was dependent on the concentrations of both beta 2m and of AGE contained in collagen (P < 0.01). Unmodified beta 2m was more avidly bound to collagen-AGE or collagen in comparison to AGE-modified beta 2m (P < 0.0001). beta 2m bound to collagen-AGE could be modified further by nonenzymatic glycosylation during three weeks of incubation with physiologic concentrations of glucose. Similar processes in vivo may be important in the pathobiology of DRA.

[Is the renal dopamine involved in the sodium retention in the nephrotic syndrome?].

Urinary dopamine (DA) and sodium excretion in patients with nephrotic syndrome (NS) were studied under various sodium loading in metabolic ward. Twenty patients and 10 age-matched normal volunteers were enrolled in this study. When they were on a low-salt diet (34 mmol/d), urinary excretion of DA and sodium in patients with heavy edema were much lower than that in normal controls, while in patients with mild or without edema, urine DA and sodium excretion did not decrease significantly, but were not mobilized on sodium loading (170 mmol/d), and the plasma renin activity and aldosterone were not completely suppressed as well. The decrement of urine DA excretion was independent of Ccr or the severity of renal tubule lesions, but was associated with the severity of proteinuria. When the proteinuria reduced, urine DA and sodium excretion increased. From the above observations, we might assume that the abnormal retention of sodium and water in NS was due partly to a failure to mobilize DA in the kidney and the change of the physical environment in renal tubule caused by heavy proteinuria was responsible for it.

[Behavior of beta 2-microglobulin in patients undergoing blood purification].

The Behavior of beta 2-microglobulin (beta 2m) in various modes of blood purification was studied. Serum beta 2m concentration was 26.1 +/- 9.9 mg/L in 39 patients on maintained hemodialysis (HD) and 20.6 +/- 5.3 mg/L in 26 patients on CAPD. In both groups, beta 2m was significantly higher than normal (P less than 0.01) and it was positively correlated with the duration of dialysis. However, patients surviving more than three years would show lower beta 2m in the CAPD group than those in the HD group. The mean value of beta 2m clearance was 108 mg during hemofiltration (HF) with polyamide membrane and 38.5 +/- 14.4 mg daily in CAPD. Not only very little. beta 2m could be removed on HD but a rise of beta 2m was observed after the procedure, even if it was corrected for hemoconcentration. A negative correlation was found between delta beta 2m and delta osmolality after HD (r = 0.58, P less than 0.05), but delta beta 2m would be decreasing if a cuprophan membrane dialyzer was reused for HD, and the result was the same if using the same membrane in isolated ultrafiltration (P less than 0.01 in all). These results suggested that the rise of beta 2m during HD with cellulose membrane may be due to increased release of beta 2m owing to the poor biocompatibility of the membrane as well as the generation of beta 2m on account of reduced plasma osmolality.

Fibronectin in patients with chronic renal failure undergoing dialysis.

Fibronectin (FN) levels were determined in 64 cases with chronic renal failure (CRF), some of whom were undergoing dialysis. FN levels were 14.9 +/- 7.6 mg/dl in CRF (n = 20), 13.4 +/- 4.3 mg/dl in patients on continuous ambulatory peritoneal dialysis (CAPD) (n = 20) and 16.7 +/- 7.2 mg/dl in patients on hemodialysis (HD) (n = 24). All the levels were significantly lower than in normal subjects (23.1 +/- 4.6 mg/dl). Serum FN was compared with some nutritional indices. Positive correlations were found between serum FN and nitrogen balance (BN), serum prealbumin (PreA) and transferrin (Tf) in all the patients. With serum albumin (Alb), however, this correlation was only found in patients undergoing dialysis. Negative correlations were found between serum FN and the ratio of serum urea to serum creatinine (Surea/Scr) in CAPD and HD patients. In 10 CAPD patients, the low serum FN levels went up after increased protein intake. This indicates that it was the result of malnutrition due to decreased protein intake. Serum FN level reflects a negative BN earlier and better than serum PreA, Tf and Alb. It is a sensitive, reliable and simple index for judging the nutritional protein status and the effect of nutritional treatment in patients with CRF undergoing dialysis.