Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

Glucocorticoids inhibit nontypeable Haemophilus influenzae-induced MUC5AC mucin expression via MAPK phosphatase-1-dependent inhibition of p38 MAPK.

Glucocorticoids are highly effective in the control of many inflammatory and immune diseases. Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, the molecular basis for the inhibitory effect of glucocorticoids on mucin overproduction, a hallmark of chronic respiratory diseases, still remains unclear. Here we show that glucocorticoids markedly inhibit up-regulation of MUC5AC induced by NTHi, a major human bacterial pathogen causing chronic obstructive pulmonary disease and otitis media. Inhibition of NTHi-induced MUC5AC expression by dexamethasone occurs at the level of p38 MAPK via glucocorticoid receptor. Moreover, glucocorticoids up-regulate MKP-1 expression, which in turn leads to p38 dephosphorylation and the subsequent inhibition of NTHi-induced MUC5AC expression. These studies provide new insight into the molecular mechanism underlying glucocorticoid therapy and may lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with NTHi infections.

Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzae-induced expression of toll-like receptor 2.

Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. We have demonstrated recently that glucocorticoids synergistically enhance nontypeable Haemophilus influenzae (NTHi)-induced expression of Toll-like receptor 2 (TLR2), an important TLR family member that has been shown to play a critical role in host immune and defense response. However, the molecular mechanisms underlying the glucocorticoid-mediated enhancement of TLR2 induction still remain unknown. Here we show that glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAPK phosphatase-1 (MKP-1) that, in turn, leads to dephosphorylation and inactivation of p38 MAPK, the negative regulator for TLR2 expression. Moreover, increased expression of TLR2 in epithelial cells greatly enhances the NTHi-induced expression of several key cytokines, including tumor necrosis factor-alpha and interleukins 1beta and 8, thereby contributing significantly to host immune and defense response. These studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPKs.

Amyloid formation in rat transthyretin: effect of oxidative stress.

BACKGROUND: Transgenic mice carrying a human mutant transthyretin (TTR) gene are too small for in vivo experiments. It is necessary to have rat TTR protein and its antibody to overcome this problem. METHODS: Posttranslational modification of purified TTR was analyzed by means of matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). Production of amyloid fibrils in vitro was confirmed by thioflavin T test and electron microscopy. Amyloidogenicity of rat TTR from rats with or without challenging paraquat was compared in vitro by thioflavin T test. RESULTS: MALDI/TOF-MS for rat TTR revealed three major modified forms-sulfate-conjugated, Cys-conjugated and glutathione-conjugated-in addition to the unconjugated (free) form of TTR. Although rat TTR in buffer of pH 7.0 could not make amyloid fibrils, rat TTR at pH 2.0-3.5 significantly formed amyloid fibrils, as confirmed by the thioflavin T test and electron microscopy. TTR purified from rats administered 4 mg/kg of paraquat formed much more amyloid fibrils than that from normal rats at pH 2.0-3.5 and significant amyloid fibrils were confirmed even at pH 7.0. CONCLUSIONS: Rat TTR may be a valuable experimental tool for examination of the amyloidogenicity of senile systemic amyloidosis (SSA) as well as familial amyloidotic polyneuropathy (FAP) both in vitro and in vivo.

Glucocorticoids synergistically enhance nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via a negative cross-talk with p38 MAP kinase.

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be up-regulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeable Hemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKKbeta-IkappaBalpha-dependent NF-kappaB pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38alpha/beta pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.