Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.

Smad7 is a critical negative regulator of TGF-beta/Smad2/3 and NF-kappaB signalling and plays a negative regulating role in both renal fibrosis and inflammation after UUO. Results from this study further support the notion that Smad7 may be a therapeutic agent for kidney diseases.

Despite the critical role that TGF-␤ plays in renal fibrosis, transgenic mice that overexpress human latent TGF-␤1 in the skin exhibit normal renal histology and function even though circulating levels of latent TGF-␤1 are an order of magnitude higher than wild-type animals. In fact, latent TGF-␤1 seems to protect against renal inflammation in a model of ureteral obstruction. It is unknown, however, whether latent TGF-␤1 also has this effect in immunologically mediated forms of renal disease such as anti-GBM crescentic glomerulonephritis. We induced anti-GBM disease in wild-type and transgenic mice overexpressing latent TGF-␤1 in keratinocytes. After 14 days, wild-type mice developed progressive crescentic glomerulonephritis with severe renal inflammation and fibrosis. In transgenic mice, proteinuria was reduced by 50%, renal function was preserved, and the formation of glomerular crescents was suppressed by 70%. In addition, transgenic animals had reduced renal inflammation, evidenced by a 70% decrease in the accumulation of T cells and macrophages, and reduced expression of renal IL-1␤, TNF␣, and MCP-1 by 70 to 80%. Progressive renal fibrosis was also prevented in the transgenic mice, and these protective effects were associated with elevated levels of latent, but not active, TGF-␤1 in plasma and renal tissue. Renal Smad7 was up-regulated and both NF-B and TGF-␤/Smad2/3 activation were suppressed. In conclusion, mice overexpressing latent TGF-␤1 in the skin were protected against anti-GBM crescentic glomerulonephritis, possibly via Smad 7-mediated inhibition of NF-B-dependent renal inflammation and TGF-␤/Smad2/3-dependent fibrosis.

Ingestion of aristolochic acid (AA) can cause AA nephropathy (AAN), in which excessive death of tubular epithelial cells (TECs) characterize the acute phase. AA forms adducts with DNA, which may lead to TEC apoptosis via p53-mediated signaling. We tested this hypothesis both by studying p53-deficient mice and by blocking p53 in TECs with its inhibitor pifithrin-␣. AA induced acute AAN in wild-type mice, resulting in massive apoptotic and necrotic TEC death and acute renal failure; p53 deficiency or pharmacologic inhibition attenuated this injury. In vitro, AA induced apoptotic and necrotic death of TEC in a time-and dosage-dependent manner, with apoptosis marked by a 10-fold increase in cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling-positive/Annexin V-positive propidium iodide-negative TECs (all P Ͻ 0.001). AA induced dephosphorylation of STAT3 and the subsequent activation of p53 and TEC apoptosis. In contrast, overexpression of STAT3, p53 inhibition, or p53 knockdown with small interfering RNA all attenuated AA-induced TEC apoptosis. Taken together, these results suggest that AA induces TEC death via apoptosis by dephosphorylation of STAT3 and posttranslational activation of p53, supporting the hypothesis that p53 promotes renal injury in acute AAN.