Impact of humanized vancomycin infusion on kidney function and kidney injury in a translational rat model.

Allometric dose scaling aims to create isometric exposures between animals and humans and is often employed in preclinical pharmacokinetic/pharmacodynamic models. Bolus-administration with allometric scaling is the most simple and commonly used strategy in pre-clinical kidney injury studies; however, it is possible to humanize drug exposures. Currently, it is unknown if dose-matched, bolus-administration with allometric scaling results in similar outcomes compared to humanized infusions in the vancomycin induced kidney injury model. We utilized a preclinical Sprague-Dawley rat model to compare traditional allometrically-scaled, dose-matched, bolus-administration of vancomycin to an infusion-pump controlled, humanized infusion scheme to assess for differences in iohexol-measured kidney function and urinary kidney injury biomarkers. Following 24 h of vancomycin administration, rats in the humanized infusion group had equivalent area under the curve exposures to animals in the dose-matched bolus group (93.7 mg·h/L [IQR 90.2-97.2] vs. 99.5 mg·h/L [IQR 95.1-104.0], P = 0.07). No significant differences in iohexol-measured kidney function nor meaningful differences in urinary kidney injury biomarkers, kidney injury molecule-1, clusterin, and osteopontin, were detected. Administration of intravenous vancomycin as either a humanized infusion or dose-matched bolus resulted in similar vancomycin exposures. No differences in iohexol-measured GFR nor meaningful differences in urinary kidney injury biomarkers were observed among male Sprague-Dawley rats.

Toward patient-centered treatment goals for duchenne muscular dystrophy: insights from the "Your Voice" study.

BACKGROUND: Patient-centered research has emerged as critically important for understanding the impact of treatments on key stakeholders. The subjective experience of quality of life (QOL) is increasingly recognized as fundamental to delineating treatment goals. The present study utilized content analysis of qualitative data and quantitative analysis to highlight important domains of disease burden and underlying reasons for their importance, and to characterize goals for new treatments for Duchenne Muscular Dystrophy (DMD). RESULTS: The study sample reflected the perspectives of DMD patients and caregivers representing ambulatory, transitional, and non-ambulatory stages of disability progression (n = 20 per category). Open-ended interviews were content-analyzed and non-parametric statistical tests were used to compare ambulation groups. As patients progressed in disability, the noted DMD burdens reflected some differences in functional areas. While daily functioning and sports/recreation remained the most important priority areas across ambulation groups, "health" became less prominent as the disability progressed from ambulatory to transitional to non-ambulatory phases of disability; whereas relationships became more prominent as one progressed to the non-ambulatory phase from the ambulatory or transitional phases (Kruskall Wallis H = 12.24 and 5.28, p = 0.002 and 0.02, respectively). When asked why their burdens were important to them and how it impacted their or their child's life, self-esteem/confidence was most important for ambulatory patients, and became less prominent for patients in the transitional and non-ambulatory phases of disability (Kruskall Wallis H = 9.46, p = 0.009). In contrast, independence was less important for ambulatory patients, and became increasing prominent for patients in the transitional and non-ambulatory phases of disability (Kruskall Wallis H = 7.35, p = 0.025). Emotional functioning was most prominent for all ambulation groups on their best and worst days. Goals for new DMD treatments focused on functional goals, general QOL goals, and concerns about safety, ease of use, and effectiveness. CONCLUSION: This study provides useful information about treatment goals for DMD from the perspective of patients and their caregivers. It highlights some consistent values across the disability trajectory, as well as introducing an evolution of priorities as the person with DMD becomes more disabled. Results provide a roadmap for patient-centered DMD drug development.

Impact of Vancomycin Loading Doses and Dose Escalation on Glomerular Function and Kidney Injury Biomarkers in a Translational Rat Model.

Vancomycin-induced kidney injury is common, and outcomes in humans are well predicted by animal models. This study employed our translational rat model to investigate temporal changes in the glomerular filtration rate (GFR) and correlations with kidney injury biomarkers related to various vancomycin dosing strategies. First, Sprague-Dawley rats received allometrically scaled loading doses or standard doses. Rats that received a loading dose had low GFRs and increased urinary injury biomarkers (kidney injury molecule 1 [KIM-1] and clusterin) that persisted through day 2 compared to those that did not receive a loading dose. Second, we compared low and high allometrically scaled vancomycin doses to a positive acute kidney injury control of high-dose folic acid. Rats in both the low- and high-dose vancomycin groups had higher GFRs on all dosing days than the positive-control group. When the two vancomycin groups were compared, rats that received the low dose had significantly higher GFRs on days 1, 2, and 4. Compared to low-dose vancomycin, the KIM-1 was elevated among rats in the high-dose group on dosing day 3. The GFR correlated most closely with the urinary injury biomarker KIM-1 on all experimental days. Vancomycin loading doses were associated with significant losses of kidney function and elevations of urinary injury biomarkers. In our translational rat model, both the degree of kidney function decline and urinary biomarker increases corresponded to the magnitude of the vancomycin dose (i.e., a higher dose resulted in worse outcomes).

Healthcare access, satisfaction, and health-related quality of life among children and adults with rare diseases.

BACKGROUND: Research in a variety of countries indicates that healthcare access and health-related quality of life are challenged among people with a variety of rare diseases (RDs). However, there has been little systematic research on the experiences of children and adults with RDs in the American healthcare system that identifies commonalities across RDs. This research aimed to: (1) Describe demographics, disease characteristics, diagnostic experiences, access to healthcare, knowledge about RDs, support from healthcare professionals, and patient satisfaction among people with RDs and their caregivers; (2) examine predictors of patient satisfaction among adults with RDs; (3) compare health-related quality of life and stigma to US population norms; 4) examine predictors of anxiety and depression among adults and children with RDs. RESULTS: This large-scale survey included (n = 1128) adults with RD or parents or caregivers of children with RDs representing 344 different RDs. About one third of participants waited four or more years for a diagnosis and misdiagnosis was common. A subset of participants reported experiencing insurance-related delays or denials for tests, treatments, specialists, or services. Approximately half of participants felt their medical and social support was sufficient, yet less than a third had sufficient dental and psychological support. Patients were generally neither satisfied or dissatisfied with their healthcare providers. Major predictors of satisfaction were lower stigma, lower anxiety, shorter diagnostic odyssey, greater physical function, and less pain interference. Adults and children with RDs had significantly poorer health-related quality of life and stigma in all domains compared to US norms. Predictors of both anxiety and depression were greater stigma/poor peer relationships, fatigue, sleep disturbance, limited ability to participate in social roles, and unstable disease course. CONCLUSIONS: People in the U.S. with RDs have poor health-related quality of life and high stigma. These factors are related to patient satisfaction and healthcare access, including diagnostic delays and misdiagnosis. Advocacy work is needed in order to improve healthcare access and ultimately health-related quality of life for children and adults with RDs.

Tropomyosin dephosphorylation results in compensated cardiac hypertrophy.

Phosphorylation of tropomyosin (Tm) has been shown to vary in mouse models of cardiac hypertrophy. Little is known about the in vivo role of Tm phosphorylation. This study examines the consequences of Tm dephosphorylation in the murine heart. Transgenic (TG) mice were generated with cardiac specific expression of α-Tm with serine 283, the phosphorylation site of Tm, mutated to alanine. Echocardiographic analysis and cardiomyocyte cross-sectional area measurements show that α-Tm S283A TG mice exhibit a hypertrophic phenotype at basal levels. Interestingly, there are no alterations in cardiac function, myofilament calcium (Ca(2+)) sensitivity, cooperativity, or response to ß-adrenergic stimulus. Studies of Ca(2+) handling proteins show significant increases in sarcoplasmic reticulum ATPase (SERCA2a) protein expression and an increase in phospholamban phosphorylation at serine 16, similar to hearts under exercise training. Compared with controls, the decrease in phosphorylation of α-Tm results in greater functional defects in TG animals stressed by transaortic constriction to induce pressure overload-hypertrophy. This is the first study to investigate the in vivo role of Tm dephosphorylation under both normal and cardiac stress conditions, documenting a role for Tm dephosphorylation in the maintenance of a compensated or physiological phenotype. Collectively, these results suggest that modification of the Tm phosphorylation status in the heart, depending upon the cardiac state/condition, may modulate the development of cardiac hypertrophy.

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments.

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca(2+)-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca(2+)-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca(2+) and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.

Structural basis for the regulation of muscle contraction by troponin and tropomyosin.

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca2+ binding to TnC (the Ca2+ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the "inhibitory" subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.

Identification of a region of troponin I important in signaling cross-bridge-dependent activation of cardiac myofilaments.

Force generating strong cross-bridges are required to fully activate cardiac thin filaments, but the molecular signaling mechanism remains unclear. Evidence demonstrating differential extents of cross-bridge-dependent activation of force, especially at acidic pH, in myofilaments in which slow skeletal troponin I (ssTnI) replaced cardiac TnI (cTnI) indicates the significance of a His in ssTnI that is an homologous Ala in cTnI. We compared cross-bridge-dependent activation in myofilaments regulated by cTnI, ssTnI, cTnI(A66H), or ssTnI(H34A). A drop from pH 7.0 to 6.5 induced enhanced cross-bridge-dependent activation in cTnI myofilaments, but depressed activation in cTnI(A66H) myofilaments. This same drop in pH depressed cross-bridge-dependent activation in both ssTnI myofilaments and ssTnI(H34A) myofilaments. Compared with controls, cTnI(A66H) myofilaments were desensitized to Ca(2+), whereas there was no difference in the Ca(2+)-force relationship between ssTnI and ssTnI(H34A) myofilaments. The mutations in cTnI and ssTnI did not affect Ca(2+) dissociation rates from cTnC at pH 7.0 or 6.5. However, at pH 6.5, cTnI(A66H) had lower affinity for cTnT than cTnI. We also probed cross-bridge-dependent activation in myofilaments regulated by cTnI(Q56A). Myofilaments containing cTnI(Q56A) demonstrated cross-bridge-dependent activation that was similar to controls containing cTnI at pH 7.0 and an enhanced cross-bridge-dependent activation at pH 6.5. We conclude that a localized N-terminal region of TnI comprised of amino acids 33-80, which interacts with C-terminal regions of cTnC and cTnT, is of particular significance in transducing signaling of thin filament activation by strong cross-bridges.

Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart.

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.