Hemopexin accumulates in kidneys and worsens acute kidney injury by causing hemoglobin deposition and exacerbation of iron toxicity in proximal tubules.

Hemopexin, a heme scavenging protein, accumulates in the kidneys during acute kidney injury (AKI). However, the function of this accumulated hemopexin in the kidney is unclear. In both the cisplatin-induced and the unilateral kidney ischemia-reperfusion injury models of AKI, we found accumulation of hemoglobin and hemopexin in the kidneys localized to the proximal tubules. Next, hemopexin wild-type and knockout mice were compared in both AKI models and hemopexin wild type mice had significantly worse kidney injury. Furthermore, there was increased kidney expression of kidney injury molecule-1 (a biomarker of AKI) and heme oxygenase-1 (an indicator of oxidative stress) in hemopexin wild type compared with knockout mice in both models of AKI. Next, the interaction of hemopexin and hemoglobin in vitro was investigated using cultured proximal tubular cells. Co-incubation of hemopexin with hemoglobin resulted in hemoglobin deposition and exaggerated hemoglobin-induced injury. Deferoxamine, an iron chelator, and ferrostatin-1, a ferroptosis inhibitor, inhibited this deleterious effect of hemoglobin and hemopexin in proximal tubular cells, implicating iron toxicity in the mechanism of hemopexin mediated injury. Furthermore, the protective effect of deferoxamine in cisplatin-induced AKI was apparent in hemopexin wild type, but not in hemopexin knockout mice, further implicating hemopexin as a mediator of iron toxicity in AKI. Thus, our findings demonstrate that hemopexin accumulates in the kidneys and worsens kidney injury in AKI by increasing hemoglobin deposition on proximal tubular cells to exaggerate hemoglobin-induced cell injury.

A strategic expression method of miR-29b and its anti-fibrotic effect based on RNA-sequencing analysis.

Tissue fibrosis is a significant health issue associated with organ dysfunction and failure. Increased deposition of collagen and other extracellular matrix (ECM) proteins in the interstitial area is a major process in tissue fibrosis. The microRNA-29 (miR-29) family has been demonstrated as anti-fibrotic microRNAs. Our recent work showed that dysregulation of miR-29 contributes to the formation of cardiac fibrosis in animal models of uremic cardiomyopathy, whereas replenishing miR-29 attenuated cardiac fibrosis in these animals. However, excessive overexpression of miR-29 is a concern because microRNAs usually have multiple targets, which could result in unknown and unexpected side effect. In the current study, we constructed a novel Col1a1-miR-29b vector using collagen 1a1 (Col1a1) promoter, which can strategically express miR-29b-3p (miR-29b) in response to increased collagen synthesis and reach a dynamic balance between collagen and miR-29b. Our experimental results showed that in mouse embryonic fibroblasts (MEF cells) transfected with Col1a1-miR-29b vector, the miR-29b expression is about 1000 times less than that in cells transfected with CMV-miR-29b vector, which uses cytomegalovirus (CMV) as a promoter for miR-29b expression. Moreover, TGF-ß treatment increased the miR-29b expression by about 20 times in cells transfected with Col1a1-miR-29b, suggesting a dynamic response to fibrotic stimulation. Western blot using cell lysates and culture media demonstrated that transfection of Col1a1-miR-29b vector significantly reduced TGF-ß induced collagen synthesis and secretion, and the effect was as effective as the CMV-miR-29b vector. Using RNA-sequencing analysis, we found that 249 genes were significantly altered (180 upregulated and 69 downregulated, at least 2-fold change and adjusted p-value <0.05) after TGF-ß treatment in MEF cells transfected with empty vector. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using GAGE R-package showed that the top 5 upregulated pathways after TGF-ß treatment were mostly fibrosis-related, including focal adhesion, ECM reaction, and TGF-ß signaling pathways. As expected, transfection of Col1a1-miR-29b or CMV-miR-29b vector partially reversed the activation of these pathways. We also analyzed the expression pattern of the top 100 miR-29b targeting genes in these cells using the RNA-sequencing data. We identified that miR-29b targeted a broad spectrum of ECM genes, but the inhibition effect is mostly moderate. In summary, our work demonstrated that the Col1a1-miR-29b vector can be used as a dynamic regulator of collagen and other ECM protein expression in response to fibrotic stimulation, which could potentially reduce unnecessary side effect due to excessive miR-29b levels while remaining an effective potential therapeutic approach for fibrosis.