Vasopressin for Post-kidney Transplant Hypotension.

Introduction: Hypotension after deceased donor kidney transplant (DDKT) is a risk factor for delayed graft function (DGF) and poor graft survival (GS). We hypothesize that vasopressin use in hypotensive DDKT recipients (DDKTRs) to increase blood pressure (BP) reduces DGF rates and is safe without increasing mortality. Methods: Group with vasopressin "study group" (n = 45) was defined as DDKTRs between 2012 and 2017 who required vasopressin for hypotension systolic BP (SBP) <120 mm Hg or diastolic BP (DBP) <60 mm Hg. DDKTRs with no-vasopressin "comparison group" (n = 90) were propensity score-matched DDKTRs between 2012 and 2017 without vasopressin use. Primary outcomes were GS, creatinine and allograft biopsy rate at 1 year, DGF rate, and death during transplant hospitalization. Results: Vasopressin group had lower mean maximum and minimum SBP and DBP in the operating room (OR). Median vasopressin start time post-DDKT was 2 hours (interquartile range [IQR] 1-6), and duration of use was 42 hours (IQR 24-63). DGF, creatinine at 1 year, and allograft biopsy rates were comparable. No deaths occurred during transplant hospitalization. Multivariable analysis did not find an effect of vasopressin use on GS. Conclusion: Treatment of hypotensive DDKTRs with vasopressin is safe and facilitated similar graft function and survival with that of nonhypotensive patients. In the absence of a randomized control trial, our study supports the safety of vasopressin therapy to prevent the adverse effects of hypotension.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be interconverted.

The first exon of the huntingtin protein (HTTex1) important in Huntington's disease (HD) can form cross-ß fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-ß fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

Effects of Prolonged GRP78 Haploinsufficiency on Organ Homeostasis, Behavior, Cancer and Chemotoxic Resistance in Aged Mice.

GRP78, a multifunctional protein with potent cytoprotective properties, is an emerging therapeutic target to combat cancer development, progression and drug resistance. The biological consequences of prolonged reduction in expression of this essential chaperone which so far has been studied primarily in young mice, was investigated in older mice, as older individuals are likely to be important recipients of anti-GRP78 therapy. We followed cohorts of Grp78+/+ and Grp78+/- male and female mice up to 2 years of age in three different genetic backgrounds and characterized them with respect to body weight, organ integrity, behavioral and memory performance, cancer, inflammation and chemotoxic response. Our results reveal that body weight, organ development and integrity were not impaired in aged Grp78+/- mice. No significant effect on cancer incidence and inflammation was observed in aging mice. Interestingly, our studies detected some subtle differential trends between the WT and Grp78+/- mice in some test parameters dependent on gender and genetic background. Our studies provide the first evidence that GRP78 haploinsufficiency for up to 2 years of age has no major deleterious effect in rodents of different genetic background, supporting the merit of anti-GRP78 drugs in treatment of cancer and other diseases affecting the elderly.

Regulation of mammalian cone phototransduction by recoverin and rhodopsin kinase.

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.

Impact of tacrolimus-sirolimus maintenance immunosuppression on proteinuria and kidney function in pancreas transplant alone recipients.

BACKGROUND: Nephrotoxicity is a major complication with immunosuppression regimens used in transplantation. Calcineurin inhibitor-sparing or reduction regimens using sirolimus (SRL) have shown variable success in kidney transplantation. There is limited data on the role of SRL on native kidney function in pancreas transplantation. METHODS: All patients undergoing pancreas transplantation from 2003 to 2010 were enrolled in this study (n=65). Patient demographic characteristics were identified and divided into two groups: those receiving tacrolimus (Tac) in combination with mycophenolate mofetil (MMF) and those maintained on a regimen of Tac and SRL with or without MMF. The slopes for estimated glomerular filtration rate (eGFR), serum creatinine level (sCr), and proteinuria changes over time were assessed between groups. Urine protein and creatinine ratio (uPr/uCr) was used to assess proteinuria. RESULTS: There was no difference in baseline demographic characteristics. Patients were followed for a median of 3 years. Baseline sCr and eGFR were similar between groups. Differences in uPr/uCr and rate of change in sCr and eGFR were not significant between the groups overall or for any specific time. There was worsening of sCr, eGFR, and uPr/uCr within the groups over the period of study. There were no significant differences when groups were split by age or gender or when the SRL group was split further based on MMF inclusion. CONCLUSIONS: Our study findings suggest that using a Tac-SRL regimen in patients with pancreas alone transplantation is a safe approach and may not lead to worsening proteinuria and kidney function when compared with regimens using Tac with MMF.

Enhanced arrestin facilitates recovery and protects rods lacking rhodopsin phosphorylation.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in every cell in the body and are targeted by the majority of clinically used drugs [1]. GPCR signaling, including rhodopsin-driven phototransduction, is terminated by receptor phosphorylation followed by arrestin binding [2]. Genetic defects in receptor phosphorylation and excessive signaling by overactive GPCR mutants result in a wide variety of diseases, from retinal degeneration to cancer [3-6]. Here, we tested whether arrestin1 mutants with enhanced ability to bind active unphosphorylated rhodopsin [7-10] can suppress uncontrolled signaling, bypassing receptor phosphorylation by rhodopsin kinase (RK) and replacing this two-step mechanism with a single-step deactivation in rod photoreceptors. We show that in this precisely timed signaling system with single-photon sensitivity [11], an enhanced arrestin1 mutant partially compensates for defects in rhodopsin phosphorylation, promoting photoreceptor survival, improving functional performance, and facilitating photoresponse recovery. These proof-of-principle experiments demonstrate the feasibility of functional compensation in vivo for the first time, which is a promising approach for correcting genetic defects associated with gain-of-function mutations. Successful modification of protein-protein interactions by appropriate mutations paves the way to targeted redesign of signaling pathways to achieve desired functional outcomes.

Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin.

BACKGROUND: The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD), age-related macular degeneration (AMD), atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. RESULTS: We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE) cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. CONCLUSION: These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells.

Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.

Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions.

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

Recoverin improves rod-mediated vision by enhancing signal transmission in the mouse retina.

Vision in dim light requires that photons absorbed by rod photoreceptors evoke signals that reliably propagate through the retina. We investigated how a perturbation in rod physiology affects propagation of those signals in the retina and ultimately visual sensitivity. Recoverin is a protein in rods that prolongs phototransduction and enhances visual sensitivity. It is not present in neurons postsynaptic to rods, yet we found that light-evoked responses of rod bipolar and ganglion cells were shortened when measured in recoverin-deficient retinas. Unexpectedly, the effect of recoverin on postsynaptic signals could not be explained by its effect on phototransduction. Instead, it is an effect of recoverin downstream of phototransduction in rods that prolongs signal transmission and enhances visual sensitivity. An important implication of our findings is that the recovery phase of the rod photoresponse does not contribute significantly to visual sensitivity near absolute threshold.

Structure of membrane-bound alpha-synuclein studied by site-directed spin labeling.

Many of the proposed physiological functions of alpha-synuclein, a protein involved in the pathogenesis of Parkinson's disease, are related to its ability to interact with phospholipids. To better understand the conformational changes that occur upon membrane binding of monomeric alpha-synuclein, we performed EPR analysis of 47 singly labeled alpha-synuclein derivatives. We show that membrane interaction is mediated by major conformational changes within seven N-terminal 11-aa repeats, which reorganize from a highly dynamic structure into an elongated helical structure devoid of significant tertiary packing. Furthermore, we find that analogous positions from different repeats are in equivalent locations with respect to membrane proximity. These and other findings suggest a curved membrane-dependent alpha-helical structure, wherein each 11-aa repeat takes up three helical turns. Similar helical structures could also apply to apolipoproteins and other lipid-interacting proteins with related 11-aa repeats.

Bax is not the heterodimerization partner necessary for sustained anti-photoreceptor-cell-death activity of Bcl-2.

PURPOSE: Ectopic expression of Bcl-2 in photoreceptors of certain mouse models of retinitis pigmentosa (RP) temporarily slows disease progression. The temporary effect produced by Bcl-2 may result from insufficient levels of functional complexes between Bcl-2 and additional proteins necessary for maintaining the anti-apoptotic activity of Bcl-2. Although the overexpression of Bax generally induces apoptosis, Bax exerts anti-apoptotic properties when complexed with Bcl-2 in certain cell culture systems. These studies were designed to determine whether coexpression of Bcl-2 and Bax would improve the neuroprotective effect provided by Bcl-2 alone in photoreceptors of mice with autosomal dominant RP (adRP). METHODS: Transgenic mice were produced that overexpressed Bax and Bcl-2 specifically in photoreceptor cells, using the murine opsin promoter to drive transgene expression. These mice were crossed with an adRP mouse model to assess the effect of coexpression of Bax and Bcl-2 on retinal degeneration. Morphologic analysis was performed on retinas isolated at various developmental times to monitor disease progression. RESULTS: Ectopic expression of Bax in photoreceptors resulted in extensive rod cell death dependent on the level of Bax transgene expression. Although Bcl-2 was able to inhibit Bax-induced photoreceptor cell death, the coexpression of Bcl-2 and Bax in photoreceptors of mice with adRP did not enhance the protective effect against photoreceptor cell death exerted by Bcl-2 alone. CONCLUSIONS: Coexpression of Bax and Bcl-2, at the levels produced in the transgenic lines, does not extend the temporary neuroprotective effect produced by Bcl-2 in photoreceptors of mice with adRP.