Travis D. Hull

In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation but whether such control occurs in vivo is not known. To enable such analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome Conformation Capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases.

The Mononuclear Phagocyte System in Homeostasis and Disease: A Role for Heme Oxygenase-1

SIGNIFICANCE Heme oxygenase-1 (HO-1) is a potential therapeutic target in many diseases, especially those mediated by oxidative stress and inflammation. HO-1 expression appears to regulate the homeostatic activity and distribution of mononuclear phagocytes (MP) in lymphoid tissue under physiological conditions. It also regulates the ability of MP to modulate the inflammatory response to tissue injury. RECENT ADVANCES The induction of HO-1 within MP-particularly macrophages and dendritic cells-modulates the effector functions that they acquire after activation. These effector functions include cytokine production, surface receptor expression, maturation state, and polarization toward a pro- or anti-inflammatory phenotype. The importance of HO-1 in MP is emphasized by their expression of specific receptors that primarily function to ingest heme-containing substrate and deliver it to HO-1. CRITICAL ISSUES MP are the first immunological responders to tissue damage. They critically affect the outcome of injury to many organ systems, yet few therapies are currently available to specifically target MP during disease pathogenesis. Elucidation of the role of HO-1 expression in MP may help to direct broadly applicable therapies to clinical use that are based on the immunomodulatory capabilities of HO-1. FUTURE DIRECTIONS Unraveling the complexities of HO-1 expression specifically within MP will more completely define how HO-1 provides cytoprotection in vivo. The use of models in which HO-1 expression is specifically modulated in bone marrow-derived cells will allow for a more complete characterization of its immunoregulatory properties.

Heme Oxygenase-1 Regulates Myeloid Cell Trafficking in AKI

Renal ischemia-reperfusion injury is mediated by a complex cascade of events, including the immune response, that occur secondary to injury to renal epithelial cells. We tested the hypothesis that heme oxygenase-1 (HO-1) expression, which is protective in ischemia-reperfusion injury, regulates trafficking of myeloid-derived immune cells in the kidney. Age-matched male wild-type (HO-1(+/+)), HO-1-knockout (HO-1(-/-)), and humanized HO-1-overexpressing (HBAC) mice underwent bilateral renal ischemia for 10 minutes. Ischemia-reperfusion injury resulted in significantly worse renal structure and function and increased mortality in HO-1(-/-) mice. In addition, there were more macrophages (CD45(+) CD11b(hi)F4/80(lo)) and neutrophils (CD45(+) CD11b(hi) MHCII(-) Gr-1(hi)) in HO-1(-/-) kidneys than in sham and HO-1(+/+) control kidneys subjected to ischemia-reperfusion. However, ischemic injury resulted in a significant decrease in the intrarenal resident dendritic cell (DC; CD45(+)MHCII(+)CD11b(lo)F4/80(hi)) population in HO-1(-/-) kidneys compared with controls. Syngeneic transplant experiments utilizing green fluorescent protein-positive HO-1(+/+) or HO-1(-/-) donor kidneys and green fluorescent protein-negative HO-1(+/+) recipients confirmed increased migration of the resident DC population from HO-1(-/-) donor kidneys, compared to HO-1(+/+) donor kidneys, to the peripheral lymphoid organs. This effect on renal DC migration was corroborated in myeloid-specific HO-1(-/-) mice subjected to bilateral ischemia. These mice also displayed impaired renal recovery and increased fibrosis at day 7 after injury. These results highlight an important role for HO-1 in orchestrating the trafficking of myeloid cells in AKI, which may represent a key pathway for therapeutic intervention.

The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins

The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinsons disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.

Heme oxygenase-1 expression protects the heart from acute injury caused by inducible Cre recombinase

The protective effect of heme oxygenase-1 (HO-1) expression in cardiovascular disease has been previously demonstrated using transgenic animal models in which HO-1 is constitutively overexpressed in the heart. However, the temporal requirements for protection by HO-1 induction relative to injury have not been investigated, but are essential to employ HO-1 as a therapeutic strategy in human cardiovascular disease states. Therefore, we generated mice with cardiac-specific, tamoxifen (TAM)-inducible overexpression of a human HO-1 (hHO-1) transgene (myosin heavy chain (MHC)-HO-1 mice) by breeding mice with cardiac-specific expression of a TAM-inducible Cre recombinase (MHC-Cre mice), with mice containing an hHO-1 transgene preceded by a floxed-stop signal. MHC-HO-1 mice overexpress HO-1 mRNA and the enzymatically active protein following TAM administration (40 mg/kg body weight on 2 consecutive days). In MHC-Cre controls, TAM administration leads to severe, acute cardiac toxicity, cardiomyocyte necrosis, and 80% mortality by day 3. This cardiac toxicity is accompanied by a significant increase in inflammatory cells in the heart that are predominantly neutrophils. In MHC-HO-1 mice, HO-1 overexpression ameliorates the depression of cardiac function and high mortality rate observed in MHC-Cre mice following TAM administration and attenuates cardiomyocyte necrosis and neutrophil infiltration. These results highlight that HO-1 induction is sufficient to prevent the depression of cardiac function observed in mice with TAM-inducible Cre recombinase expression by protecting the heart from necrosis and neutrophil infiltration. These findings are important because MHC-Cre mice are widely used in cardiovascular research despite the limitations imposed by Cre-induced cardiac toxicity, and also because inflammation is an important pathological component of many human cardiovascular diseases.

Macrophage and epithelial cell H-ferritin expression regulates renal inflammation

Inflammation culminating in fibrosis contributes to progressive kidney disease. Crosstalk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice; a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Utilizing transgenic mice with conditional deletion of FtH in the proximal tubules (FtHPT−/−) or myeloid cells (FtHLysM−/−), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared to wild-type (FtH+/+) controls. However, tubular FtH deletion led to a marked increase in pro-inflammatory macrophages. Furthermore, injured kidneys from FtHPT−/− mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared to kidneys from FtH+/+ and FtHLysM−/− mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscores the critical role of FtH in tubular-macrophage crosstalk during kidney injury.

Cyclic Di-GMP phosphodiesterases RmdA and RmdB are involved in regulating colony morphology and development in Streptomyces coelicolor.

Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes.

Bilirubin: A Potential Biomarker and Therapeutic Target for Diabetic Nephropathy

Diabetic nephropathy (DN), which is a major end-organ complication in diabetes, continues to be the most common cause of end-stage renal disease and accounts for >40% of patients on renal replacement therapy (1). Currently available factors that are routinely used in clinical practice to predict and monitor the progression of DN include degree of proteinuria and both glycemic and blood pressure control. In this issue of Diabetes , Riphagen et al. (2) performed a post hoc analysis of two large clinical trials—Reduction of Endpoints in NIDDM With the Angiotensin II Antagonist Losartan (RENAAL) and Irbesartan Diabetic Nephropathy Trial (IDNT)—and demonstrated that serum bilirubin levels are inversely correlated with progression of DN. These findings are clinically important because they identify a potential biomarker and/or therapeutic target for DN, a disease that causes significant morbidity and mortality. Bilirubin is generated when heme oxygenase (HO) catalyzes the degradation of heme (derived from heme proteins). This results in formation of biliverdin, which is rapidly converted into bilirubin by biliverdin reductase (Fig. 1) (3). Further processing of bilirubin occurs in hepatocytes, where unconjugated (lipid-soluble) bilirubin is conjugated by uridine diphosphate–glucuronosyl transferase (UDP-GT) to a water-soluble form for excretion. Total bilirubin is the sum of conjugated (direct) and unconjugated (indirect) bilirubin and generally ranges from 0.3 to 1.2 mg/dL in healthy individuals. In conditions such as erythroblastosis fetalis, hemolysis results in markedly elevated bilirubin, which causes kernicterus and neurological damage in neonates. Furthermore, genetic deficiency of UDP-GT in Crigler-Najjar syndrome type I results in severely elevated total bilirubin and is incompatible with life. Given the outcomes of these conditions, it is not surprising that bilirubin was long considered to be merely a toxic byproduct of heme degradation. However, unconjugated bilirubin levels are positively correlated with plasma antioxidant capacity (4), and moderate elevations in serum total bilirubin …

Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

New Ultrasound Techniques Promise Further Advances in AKI and CKD

AKI and CKD are important clinical problems because they affect many patients and the associated diagnostic and treatment paradigms are imperfect. Ultrasound is a cost-effective, noninvasive, and simple imaging modality that offers a multitude of means to improve the diagnosis, monitoring, and treatment of both AKI and CKD, especially considering recent advances in this technique. Ultrasound alone can attenuate AKI and prevent CKD by stimulating the splenic cholinergic anti-inflammatory pathway. Additionally, microbubble contrast agents are improving the sensitivity and specificity of ultrasound for diagnosing kidney disease, especially when these agents are conjugated to ligand-specific mAbs or peptides, which make the dynamic assessment of disease progression and response to treatment possible. More recently, drug-loaded microbubbles have been developed and the load release by ultrasound exposure has been shown to be a highly specific treatment modality, making the potential applications of ultrasound even more promising. This review focuses on the multiple strategies for using ultrasound with and without microbubble technology for enhancing our understanding of the pathophysiology of AKI and CKD.