Rational design and topochemical synthesis of polymorphs of a polymer.

Packing a polymer in different ways can give polymorphs of the polymer having different properties. ß-Turn forming peptides such as 2-aminoisobutyric acid (Aib)-rich peptides adopt several conformations by varying the dihedral angles. Aiming at this, a ß-turn-forming peptide monomer would give different polymorphs and these polymorphs upon topochemical polymerization would yield polymorphs of the polymer, we designed an Aib-rich monomer N3-(Aib)3-NHCH2-C[triple bond, length as m-dash]CH. This monomer crystallizes as two polymorphs and one hydrate. In all forms, the peptide adopts ß-turn conformations and arranges in a head-to-tail manner with their azide and alkyne units proximally placed in a ready-to-react alignment. On heating, both the polymorphs undergo topochemical azide-alkyne cycloaddition polymerization. Polymorph I polymerized in a single-crystal-to-single-crystal (SCSC) fashion and the single-crystal X-ray diffraction analysis of the polymer revealed its screw-sense reversing helical structure. Polymorph II maintains its crystallinity during polymerization but gradually becomes amorphous upon storage. The hydrate III undergoes a dehydrative transition to polymorph II. Nanoindentation studies revealed that different polymorphs of the monomer and the corresponding polymers exhibited different mechanical properties, in accordance with their crystal packing. This work demonstrates the promising future of the marriage of polymorphism and topochemistry for obtaining polymorphs of polymers.

S100/Calgranulin-mediated inflammation accelerates left ventricular hypertrophy and aortic valve sclerosis in chronic kidney disease in a receptor for advanced glycation end products-dependent manner.

OBJECTIVE: S100A12 and fibroblast growth factor 23 are biomarkers of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). We tested the hypothesis that human S100/calgranulin would accelerate cardiovascular disease in mice subjected to CKD. APPROACH AND RESULTS: A bacterial artificial chromosome of the human S100/calgranulin gene cluster containing the genes and regulatory elements for S100A8, S100A9, and S100A12 was expressed in C57BL/6J mouse (hBAC-S100) to generate a novel humanized mouse model. CKD was induced by ureteral ligation, and hBAC-S100 mice and wild-type mice were studied after 10 weeks of chronic uremia. hBAC-S100 mice with CKD showed increased fibroblast growth factor 23 in the hearts, left ventricular hypertrophy, diastolic dysfunction, focal cartilaginous metaplasia, and calcification of the mitral and aortic valve annulus together with aortic valve sclerosis. This phenotype was not observed in wild-type mice with CKD or in hBAC-S100 mice lacking the receptor for advanced glycation end products with CKD, suggesting that the inflammatory milieu mediated by S100/receptor for advanced glycation end products promotes pathological cardiac hypertrophy in CKD. In vitro, inflammatory stimuli including interleukin-6, tumor necrosis factor-α, lipopolysaccarides, or serum from hBAC-S100 mice upregulated fibroblast growth factor 23 mRNA and protein in primary murine neonatal and adult cardiac fibroblasts. CONCLUSIONS: Myeloid-derived human S100/calgranulin is associated with the development of cardiac hypertrophy and ectopic cardiac calcification in a receptor for advanced glycation end products-dependent manner in a mouse model of CKD. We speculate that fibroblast growth factor 23 produced by cardiac fibroblasts in response to cytokines may act in a paracrine manner to accelerate left ventricular hypertrophy and diastolic dysfunction in hBAC-S100 mice with CKD.

Longitudinal changes in MRI markers in a reversible unilateral ureteral obstruction mouse model: preliminary experience.

PURPOSE: To evaluate longitudinal changes in renal oxygenation and diffusion measurements in a model of reversible unilateral ureteral obstruction (rUUO) which has been shown to induce chronic renal functional deficits in a strain dependent way. C57BL/6 mice show higher degree of functional deficit compared with BALB/c mice. Because hypoxia and development of fibrosis are associated with chronic kidney diseases and are responsible for progression, we hypothesized that MRI measurements would be able to monitor the longitudinal changes in this model and will show strain dependent differences in response. Here blood oxygenation level dependent (BOLD) and diffusion MRI measurements were performed at three time points over a 30 day period in mice with rUUO. MATERIALS AND METHODS: The studies were performed on a 4.7T scanner with the mice anesthetized with isoflurane before UUO, 2 and 28 days postrelease of 6 days of obstruction. RESULTS: We found at the early time point (∼2 days after releasing the obstruction), the relative oxygenation in C57Bl/6 mice were lower compared with BALB/c. Diffusion measurements were lower at this time point and reached statistical significance in BALB/c CONCLUSION: These methods may prove valuable in better understanding the natural progression of kidney diseases and in evaluating novel interventions to limit progression.

Contrasting effects of systemic monocyte/macrophage and CD4+ T cell depletion in a reversible ureteral obstruction mouse model of chronic kidney disease.

Using a reversible UUO model (rUUO), we have demonstrated that C57BL/6 mice are susceptible to development of CKD after obstruction-mediated kidney injury while BALB/c mice are resistant. We hypothesized that selective systemic depletion of subpopulations of inflammatory cells during injury or repair might alter the development of CKD. To investigate the impact of modification of Th-lymphocytes or macrophage responses on development of CKD after rUUO, we used an anti-CD4 antibody (GK1.5) or liposomal clodronate to systemically deplete CD4(+) T cells or monocyte/macrophages, respectively, prior to and throughout the rUUO protocol. Flow cytometry and immunohistochemistry confirmed depletion of target cell populations. C57BL/6 mice treated with the GK1.5 antibody to deplete CD4(+) T cells had higher BUN levels and delayed recovery from rUUO. Treatment of C57BL/6 mice with liposomal clodronate to deplete monocyte/macrophages led to a relative protection from CKD as assessed by BUN values. Our results demonstrate that modulation of the inflammatory response during injury and repair altered the susceptibility of C57BL/6 mice to development of CKD in our rUUO model.

Vascular remodeling and arterial calcification are directly mediated by S100A12 (EN-RAGE) in chronic kidney disease.

BACKGROUND: The proinflammatory cytokine S100A12 (also known as EN-RAGE) is associated with cardiovascular morbidity and mortality in hemodialysis patients. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in nonatherosclerosis-prone C57BL/6J mice on normal rodent chow diet, but exposed to the metabolic changes of chronic kidney disease (CKD), would develop vascular disease resembling that observed in patients with CKD. METHODS: CKD was induced in S100A12 transgenic mice and wild-type littermate mice not expressing human S100A12 by surgical ligation of the ureters. The aorta was analyzed after 7 weeks of elevated BUN (blood urea nitrogen), and cultured aortic smooth muscle cells were studied. RESULTS: We found enhanced vascular medial calcification in S100A12tg mice subjected to CKD. Vascular calcification was mediated, at least in part, by activation of the receptor for S100A12, RAGE (receptor for advanced glycation endproducts), and by enhanced oxidative stress, since inhibition of NADPH-oxidase Nox1 and limited access of S100A12 to RAGE attenuated the calcification and gene expression of osteoblastic genes in cultured vascular smooth muscle cells. CONCLUSION: S100A12 augments CKD-triggered osteogenesis in murine vasculature, reminiscent of features associated with enhanced vascular calcification in patients with chronic and end-stage kidney disease.

Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background.

Chronic kidney disease (CKD) begins with renal injury; the progression thereafter depends upon a number of factors, including genetic background. Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and as such is considered a model of CKD. We used an improved reversible unilateral ureteral obstruction (rUUO) model in mice to study the strain dependence of development of CKD after obstruction-mediated injury. C57BL/6 mice developed CKD after reversal of three or more days of ureteral obstruction as assessed by blood urea nitrogen (BUN) measurements (>40 mg/dl). In contrast, BALB/c mice were resistant to CKD with up to 10 days ureteral obstruction. During rUUO, C57BL/6 mice exhibited pronounced inflammatory and intrinsic proliferative cellular responses, disruption of renal architecture, and ultimately fibrosis. By comparison, BALB/c mice had more controlled and measured extrinsic and intrinsic responses to injury with a return to normal within several weeks after release of ureteral obstruction. Our findings provide a model that allows investigation of the genetic basis of events during recovery from injury that contribute to the development of CKD.

Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle.

The biological response of muscle to eccentric contractions (ECs) results in strengthening and protection from further injury. However, the cellular basis for this response remains unclear. Previous studies identified the muscle ankyrin repeat protein (MARP) family, consisting of cardiac ankyrin repeat protein (CARP), ankyrin repeat domain 2/ankyrin repeat protein with PEST and proline-rich region (Ankrd2/Arpp), and diabetes-associated ankyrin repeat protein (DARP), as rapidly and specifically upregulated in mice after a single bout of EC. To determine the role of these genes in skeletal muscle, a survey of skeletal muscle structural and functional characteristics was performed on mice lacking all three MARP family members (MKO). There was a slight trend toward MKO muscles having a slower fiber type distribution but no differences in muscle fiber size. Single MKO fibers were less stiff, tended to have longer resting sarcomere lengths, and expressed a longer isoform of titin than their wild-type counterparts, indicating that these proteins may play a role in the passive mechanical behavior of muscle. Finally, MKO mice showed a greater degree of torque loss after a bout of ECs compared with wild-type mice, although they recovered from the injury with the same or even improved time course. This recovery was associated with enhanced expression of the muscle regulatory genes MyoD and muscle LIM protein (MLP), suggesting that the MARP family may play both important structural and gene regulatory roles in skeletal muscle.

Stress-dependent and -independent expression of the myogenic regulatory factors and the MARP genes after eccentric contractions in rats.

The relationship between muscle mechanical conditions and gene expression was investigated by varying both stress and contraction mode imposed upon rat dorsiflexors (n= 25), activating them at high or low frequencies (150 Hz or 40 Hz) either eccentrically or isometrically. Muscle physiological, immunohistochemical and gene expression changes were then measured 24 h after the exercise bout. Peak stress was the best predictor of muscle injury, independent of contraction mode (i.e. eccentric or isometric). When peak stresses were matched, no physiological or immunohistochemical differences were detected between isometric and eccentric contractions. The expression of certain myogenic regulatory and muscle ankyrin repeat protein (MARP) genes (myoD, myogenin, MLP and CARP) depended both on peak muscle stress achieved during contraction and contraction mode. In contrast, Arpp/Ankrd2 was dramatically upregulated only by eccentric contractions, but not by isometric contractions, even though the stress level of the eccentric contractions varied over a three-fold range and overlapped with that of the isometric group. The role that Arpp/Ankrd2 upregulation plays in the biological response to eccentric contraction remains to be determined, as does the control mechanism whereby the expression of certain genes (such as myoD, myogenin, MLP and CARP) is sensitive to muscle stress while another (Arpp/Ankrd2) is sensitive only to contraction mode.

Muscle LIM protein plays both structural and functional roles in skeletal muscle.

Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles.

Rapid muscle-specific gene expression changes after a single bout of eccentric contractions in the mouse.

Eccentric contractions (ECs), in which a muscle is forced to lengthen while activated, result in muscle injury and, eventually, muscle strengthening and prevention of further injury. Although the mechanical basis of EC-induced injury has been studied in detail, the biological response of muscle is less well characterized. This study presents the development of a minimally invasive model of EC injury in the mouse, follows the time course of torque recovery after an injurious bout of ECs, and uses Affymetrix microarrays to compare the gene expression profile 48 h after ECs to both isometrically stimulated muscles and contralateral muscles. Torque dropped by approximately 55% immediately after the exercise bout and recovered to initial levels 7 days later. Thirty-six known genes were upregulated after ECs compared with contralateral and isometrically stimulated muscles, including five muscle-specific genes: muscle LIM protein (MLP), muscle ankyrin repeat proteins (MARP1 and -2; also known as cardiac ankyrin repeat protein and Arpp/Ankrd2, respectively), Xin, and myosin binding protein H. The time courses of MLP and MARP expression after the injury bout (determined by quantitative real-time polymerase chain reaction) indicate that these genes are rapidly induced, reaching a peak expression level of 6-11 times contralateral values 12-24 h after the EC bout and returning to baseline within 72 h. Very little gene induction was seen after either isometric activation or passive stretch, indicating that the MLP and MARP genes may play an important and specific role in the biological response of muscle to EC-induced injury.

Asynchronous functional, cellular and transcriptional changes after a bout of eccentric exercise in the rat.

Thirty eccentric contractions (ECs) were imposed upon rat dorsiflexors (n = 46) by activating the peroneal nerve and plantarflexing the foot ~40 deg, corresponding to a sarcomere length change over the range 2.27-2.39 microm for the tibialis anterior and 2.52-2.66 microm for the extensor digitorum longus. Animals were allowed to recover for one of 10 time periods ranging from 0.5 to 240 h, at which time muscle contractile properties, immunohistochemical labelling and gene expression were measured. Peak isometric torque dropped significantly by ~40 % from an initial level of 0.0530 +/- 0.0009 Nm to 0.0298 +/- 0.0008 Nm (P < 0.0001) immediately after EC, and then recovered in a linear fashion to control levels 168 h later. Immunohistochemical labelling of cellular proteins revealed a generally asynchronous sequence of events at the cellular level, with the earliest event measured being loss of immunostaining for the intermediate filament protein, desmin. Soon after the first signs of desmin loss, infiltration of inflammatory cells occurred, followed by a transient increase in membrane permeability, manifested as inclusion of plasma fibronectin. The quantitative polymerase chain reaction (QPCR) was used to measure transcript levels of desmin, vimentin, embryonic myosin heavy chain (MHC), myostatin, myoD and myogenin. Compared to control levels, myostatin transcripts were significantly elevated after only 0.5 h, myogenic regulatory factors significantly elevated after 3 h and desmin transcripts were significantly increased 12 h after EC. None of the measured parameters provide a mechanistic explanation for muscle force loss after EC. Future studies are required to investigate whether there is a causal relationship among desmin loss, increased cellular permeability, upregulation of the myoD and desmin genes, and, ultimately, an increase in the desmin content per sarcomere of the muscle.