C-reactive Protein Transgenic Mice Research Articles

#4208 CRP EXACERBATES SARS-COV-2-NUCLEOCAPSID PROTEIN-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims COVID-19, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), is a progressive viral pneumonia with a broad spectrum of clinical manifestations involving multiple organs. Acute kidney injury (AKI) is common in COVID-19, especially the critically ill patients. Our previous study proved that kidney-specifically overexpressing SARS-CoV-2 N protein induces AKI, which become worsen in mice under ischemic condition. Clinically, CRP is a risk factor for AKI in COVID-19 patients. Recent clinical study showed that CRP Apheresis is able to effectively remove the plasma CRP and thus improve the clinical outcome in critically ill COVID-19 patients. We thus hypothesize that CRP may not only be a biomarker or risk factor, but also a potential mediator or novel therapeutic target for COVID-19 AKI. Method The role of CRP in COVID-19 AKI was investigated in a mouse model of AKI induced by ultrasound-microbubble-mediated kidney-specifically transferring SARS-CoV-2 N-expressing plasmids in CRP wide-type (WT) and Transgenic (Tg) mice. We also developed a novel therapy for SARS-CoV-2 N-induced by targeting CRP signaling with a neutralizing anti-CD32 antibody both in vivo and in vitro. Results In COVID-19 patients with AKI, serum levels of CRP were significantly elevated when compared with non-AKI group. In mice, kidney-specifically overexpressing SARS-CoV-2 N protein caused AKI including tubular necrosis and elevated levels of serum creatinine and BUN in CRP WT mice, which became more severe in CRP Tg mice. Importantly, targeting CRP-CD32 signaling with a neutralizing antibody to CD32 significantly inhibited SARS-CoV-2 N protein-induced AKI in CRP Tg mice. Mechanistically, we uncovered that CRP promoted SARS-CoV-2-N protein-induced AKI via the Smad3-p21-dependent G1 cell cycle arrest and NF-κB/p-p65-driven renal inflammation, which was blocked by anti-CD32 antibody in vivo and in vitro. Conclusion CRP is not only a biomarker or risk factor, but also a mediator in SARS-CoV-2 N protein-induced AKI. CRP signals through CD32 mediate SARS-CoV-2 N-induced AKI via mechanisms associated with the Smad3-p21-dependent G1 cell-cycle arrest and NF-κB/p-p65-driven renal inflammation. Targeting CRP-CD32 signaling may represent as a novel therapy for COVID-19-associated AKI.

Deficiency of C-reactive protein or human C-reactive protein transgenic treatment aggravates influenza A infection in mice.

C-reactive protein (CRP) has been shown to be a potential candidate target in the immunotherapy of severe influenza A infection. However, it is unclear on the pathogenesis associated with CRP in influenza infections. Here, we used influenza A H1N1 CA04 to infect human CRP transgenic mice (KI), CRP knockout mice (KO), and wild-type mice (WT), respectively, and compared the viral pathogenicity and associated immune response in those mice. The results showed that CA04 infection resulted in 100%, 80%, and 60% death in KO, KI, and WT mice, respectively. Compared to WT mice, CA04 infection resulted in higher TCID50 in lungs on day 3 after infection but lowered HI antibody titers in sera of survivors on day 21 after infection in KI mice. ELISA assay showed that IFN-γ concentration was significantly increased in sera of WT, KI, or KO mice on day 7 after infection, and IL-17 was remarkably increased in sera of WT mice but decreased in sera of KI mice while no significant change in sera of KO mice on day 3 or 7 after infection. Quantitative RT-PCR showed that the relative expression levels of immune checkpoint CTLA-4, LAIR-1, GITR, BTLA, TIM-3, or PD-1 mRNA in the lung presented decreased levels on day 3 or 7 after infection in KI or KO mice. The correlation analysis showed that mRNA expression levels of the 6 molecules positively correlated with viral TICD50 in WT mice but negatively correlated with viral TCID50 in KI or KO mice. However, only LAIR-1 presented a significant correlation in each lung tissue of WT, KI, or KO mice with CA07 infection statistically. IHC results showed that LAIR-1 positive cells could be found in WT, KO, or KI mice lung tissues with CA04 infection, and the positive cells were mainly distributed in an inflammatory dense area. Our results suggested that deficiency of CRP or human CRP transgenic treatment aggravates influenza A virus infection in mice. CRP is a double sword in immune regulation of influenza infection in which IL-17 and immune checkpoint may be involved.

Therapeutic Lowering of C-Reactive Protein.

In the blood of healthy individuals C-reactive protein (CRP) is typically quite scarce, whereas its blood concentration can rise robustly and rapidly in response to tissue damage and inflammation associated with trauma and infectious and non-infectious diseases. Consequently, CRP plasma or serum levels are routinely monitored in inpatients to gauge the severity of their initial illness and injury and their subsequent response to therapy and return to health. Its clinical utility as a faithful barometer of inflammation notwithstanding, it is often wrongly concluded that the biological actions of CRP (whatever they may be) are manifested only when blood CRP is elevated. In fact over the last decades, studies done in humans and animals (e.g. human CRP transgenic and CRP knockout mice) have shown that CRP is an important mediator of biological activities even in the absence of significant blood elevation, i.e. even at baseline levels. In this review we briefly recap the history of CRP, including a description of its discovery, early clinical use, and biosynthesis at baseline and during the acute phase response. Next we overview evidence that we and others have generated using animal models of arthritis, neointimal hyperplasia, and acute kidney injury that baseline CRP exerts important biological effects. In closing we discuss the possibility that therapeutic lowering of baseline CRP might be a useful way to treat certain diseases, including cancer.

SAT-120 RESTORATION OF PGC-1α ACTIVITY BY DELETION OF SMAD3 PROTECTS AGAINST KIDNEY INJURY IN CRP TG MICE WITH UNILATERAL URETERAL OBSTRUCTION (UUO)

Sustained elevated plasma levels of C-reactive protein (CRP) is closely associated with increased risk of chronic kidney disease. Studies have shown that kidney injury is exacerbated in CRP transgenic mice (CRP Tg) with UUO and attenuated in Samd3 deficiency mice (Smad3 KO). In this study, we investigated the signalling mechanisms of CRP promoting renal fibrosis and inflammation in UUO by using CRP Tg, Smad3 KO, and CRP Tg/Smad3 KO mice. CRP Tg/ Smad3 KO mice and wild type littermates were generated by Smad3 heterozygous X CRP Tg mice. UUO was induced in CRP wild-type (Wt)/Smad3 Wt; CRP Wt/Smad3 KO, CRP Tg/Smad3 Wt, and CRP Tg/Smad3 KO mice.A rat tubular epithelia cell line with stable Smad3 knockdown (NRK52E) was used for in vitro study. Our results show that renal inflammation and fibrosis induced by UUO was greater for CRP Tg than Wt. In contrast in Smad3 KO mice, whether CRP Wt or CRP Tg, exhibited attenuated kidney injury including: 1) obviously reduced histological injury revealed by Masson‘s Trichrome &HEStaining; 2) marked down-regulation of pro-inflammatory cytokines (IL-1ß and TNF-a); 3) reduced renal fibrosis as demonstrated by significantly down-regulation of extracellular matrix deposition, such as a-smooth muscle actin (a-SMA) ,collagen 1 and fibronectin. Exacerbation of UUO-induced kidney injury in CRP Tg was associate with increased TGF-ß/Smad3 signaling and decreased expression of peroxisomal proliferation-coactivator (PGC-1a). On the other hand, protection from UUO-induced kidney injury in Smad3 KO mice was associated with restored expression of PGC-1a. In vitrostudies using Smad3 KO NRK52E TECs showed that CRP activates CD32-Smad3 and inhibits PGC-1a expression, restored activity of PGC-1a by deletion of Smad3 protects against CRP-induced extracellular matrix deposition (collagen I, and fibronectin). In conclusion, deletion of Smad3 can protect against CRP exacerbated kidney injury after UUO, at least partly the results of CD32-Smad3 driven inhibition of the PGC-1a expression. Targeting Smad3-regulating PGC-1a activity may offer a new treatment approach for obstructive nephropathy.

C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance.

C-reactive protein (CRP) is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE) in CRP transgenic mice by shifting CD4+ T cells away from the TH1 and toward the TH2 subset. Notably, this action required the inhibitory Fcγ receptor IIB (FcγRIIB), but did not require high levels of human CRP. Herein, we sought to determine if CRP’s influence in EAE might be explained by CRP acting on dendritic cells (DC; antigen presenting cells known to express FcγRIIB). We found that CRP (50 µg/ml) reduced the yield of CD11c+ bone marrow-derived DCs (BMDCs) and CRP (≥5 μg/ml) prevented their full expression of major histocompatibility complex class II and the co-stimulatory molecules CD86 and CD40. CRP also decreased the ability of BMDCs to stimulate antigen-driven proliferation of T cells in vitro. Importantly, if the BMDCs were genetically deficient in mouse FcγRIIB then (i) the ability of CRP to alter BMDC surface phenotype and impair T cell proliferation was ablated and (ii) CD11c-driven expression of a human FCGR2B transgene rescued the CRP effect. Lastly, the protective influence of CRP in EAE was fully restored in mice with CD11c-driven human FcγRIIB expression. These findings add to the growing evidence that CRP has important biological effects even in the absence of an acute phase response, i.e., CRP acts as a tonic suppressor of the adaptive immune system. The ability of CRP to suppress development, maturation, and function of DCs implicates CRP in the maintenance of peripheral T cell tolerance.

Protective effect of rosiglitazone on kidney function in high-fat challenged human-CRP transgenic mice: a possible role for adiponectin and miR-21?

Obesity-related albuminuria is associated with decline of kidney function and is considered a first sign of diabetic nephropathy. Suggested factors linking obesity to kidney dysfunction include low-grade inflammation, insulin resistance and adipokine dysregulation. Here, we investigated the effects of two pharmacological compounds with established anti-inflammatory properties, rosiglitazone and rosuvastatin, on kidney dysfunction during high-fat diet (HFD)-induced obesity. For this, human CRP transgenic mice were fed standard chow, a lard-based HFD, HFD+rosuvastatin or HFD+rosiglitazone for 42 weeks to study effects on insulin resistance; plasma inflammatory markers and adipokines; and renal pathology. Rosiglitazone but not rosuvastatin prevented HFD-induced albuminuria and renal fibrosis and inflammation. Also, rosiglitazone prevented HFD-induced KIM-1 expression, while levels were doubled with rosuvastatin. This was mirrored by miR-21 expression, which plays a role in fibrosis and is associated with renal dysfunction. Plasma insulin did not correlate with albuminuria. Only rosiglitazone increased circulating adiponectin concentrations. In all, HFD-induced albuminuria, and renal inflammation, injury and fibrosis is prevented by rosiglitazone but not by rosuvastatin. These beneficial effects of rosiglitazone are linked to lowered miR-21 expression but not connected with the selectively enhanced plasma adiponectin levels observed in rosiglitazone-treated animals.

C-reactive protein modulates myeloid derived suppressor cell function and signaling.

Abstract Recently, we provided evidence that C-reactive protein (CRP) plays a causal role in acute kidney injury (AKI) i.e., human CRP exacerbates AKI in CRP transgenic mice (CRPtg) and AKI severity is lessened in CRP knockout mice. The CRP effect in CRPtg was associated with significantly increased renal infiltration by myeloid derived suppressor cells (MDSC). Subsequently, we showed that the AKI exacerbating influence of CRP in CRPtg could be blocked by depletion of MDSCs. In the present work we sought to define the impact of CRP on MDSC ontogeny and function. Using wild type mouse bone marrow (BM) we derived MDSCs and established that CRP enhances their ability to suppress CD3ɛ/CD28-stimulated T cell proliferation in vitro. By flow cytometry we verified that mouse BM-MDSCs express variable amounts of three CRP receptors: LOX-1, FcγRI, and FcγRIIB. Expression of the latter on BM-MDSCs was upregulated upon CRP exposure, as demonstrated by flow cytometry. Furthermore, preliminary data obtained from studies using BM-MDSCs derived from FcγRIIB deficient mice suggest that FcγRIIB participates in CRP-mediated signaling. In their sum, these data suggest that CRP-mediated augmentation of MDSC suppressive function is FcγRIIB-dependent. Ongoing work seeks to define the consequences of CRP-mediated modulation of MDSC functions in vivo and to explore whether FcγRIIB and other CRP receptors are involved.

Peritoneal inflammation and fibrosis in C-reactive protein transgenic mice undergoing peritoneal dialysis solution treatment.

C-reactive protein (CRP) is a mediator of systemic inflammation. Peritoneal dialysis (PD) is known to cause peritoneal inflammation and fibrosis. We compare the degree of peritoneal inflammation and fibrosis in wild-type (WT) and CRP-transgenic (Tg) mice after PD treatment. WT (n = 7) and CRP-Tg (n = 10) C57BL/6 J mice (all male, 10-12 weeks old) were injected intra-peritoneally with 4.25% dextrose PD solution (3 mL/mouse) daily for 28 days, followed by a 2-h peritoneal equilibration test (PET). The mice were then killed. Parietal peritoneal and omental tissues were collected for the assessment of inflammation and fibrosis. After 28 days of PD treatment, CRP-Tg mice had higher dialysate-to-plasma (D/P) creatinine ratio than that of WT mice. Parietal peritoneum of the CRP-Tg mice was more cellular and thicker than that of the WT mice. CRP-Tg mice also had higher connective tissue growth factor (CTGF), intercellular adhesion molecule 1 (ICAM1) and tumor necrosis factor α (TNFα) RNA expressions as well as immunohistochemical staining in the parietal peritoneum than that of the WT mice. CRP-Tg mice have significantly more inflammation and fibrosis than WT mice after PD treatment. Our results suggest that CRP play a role in inflammation and fibrosis induced by PD. The implication of our results to human PD therapy needs further investigations.

C-reactive protein promotes acute kidney injury via Smad3-dependent inhibition of CDK2/cyclin E

Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-β/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition oftubular epithelial cells. Invitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-β-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. Invivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach forAKI.

Abstract 218: Endothelial Fc gamma Receptor IIB Activation Blunts Insulin Transport to Skeletal Muscle to Cause Insulin Resistance in Mice

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance, and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. Having determined that FcγRIIB is expressed in endothelium, here we investigated how endothelial FcγRIIB influences glucose homeostasis. Floxed FcγRIIB mice were crossed with VECadherin-driven Cre recombinase and CRP transgenic mice to yield mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated skeletal muscle insulin delivery. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and endothelial NO synthase (eNOS) antagonism. Furthermore, in knock-in mice harboring constitutively-active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin transport to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin transport has been identified that may contribute to type 2 diabetes pathogenesis.

Human C-reactive protein impedes entry of leptin into the CNS and attenuates its physiological actions in the CNS.

Defective central leptin signalling and impaired leptin entry into the CNS (central nervous system) represent two important aspects of leptin resistance in obesity. In the present study, we tested whether circulating human CRP (C-reactive protein) not only diminishes signalling of leptin within the CNS, but also impedes this adipokine's access to the CNS. Peripheral infusion of human CRP together with co-infused human leptin was associated with significantly decreased leptin content in the CSF of ob/ob mice. Furthermore, following peripheral infusion of human leptin, the CSF (cerebrospinal fluid) concentration of leptin in transgenic mice overexpressing human CRP was sharply lower than that achieved in similarly infused wild-type mice. Administration of LPS (lipopolysaccharide) to human CRP-transgenic mice dramatically elevated the concentrations of human CRP in the CSF. The i.c.v. (intracerebroventricular) delivery of human CRP into the lateral ventricles of ob/ob mice blocked the satiety and weight-reducing actions of human leptin, but not those of mouse leptin. I.c.v. injection of human CRP abolished hypothalamic signalling by human leptin, and ameliorated the effects of leptin on the expression of NPY (neuropeptide Y), AgRP (Agouti-related protein), POMC (pro-opiomelanocortin) and SOCS-3 (suppressor of cytokine signalling 3). Human CRP can impede the access of leptin to the CNS, and elevation of human CRP within the CNS can have a negative impact on the physiological actions of leptin.

C-reactive protein exacerbates renal ischemia-reperfusion injury: are myeloid-derived suppressor cells to blame?

Myeloid-derived suppressor cells (MDSCs) are a CD11b(+)Gr1(+) population in mice that can be separated into granulocytic (g-MDSC) and monocytic (m-MDSC) subtypes based on their expression of Ly6G and Ly6C. Both MDSC subtypes are potent suppressors of T cell immunity, and their contribution has been investigated in a plethora of diseases including renal cancer, renal transplant, and chronic kidney disease. Whether MDSCs contribute to the pathogenesis of acute kidney injury (AKI) remains unknown. Herein, using human C-reactive protein (CRP) transgenic (CRPtg) and CRP-deficient mice (CRP(-/-)) subjected to bilateral renal ischemia-reperfusion injury (IRI), we confirm our earlier finding that CRP exacerbates renal IRI and show for the first time that this effect is accompanied in CRPtg mice by a shift in the balance of kidney-infiltrating MDSCs toward a suppressive Ly6G(+)Ly6C(low) g-MDSC subtype. In CRPtg mice, direct depletion of g-MDSCs (using an anti-Gr1 monoclonal antibody) reduced the albuminuria caused by renal IRI, confirming they play a deleterious role. Remarkably, treatment of CRPtg mice with an antisense oligonucleotide that specifically blocks the human CRP acute-phase response also led to a reduction in renal g-MDSC numbers and improved albuminuria after renal IRI. Our study in CRPtg mice provides new evidence that MDSCs participate in the pathogenesis of renal IRI and shows that their pharmacological depletion is beneficial. If ongoing investigations confirm that CRP is an endogenous regulator of MDSCs in CRPtg mice, and if this action is recapitulated in humans, then targeting CRP or/and MDSCs might offer a new approach for the treatment of AKI.

C-reactive protein directly suppresses Th1 cell differentiation and alleviates experimental autoimmune encephalomyelitis.

Human C-reactive protein (CRP) is a serum-soluble pattern recognition receptor that serves as a marker of inflammation and directly contributes to innate immunity. In this study, we show that human CRP also directly contributes to adaptive immunity, that is, native CRP binds specifically to human Jurkat T cells and to mouse naive CD4(+) T cells and modulates their Th1 and Th2 responses. In vitro both exogenously added (purified) and endogenously expressed (via transfection) human CRP inhibited Th1 differentiation and augmented Th2 differentiation of naive CD4(+) T cells. In vivo for human CRP transgenic compared with wild-type mice, a lesser proportion of the T cells recovered from the spleens of healthy animals were Th1 cells. Moreover, in both CRP transgenic mice and in wild-type mice treated with human CRP, during myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis both the Th1 cell response and disease severity were inhibited. These pattern recognition-independent actions of CRP directly on T cells highlights the potential for this soluble pattern recognition receptor to act as a tonic regulator of immunity, shaping global adaptive immune responses during both homeostasis and disease.

Hepatic but Not CNS-Expressed Human C-Reactive Protein Inhibits Experimental Autoimmune Encephalomyelitis in Transgenic Mice

We recently demonstrated that human C-reactive protein (CRP), expressed hepatically in transgenic mice (CRPtg), improved the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The liver is the primary site of CRP synthesis in humans and in CRPtg mice but is also expressed by both at low levels in the CNS. To determine if CNS expression of human CRP is sufficient to impact EAE, we generated neuronal CRP transgenic mice (nCRPtg) wherein human CRP expression is driven by the neuron-specific Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) gene promoter. We found that hepatically expressed/blood-borne CRP, but not CNS expressed CRP, lessened EAE severity. These outcomes indicate that the protective actions of human CRP in EAE are manifested in the periphery and not in the CNS and reveal a previously unappreciated site specificity for the beneficial actions of CRP in CNS disease.

THU0539 Isis-Crp Rx: Pharmacokinetics, Pharmacodynamics, Safety and Tolerability of an Antisense Oligonucleotide Specific for Inhibition of CRP in Inflammation

BackgroundCRP is elevated in many diseases with inflammatory components such as rheumatoid arthritis, acute coronary syndrome and atrial fibrillation. Elevated CRP levels are associated with increased disease burden and are...

Central nervous system-specific expression of C-reactive protein exacerbates experimental autoimmune encephalomyelitis. (BA3P.202)

Abstract Long considered merely a marker of inflammation, new evidence indicates that C-reactive protein (CRP) may also affect the maintenance of health and propagation of disease. Our lab previously showed that human CRP, hepatically synthesized by human CRP transgenic mice (CRPtg), delays onset and reduces severity of Experimental Autoimmune Encephalomyelitis (EAE; a model of Multiple Sclerosis). Though no causal data are available for humans, research suggests that unlike CRPtg, expression/deposition of CRP in the CNS associates with disease, rather than protection. Though human CRP expression in both man and CRPtg mouse is largely hepatic, studies have shown that it is also expressed by multiple cells of the CNS, particularly during neuroinflammation. To test CNS-expressed CRP’s impact on EAE, we made transgenic mice wherein the neuron-specific Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) promoter drives human CRP expression. We confirmed that human CRP expression is restricted to the CNS in these neuronal CRPtg (nCRPtg) mice. In contrast to CRPtg, CNS-specific expression of CRP neither delays EAE onset nor improves its symptoms. As in humans, CNS-expressed CRP associates with more severe disease. In summary, observations made with CRPtg vs. nCRPtg reveal a previously unappreciated site-specificity to the impact of CRP on the CNS. The possibility that CRP mediates beneficial vs. detrimental actions depending on the local cellular environment will be investigated.

Animal models of C-reactive protein.

As the main theme of this special issue, CRP not only is an inflammatory marker but also has diverse biological functions associated with different diseases. To investigate CRP's physiologies and their relationship with human pathological significance, it is essential to use appropriate animal models for translational research. The most popular models for the study of CRP are transgenic mice. However, researchers should be careful when extrapolating the findings derived from these animal models. This review will discuss the current concerns on CRP transgenic mice and rabbits.

Inhibiting C-reactive protein for the treatment of cardiovascular disease: promising evidence from rodent models.

Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.

C-reactive protein exacerbates renal ischemia reperfusion injury (P4021)

Abstract Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) after kidney transplantation, hypotension, and cardiovascular surgery. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild type mice (WT), human CRP transgenic mice (CRPtg) and CRP deficient mice (CRP-/-) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased ~six-fold after IRI in CRPtg (11.58 ± 1.40 µg/ml at baseline versus 65.75 ± 7.90 µg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared to WT, serum creatinine and urine albumin was increased after IRI in CRPtg mice. Histological evidence of kidney damage was greater in CRPtg than in WT, whereas CRP-/- kidneys were protected. RT-PCR analysis of mRNA isolated from whole kidneys subjected to IRI revealed that expression of FcγRs increased in CRP-/- kidneys and markers of macrophage activation indicated a stronger classically activated (M1) macrophage response in CRPtg kidneys. Our finding that CRP exacerbates IRI induced AKI by shifting the balance of beneficial and detrimental FcγRs and macrophages, makes CRP a promising therapeutic target for the treatment of AKI.

C-reactive protein exacerbates renal ischemia-reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), occurring with hypotension and cardiovascular surgery and inevitably during kidney transplantation. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild-type mice (WT) and human CRP transgenic mice (CRPtg) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased approximately sixfold after IRI in CRPtg (10.62 ± 1.31 μg/ml at baseline vs. 72.01 ± 9.41 μg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared with WT, serum creatinine, urine albumin, and histological evidence of kidney damage were increased after IRI in CRPtg mice. RT-PCR analysis of mRNA isolated from whole kidneys of CRPtg and WT subjected to IRI revealed that in CRPtg kidneys 1) upregulation of markers of macrophage classical activation (M1 markers) was blunted, 2) downregulation of markers of macrophage alternative activation (M2 markers) was more robust, and 3) expression of the activating receptor FcγRI was increased. Our finding that CRP exacerbates IRI-induced AKI, perhaps by shifting the balance of macrophage activation and FcγR expression towards a detrimental portfolio, might make CRP a promising therapeutic target for the treatment of AKI.