Jin Shuen Chen

Aberrant cytokines/chemokines production correlate with proteinuria in patients with overt diabetic nephropathy

BACKGROUND Diabetic nephropathy (DN) occurs in 20% to 30% of all patients with type 2 diabetes mellitus (DM) and is the most common cause of end-stage renal disease. However, the definite pathogenesis, especially the role of immune response, is still unclear. METHODS We studied the production and expression of Th1 (IFN-gamma, IL-2R), Th2 (IL-4, IL-10), proinflammatory cytokines (IL-1beta, and TNF-alpha), and chemokines (MCP-1, and RANTES) in patients with DN. The correlation among cytokines, chemokines, and clinical parameters were examined. RESULTS A patient with DN presented with longer disease duration, heavy proteinuria, and impaired renal function. Our results demonstrated increased proinflammatory cytokines, Th1 cytokines and chemokines, but not Th2 cytokines, in the plasma and urine of patients with DN as compared to patients with DM without overt nephropathy. Enhanced cytokine/chemokine activation in DN was also demonstrated by positive immunohistochemical staining of kidney tissue. We found a positive correlation between daily protein loss and plasma IFN-gamma and IL-2R, and urinary MCP-1, as well as a negative correlation between creatinine clearance and plasma TNF-alpha and urinary MCP-1. CONCLUSIONS There were aberrant cytokines/chemokines production correlated with the degree of proteinuria in patient with overt DN and gross proteinuria. Inflammation may be important in the pathogenesis of DN.

HO-1 induction ameliorates experimental murine membranous nephropathy: anti-oxidative, anti-apoptotic and immunomodulatory effects

BACKGROUND Therapeutic agents for membranous nephropathy (MN) remain ill-defined. Haeme oxygenase (HO)-1 is considered to play a protective role in various disorders. Here, we assessed the efficacy of HO-1 induction therapy for MN. METHODS MN was induced in BALB/c mice with intravenous injections of cationic bovine serum albumin. Three groups of mice were administered 100 micromol/kg Cobalt protoporphyrin (CoPP, a potent HO-1 inducer), Tin protoporphyrin (SnPP, a potent HO-1 inhibitor) or phosphate-buffered saline via intra-peritoneal injections once a week starting from the induction of MN. Disease severity was verified by serum and urine metabolic profiles and by renal histopathology. Cytokine profiles, immunoglobulin production, the expression of oxidative stress markers (thiobarbituric acid reactive substances, TBARS) and apoptosis, as measured by TUNEL, were also determined. RESULTS Mice treated with CoPP displayed a significant reduction in proteinuria and a marked amelioration of glomerular lesions, accompanied by attenuated immune-complex deposition. The production of immunoglobulins in MN mice treated with CoPP was significantly reduced compared with that of mice in the other two groups. TBARS in the serum and kidneys, as well as apoptosis, were also significantly reduced in CoPP-treated mice. Cytokine mRNA expression in the renal cortex indicated that CoPP not only decreased the expression of proinflammatory cytokines, but also increased the expression of anti-inflammatory cytokines (interleukin-10). CONCLUSIONS HO-1 induction therapy may ameliorate experimental MN via multiple pathways, including anti-oxidative, anti-apoptotic and immunomodulatory effects. HO-1 inducing regimens should be considered as a potential therapeutic intervention in MN in the future.

Rab 23 is expressed in the glomerulus and plays a role in the development of focal segmental glomerulosclerosis.

BACKGROUND Rab23, a member of the Rab family of small GTPase, has a function in antagonizing sonic hedgehog signal transduction. Both Rab-family and hedgehog-related proteins are involved in sclerosis and fibrosis in certain pathological states, but their roles in focal segmental glomerulosclerosis (FSGS) remain unclear. METHODS The FSGS model was established in Balb/c mice by a single injection of adriamycin. Serum, urine and mice kidneys were collected on Days 0, 7, 15 and 20. Western blot analysis was performed to detect the levels of Rab23 in the samples. Immunohistochemistry was used to examine the expressional profiles of Rab23 in kidneys. The expressions of transcripts of Rab23, extracellular matrix (ECM) proteins, and various hedgehog signalling pathway genes in kidneys or mesangial cells were evaluated by real-time RT-PCR. The effect of Rab23 on ECM protein expressions was evaluated by the knockdown or overexpression of Rab23 in mesangial cells. RESULTS Our results show that elevations of Rab23 were observed in the urine, but not in the serum, of the FSGS mice. Rab23 and hedgehog signalling pathway genes were constitutively expressed in normal kidneys and were significantly up-regulated in the kidneys of FSGS mice. The basal expression of Rab23 was identified in glomeruli, and mesangial cells displayed obvious elevation of Rab23 in the FSGS state. The knockdown or overexpression of Rab23 affected the collagen expression in cultured mesangial cells. CONCLUSIONS An autocrine loop of hedgehog signalling could be activated in mesangial cells in the FSGS state, and Rab23 may be elevated to suppress hedgehog signalling and/or influence collagen synthesis. Importantly, Rab23 could serve as a biomarker that indicates the severity of FSGS.

Experimental model of membranous nephropathy in mice : sequence of histological and biochemical events

An experimental model of membranous nephropathy (MN) has not been established fully in mice. We characterized the time course of MN in a murine MN model induced by cationic bovine serum albumin (cBSA). Preimmunized mice received cBSA intravenously for six weeks to induce MN and were then sacrificed at different times. Metabolic profiles, renal histopathology, lymphocyte subsets, serum anti-cBSA immunoglobulins (Igs), antibody subclasses and circulating immune complexes (CIC) were evaluated to study the severity and mechanisms of disease initiation and progression. Clinical symptoms of overt proteinuria, hypoalbuminaemia and hypercholesterolaemia were observed from week 4, and typical histological findings of diffuse thickening of the glomerular basement membrane and subepithelial deposition were identified after week 6. Granular fluorescent staining for IgG and complement C3 were observed as early as week 4. Total splenocyte number increased, but the percentages of CD4+ and CD8+ cells did not change as the disease progressed. The predominant isotype of anti-cBSA Igs was IgG1, suggesting a T-helper 2 cell-prone immune response in the development of MN. The strong positive immunofluorescent staining of the immune complex concomitant with higher concentrations of Igs in serum but no significant change in CIC levels before week 4 suggest the involvement of in situ deposition of immune complex in the process of MN. This murine model resembles the clinical and pathological features of human MN and may provide a tool for investigating MN; this model may also have potential applications in gene-knockout or transgenic mouse technologies.

Attenuation of Mouse Mesangial Cell Contractility by High Glucose and Mannitol: Involvement of Protein Kinase C and Focal Adhesion Kinase

Hyperglycemia and mannitol activate protein kinase C (PKC) and induce mesangial cell hypocontractility that subsequently may modulate renal function. Since focal adhesion kinase (FAK) activation is known to be linked with PKC activity, FAK may also be involved in mesangial cell contraction. To facilitate our understanding of the PKC- and FAK-modulating mechanism, we developed an in vitro model of mouse mesangial cell hypocontractility induced by hyperglycemia or mannitol. Mouse mesangial cells (CRL-1927) were exposed to: normal D-glucose (group N), high D-glucose (group H), and control groups at the same osmolality as H plus L-glucose (group L) and mannitol (group M). Changes in the planar surface area of cells in response to 1 µM phorbol 12-myristate 13-acetate (PMA) were determined. Western blot analyses for PKC, phosphorylated (p)-PKC, tyrosine phosphorylation, FAK, and p-FAK were done on each of these four groups. The effects of mannitol in various doses on cell contraction and activation of PKC and FAK were also assayed. The planar surface areas of groups H and M both showed an attenuated change in response to PMA stimulation. Before PMA stimulation, the baseline PKC expression of groups H and M showed a higher expression of p-PKCα and δ than that seen in group N (p < 0.05). Results of tyrosine phosphorylation and immunoprecipitation showed that FAK may be involved in this contraction process. The total amount of FAK showed no significant difference among the four experimental groups; however, p-FAK was found to have significantly increased in group M (p < 0.05). The use of PKC and tyrosine kinase inhibitors reduced PMA-induced mesangial cell contraction in all four groups. Activation of PKCα, δ, and FAK with the resultant inhibition of mesangial cell contraction by mannitol was found to be dose-dependent. These results may provide a correlation between increased expression of several PKC isoforms and, in particular, increased phosphorylation levels of PKCα and δ and hypocontractility induced by high glucose and mannitol treatment. Furthermore, the mannitol-induced hypocontractility involving PKC and FAK occurred in a dose-dependent manner.

Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy

OBJECTIVES Soluble urokinase plasminogen activator receptor (suPAR), derived from membrane bound uPAR, is associated with inflammatory diseases. In the present study, we explored the expression of uPAR/suPAR in common biopsy-proven kidney diseases and the relationship between suPAR and staging of type 2 diabetic nephropathy (DN). DESIGN AND METHODS Serum samples for suPAR and renal tissues for uPAR staining were investigated in various common kidney diseases. The levels of serum suPAR were measured and adequate cut-off values of different stage of DN were calculated by receiver operating characteristic (ROC) curve. RESULTS In our results, the expression of uPAR on renal tissues was pronounced in the majority of kidney diseases. Comparing of expression of uPAR among different kidney diseases, it was strongest in minimal change disease (MCD) and weakest in chronic interstitial nephritis. Serum suPAR in most kidney diseases, except of MCD, was significantly elevated and was highest in DN. As for DN and suPAR, we found that suPAR progressively increased with staging of DN. Moreover, suPAR was linearly and negatively related to estimated glomerular filtration rate and positively related to the amount of proteinuria. By ROC curve, the cut-off values of suPAR in DN for assessing development increased with the progression of the disease. CONCLUSIONS We concluded that uPAR/suPAR is elevated in most kidney diseases and that suPAR is a useful biomarker for assessing stages of DN.

Resveratrol Ameliorates Renal Damage, Increases Expression of Heme Oxygenase-1, and Has Anti-Complement, Anti-Oxidative, and Anti-Apoptotic Effects in a Murine Model of Membranous Nephropathy

Background Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and a common cause of nephrotic syndrome in adults. There are limited available treatments for MN. We assessed the efficacy of resveratrol (RSV) therapy for treatment of MN in a murine model of this disease. Methods Murine MN was experimentally induced by daily subcutaneous administration of cationic bovine serum albumin, with phosphate-buffered saline used in control mice. MN mice were untreated or given RSV. Disease severity and pathogenesis was assessed by determination of metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, apoptosis, and production of heme oxygenase-1 (HO1). Results MN mice given RSV had significantly reduced proteinuria and a marked amelioration of glomerular lesions. RSV also significantly attenuated immunofluorescent staining of C3, although there were no changes of serum immunoglobulin levels or immunocomplex deposition in the kidneys. RSV treatment of MN mice also reduced the production of reactive oxygen species (ROS), reduced cell apoptosis, and upregulated heme oxygenase 1 (HO1). Inhibition of HO1 with tin protoporphyrin IX partially reversed the renoprotective effects of RSV. The HO1 induced by RSV maybe via Nrf2 signaling. Conclusion Our results show that RSV increased the expression of HO1 and ameliorated the effects of membranous nephropathy in a mouse model due to its anti-complement, anti-oxidative, and anti-apoptotic effects. RSV appears to have potential as a treatment for MN.

Effects of calcitriol on type 5b tartrate-resistant acid phosphatase and interleukin-6 in secondary hyperparathyroidism.

Background/Aims: Secondary hyperparathyroidism (SHP) is characterized by high bone turnover and elevated serum bone remodeling markers. Elevation of serum interleukin-6 (IL-6) levels is also characteristic of end-stage renal disease. This study investigates the effects of intravenous calcitriol on serum bone resorptive markers, namely, type 5b tartrate-resistant acid phosphatase (TRACP5b) and IL-6 in patients with SHP. Methods: Intravenous calcitriol therapy was given for 16 weeks to 24 patients on maintenance hemodialysis with plasma intact parathyroid hormone (iPTH) levels >300 pg/ml. Blood was drawn at baseline and every 4 weeks for 16 weeks for determination of the levels of biochemical parameters, iPTH, IL-6 and bone remodeling markers, including bone-specific alkaline phosphatase (bAP) and TRACP5b. Results: Only 21 patients responded to the calcitriol therapy, with significant decrements in serum iPTH after 4 weeks of therapy and thereafter. After 16 weeks of calcitriol therapy, 21 patients had significant decrements in serum iPTH (707.9 ± 317.8 vs. 205.0 ± 63.1 pg/ml, p < 0.01). Prior to treatment, a significant correlation was found between increased levels of serum iPTH and IL-6 levels (r = 0.45, p < 0.05). After treatment, there was also a significant and parallel lowering of levels of serum iPTH, IL-6 (8.52 ± 3.59 vs. 7.24 ± 2.81 pg/ml, p < 0.01), bAP (54.68 ± 36.17 vs. 24.55 ± 13.84 U/l, p < 0.01) and TRACP5b (3.41 ± 1.89 vs. 1.80 ± 0.55 U/l, p < 0.01). Our results additionally showed significant positive correlationsbetween baseline levels of serum IL-6 and those of iPTH, bAP and TRACP5b. After 16 weeks of calcitriol treatment, the correlation between IL-6 and iPTH levels lost significance but levels of serum IL-6, bAP and TRACP5b remained significantly correlated. Conclusions: Elevated levels of serum IL-6 and bone remodeling markers, namely, bAP and TRACP5b which are common features of SHP, are effectively suppressed by calcitriol therapy. This indicates that hyperparathyroidism not only accelerates bone remodeling but may also aggravate inflammation in patients on maintenance hemodialysis.

Ferulic Acid, an Angelica sinensis-Derived Polyphenol, Slows the Progression of Membranous Nephropathy in a Mouse Model.

Membranous nephropathy (MN) is a leading cause of adult nephrotic syndrome but lacks adequate treatment. Different extracts of Angelica sinensis (AS) and one of its active compounds, ferulic acid (FA), were used to evaluate the therapeutic effects in a MN mouse model. The MN model was grouped into three subgroups: no treatment (N-T), treatment at induction of MN (Pre-T), and treatment after full-blown MN (Post-T). The results showed that the methanol (ME) layer of AS extract exhibited a therapeutic effect on MN-induced proteinuria. The ME layer-enriched compound, FA, improved the hypoalbuminemia, hyperlipidemia, and proteinuria in both Pre-T and Post-T groups. Ferulic acid also reduced the formation of oxidative protein products and increased the synthesis of antioxidant enzymes in groups Pre-T and Post-T. Regarding angiogenesis factors, the antiangiogenic factors in renal glomeruli were increased in group N-T, but, after FA treatment, only one of the antiangiogenic factors, thrombospondin-1, showed a significant decrease. Furthermore, the expression of Th2 predominant showed significant decrease in both Pre-T and Post-T groups when compared to that of N-T group. In summary, FA retarded the progression of MN, and the mechanisms involved the regulation of oxidative stresses, angiogenic and antiangiogenic factors, and attenuation of Th2 response.

Pathogenic Role of Effector Cells and Immunoglobulins in Cationic Bovine Serum Albumin-Induced Membranous Nephropathy

Membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis. The roles of effector cells and immunoglobulins (Igs) in the mediation of glomerular injury in MN have not been fully elucidated. MN was induced by cationic bovine serum albumin (cBSA), and passive disease was induced by transferring effector cells or serum into severe combined immunodeficient (SCID) mice. MN could not be induced in SCID mice. Transfer of serum from MN mice, but not from normal control mice, to SCID mice induced granular immune complex deposits and pathologic proteinuria. Increased immunofluorescent staining for complement, oxidative stress, terminal deoxynucleotidyl transferase-mediated nick end-labeling assay-positive cells, and augmented phospho-NF-κB staining were evident in the kidneys of MN serum recipients. However, no histological or clinical manifestations were exhibited by SCID mice that received an adoptive transfer of splenocytes. Adaptive immunity was essential for the development of MN. Specific Igs and their subsequent response contribute to the development of renal injury in cBSA-induced MN.