Danielle E. Soranno

Secondary Photocrosslinking of Injectable Shear-Thinning Dock-and-Lock Hydrogels

Shear-thinning hydrogels are useful in numerous applications, including as injectable carriers that act as scaffolds to support cell and drug therapies. Here, we describe the engineering of a self-assembling Dock-and-Lock (DnL) system that forms injectable shear-thinning hydrogels using molecular recognition interactions that also possess photo-triggerable secondary crosslinks. These DnL hydrogels are fabricated from peptide-modified hyaluronic acid (HA) and polypeptide precursors, can self-heal immediately after shear induced flow, are cytocompatible, and can be stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and erosion kinetics. Secondary crosslinked hydrogels retain self-adhesive properties and exhibit cooperative physical and chemical crosslinks with moduli as high as ∼10 times larger than moduli of gels based on physical crosslinking alone. The extent of reaction and change in properties are dependent on whether the methacrylate is incorporated either at the terminus of the peptide or directly to the HA backbone. Additionally, the gel erosion can be monitored through an incorporated fluorophore and physical-chemical gels remain intact in solution over months, whereas physical gels that are not covalently crosslinked erode completely within days. Mesenchymal stem cells exhibit increased viability when cultured in physical- chemical gels, compared with those cultured in gels based on physical crosslinks alone. The physical properties of these DnL gels may be additionally tuned by adjusting component compositions, which allows DnL gels with a wide range of physical properties to be constructed for use.

Magnitude and presentation of mechanical signals influence adult stem cell behavior in 3-dimensional macroporous hydrogels

The mechanical properties of the microenvironment are being recognized as a key contributor to stem cell behaviour, whether in the context of tissues or when designing biomaterials. While there has been considerable evidence demonstrating the effect of 2-D static mechanics on stem cells, few systems exist to investigate the influence of the 3-D presentation of mechanical signals. In this study, methacrylated hyaluronic acid (MeHA) was processed into porous (crosslinking around spherical templates) 3-D hydrogels with tunable elastic moduli ranging from ∼1.5 kPa to 12.4 kPa. Porous hydrogels were fabricated with a sequential crosslinking process (addition crosslinking of methacrylates with dithiols, followed by UV photopolymerization) where the hydrogel mechanics are controlled by the extent of UV exposure and are subsequently seeded with human mesenchymal stem cells (hMSCs). hMSCs spread within the pores and proliferated in a mechanically dependent manner as cells within the softest 1.5 kPa hydrogels were less spread initially and showed little proliferation with time, while hMSCs in stiffer hydrogels (>1.5 kPa) had higher initial spreading and a roughly two-fold increase in cell number after 14 days. In growth media, porous hydrogels supported a slight upregulation (two to eight-fold) in chondrogenic genes across all mechanics, while there was only modest upregulation in a few conditions (and in many cases downregulation) for myogenic, osteogenic, and adipogenic genes. The secretion of various cytokines and angiogenic molecules was found to be mechanically dependent in the porous system with greater secretion at day 2 in the stiffer hydrogels (3.8 kPa and 7.4 kPa). However, by day 14 there was greater secretion in the softer hydrogels (1.5 kPa and 2.6 kPa). Finally, when mechanics were temporally increased during culture (from ∼2.6 kPa to 12.4 kPa), there was a noticeable decrease in the secretion of 15 angiogenic and cytokine proteins. Thus, the influence of mechanics on stem cells within hydrogel structures appears to be dependent on the magnitude and timing of presentation.

Incidence and outcomes of neonatal acute kidney injury (AWAKEN): a multicentre, multinational, observational cohort study

Background Single-center studies suggest that neonatal acute kidney injury (AKI) is associated with poor outcomes. However, inferences regarding the association between AKI, mortality, and hospital length of stay are limited due to the small sample size of those studies. In order to determine whether neonatal AKI is independently associated with increased mortality and longer hospital stay, we analyzed the Assessment of Worldwide Acute Kidney Epidemiology in Neonates (AWAKEN) database. Methods All neonates admitted to 24 participating neonatal intensive care units from four countries (Australia, Canada, India, United States) between January 1 and March 31, 2014, were screened. Of 4273 neonates screened, 2022 (47·3%) met study criteria. Exclusion criteria included: no intravenous fluids ≥48 hours, admission ≥14 days of life, congenital heart disease requiring surgical repair at <7 days of life, lethal chromosomal anomaly, death within 48 hours, inability to determine AKI status or severe congenital kidney abnormalities. AKI was defined using a standardized definition —i.e., serum creatinine rise of ≥0.3 mg/dL (26.5 mcmol/L) or ≥50% from previous lowest value, and/or if urine output was <1 mL/kg/h on postnatal days 2 to 7. Findings Incidence of AKI was 605/2022 (29·9%). Rates varied by gestational age groups (i.e., ≥22 to <29 weeks =47·9%; ≥29 to <36 weeks =18·3%; and ≥36 weeks =36·7%). Even after adjusting for multiple potential confounding factors, infants with AKI had higher mortality compared to those without AKI [(59/605 (9·7%) vs. 20/1417 (1·4%); p< 0.001; adjusted OR=4·6 (95% CI=2·5–8·3); p=<0·0001], and longer hospital stay [adjusted parameter estimate 8·8 days (95% CI=6·1–11·5); p<0·0001]. Interpretation Neonatal AKI is a common and independent risk factor for mortality and longer hospital stay. These data suggest that neonates may be impacted by AKI in a manner similar to pediatric and adult patients. Funding US National Institutes of Health, University of Alabama at Birmingham, Cincinnati Children’s, University of New Mexico.

Assessment of Worldwide Acute Kidney Injury Epidemiology in Neonates: Design of a Retrospective Cohort Study

Introduction Acute kidney injury (AKI) affects ~30% of hospitalized neonates. Critical to advancing our understanding of neonatal AKI is collaborative research among neonatologists and nephrologists. The Neonatal Kidney Collaborative (NKC) is an international, multidisciplinary group dedicated to investigating neonatal AKI. The AWAKEN study (Assessment of Worldwide Acute Kidney injury Epidemiology in Neonates) was designed to describe the epidemiology of neonatal AKI, validate the definition of neonatal AKI, identify primary risk factors for neonatal AKI, and investigate the contribution of fluid management to AKI events and short-term outcomes. Methods and analysis The NKC was established with at least one pediatric nephrologist and neonatologist from 24 institutions in 4 countries (USA, Canada, Australia, and India). A Steering Committee and four subcommittees were created. The database subcommittee oversaw the development of the web-based database (MediData Rave™) that captured all NICU admissions from 1/1/14 to 3/31/14. Inclusion and exclusion criteria were applied to eliminate neonates with a low likelihood of AKI. Data collection included: (1) baseline demographic information; (2) daily physiologic parameters and care received during the first week of life; (3) weekly “snapshots”; (4) discharge information including growth parameters, final diagnoses, discharge medications, and need for renal replacement therapy; and (5) all serum creatinine values. Ethics and dissemination AWAKEN was proposed as human subjects research. The study design allowed for a waiver of informed consent/parental permission. NKC investigators will disseminate data through peer-reviewed publications and educational conferences. Discussion The purpose of this publication is to describe the formation of the NKC, the establishment of the AWAKEN cohort and database, future directions, and a few “lessons learned.” The AWAKEN database includes ~325 unique variables and >4 million discrete data points. AWAKEN will be the largest, most inclusive neonatal AKI study to date. In addition to validating the neonatal AKI definition and identifying risk factors for AKI, this study will uncover variations in practice patterns related to fluid provision, renal function monitoring, and involvement of pediatric nephrologists during hospitalization. The AWAKEN study will position the NKC to achieve the long-term goal of improving the lives, health, and well-being of newborns at risk for kidney disease.

Intravenous acyclovir and renal dysfunction in children: a matched case control study.

OBJECTIVES A cluster of children receiving intravenous (IV) acyclovir for meningoencephalitis developed acute renal failure in April-May 2008, which prompted a retrospective case-control study to determine the rate of and risk factors for acute nephrotoxicity during IV acyclovir treatment in children. STUDY DESIGN The percentage decrease in glomerular filtration rate in children receiving IV acyclovir who had ≥ 1 creatinine measurement after acyclovir initiation from October 2006 to January 2009 was classified as renal risk, injury, or failure according to modified Pediatric Risk Injury, Failure, Loss, End-Stage Renal Disease criteria. Univariate and multivariate matched analyses were conducted to identify risk factors contributing to nephrotoxicity. RESULTS In the selected study group, renal dysfunction was seen in 131 of 373 (35%) treatment courses studied: 81 of 373 (22%) risk, 36 of 373 (9.7%) injury, and 14 of 373 (3.8%) failure. Most renal dysfunction occurred within 48 hours of the initiation of acyclovir. Renal function returned to the normal range but not to baseline in most cases during the follow-up period. Risk factors for renal dysfunction included acyclovir dose >15 mg/kg (OR 3.81, 95% CI 1.55-9.37) for risk; cumulative exposure greater than calculated cumulative exposure based on 500 mg/m(2)/dose (OR 6.00, 95% CI 1.95-18.46) for injury; and age >8 years (OR 21.5, 95% CI 2.2, >1000) and ceftriaxone coadministration (OR 19.3, 95% CI 1.8, >1000) for failure. CONCLUSIONS Nephrotoxicity associated with IV acyclovir is common and necessitates renal function monitoring. Risk factors include greater dose, older age, and concomitant ceftriaxone administration. Outside the neonatal period, renal dysfunction may be minimized by dosing IV acyclovir below thresholds associated with nephrotoxicity (ie, ≤ 500 mg/m(2)/dose or ≤ 15 mg/kg/dose), particularly in older patients.

Local immunotherapy via delivery of interleukin-10 and transforming growth factor β antagonist for treatment of chronic kidney disease.

Obstructive nephropathy is the leading cause of kidney disease in children. The tissue injury resulting from initial dilation precipitates a deleterious cascade of macrophage infiltration, apoptosis, and fibrosis to produce a resultant dysfunctional tissue. We propose to abate this tissue remodeling process through immunotherapy administered via the local and sustained delivery of interleukin-10 (IL-10; anti-inflammatory) and anti-transforming growth factor β (anti-TGFβ; anti-fibrotic). Shear-thinning, injectable hyaluronic acid (HA) hydrogels were formed through supramolecular guest-host interactions and used to contain IL-10, anti-TGFβ, or both molecules together. Degradation assays demonstrated that diffusive molecule release was associated with concurrent hydrogel erosion and was sustained for up to 3weeks in vitro. Erosion was likewise monitored in vivo by non-invasive optical imaging, where gel localization to the affected tissue was observed with near complete clearance by day 18. Hydrogels were applied to a murine model of chronic kidney disease, with subcapsular hydrogel injections acting as a delivery depot. Quantitative histological analysis (days 7, 21, and 35) was used to evaluate treatment efficacy. Notably, results demonstrated reduced macrophage infiltration beyond day 7 in treatment groups and reduced apoptosis at day 21, relative to untreated unilateral ureteral obstruction disease model. Fibrosis was reduced at the 35day timepoint in groups treated with IL-10 or anti-TGFβ alone, but not with the combination therapy. Rather, dual delivery of IL-10 and anti-TGFβ resulted in a paradoxical hastening of fibrosis, warranting further investigation. Localized immunotherapy is a novel approach to treat kidney disease and shows promise as a translatable therapy.

Immunotherapy with injectable hydrogels to treat obstructive nephropathy

Hydrogels are gaining attention as injectable vehicles for delivery of therapeutics for a range of applications. We describe self-assembling and injectable Dock-and-Lock hydrogels for local delivery of interleukin-10 (IL-10) to abate the progression of inflammation and fibrosis that leads to chronic kidney disease. As monitored with a fluorescent tag, hydrogels degraded within a few days in vitro and matched IL-10 release profiles; however, hydrogels remained in the kidney for up to 30 days in vivo. A unilateral ureteral obstruction (UUO) mouse model was used to investigate in vivo outcomes after hydrogel injection and IL-10 delivery. Eight groups were investigated (7, 21, 35 days, n = 4): healthy, sham, healthy injected with mouse serum albumin (MSA), healthy + hydrogel, UUO, UUO + IL-10, UUO + hydrogel, UUO + hydrogel/IL-10. 15 μL of IL-10, hydrogel, or hydrogel/IL-10 was injected under the renal capsule 3 days after the UUO. Immunohistochemistry (IHC) was performed on paraffin sections to identify macrophages and apoptotic cells and trichrome staining was used to evaluate fibrosis. There were no significant differences in inflammatory markers between all control groups. With hydrogel delivery, macrophage infiltration and apoptosis were significantly reduced at days 21 and 35 compared to untreated animals. By day 35, IL-10 delivery via hydrogel reduced macrophage infiltration and apoptosis more than IL-10 injection alone. Fibrosis was decreased by day 35 in all treatment groups. This work supports the use of hydrogel delivery of IL-10 to treat chronic kidney disease.

Prolonged acute kidney injury exacerbates lung inflammation at 7 days post-acute kidney injury

Patients with acute kidney injury (AKI) have increased mortality; data suggest that the duration, not just severity, of AKI predicts increased mortality. Animal models suggest that AKI is a multisystem disease that deleteriously affects the lungs, heart, brain, intestine, and liver; notably, these effects have only been examined within 48 h, and longer term effects are unknown. In this study, we examined the longer term systemic effects of AKI, with a focus on lung injury. Mice were studied 7 days after an episode of ischemic AKI (22 min of renal pedicle clamping and then reperfusion) and numerous derangements were present including (1) lung inflammation; (2) increased serum proinflammatory cytokines; (3) liver injury; and (4) increased muscle catabolism. Since fluid overload may cause respiratory complications post‐AKI and fluid management is a critical component of post‐AKI care, we investigated various fluid administration strategies in the development of lung inflammation post‐AKI. Four different fluid strategies were tested – 100, 500, 1000, or 2000 μL of saline administered subcutaneously daily for 7 days. Interestingly, at 7 days post‐AKI, the 1000 and 2000 μL fluid groups had less severe AKI and less severe lung inflammation versus the 100 and 500 μL groups. In summary, our data demonstrate that appropriate fluid management after an episode of ischemic AKI led to both (1) faster recovery of kidney function and (2) significantly reduced lung inflammation, consistent with the notion that interventions to shorten AKI duration have the potential to reduce complications and improve patient outcomes.

Delivery of interleukin-10 via injectable hydrogels improves renal outcomes and reduces systemic inflammation following ischemic acute kidney injury in mice

Injectable hydrogels can be used to deliver drugs in situ over a sustained period of time. We hypothesized that sustained delivery of interleukin-10 (IL-10) following acute kidney injury (AKI) would mitigate the local and systemic proinflammatory cascade induced by AKI and reduce subsequent fibrosis. Wild-type C57BL/6 mice underwent ischemia-reperfusion AKI with avertin anesthesia. Three days later, mice were treated with either hyaluronic acid injectable hydrogel with or without IL-10, or IL-10 suspended in saline, injected under the capsule of the left kidney, or hydrogel with IL-10 injected subcutaneously. Untreated AKI served as controls. Serial in vivo optical imaging tracked the location and degradation of the hydrogel over time. Kidney function was assessed serially. Animals were killed 28 days following AKI and the following were evaluated: serum IL-6, lung inflammation, urine neutrophil gelatinase-associated lipocalin, and renal histology for fibroblast activity, collagen type III deposition and fibrosis via Picrosirius Red staining and second harmonic imaging. Our model shows persistent systemic inflammation, and renal inflammation and fibrosis 28 days following AKI. The hydrogels are biocompatible and reduced serum IL-6 and renal collagen type III 28 days following AKI even when delivered without IL-10. Treatment with IL-10 reduced renal and systemic inflammation, regardless of whether the IL-10 was delivered in a sustained manner via the injectable hydrogel under the left kidney capsule, as a bolus injection via saline under the left kidney capsule, or via the injectable hydrogel subcutaneously. Injectable hydrogels are suitable for local drug delivery following renal injury, are biocompatible, and help mitigate local and systemic inflammation.

Early peritoneal dialysis reduces lung inflammation in mice with ischemic acute kidney injury

Although dialysis has been used in the care of patients with acute kidney injury (AKI) for over 50 years, very little is known about the potential benefits of uremic control on systemic complications of AKI. Since the mortality of AKI requiring renal replacement therapy (RRT) is greater than half in the intensive care unit, a better understanding of the potential of RRT to improve outcomes is urgently needed. Therefore, we sought to develop a technically feasible and reproducible model of RRT in a mouse model of AKI. Models of low- and high-dose peritoneal dialysis (PD) were developed and their effect on AKI, systemic inflammation, and lung injury after ischemic AKI was examined. High-dose PD had no effect on AKI, but effectively cleared serum IL-6, and dramatically reduced lung inflammation, while low-dose PD had no effect on any of these three outcomes. Both models of RRT using PD in AKI in mice reliably lowered urea in a dose-dependent fashion. Thus, use of these models of PD in mice with AKI has great potential to unravel the mechanisms by which RRT may improve the systemic complications that have led to increased mortality in AKI. In light of recent data demonstrating reduced serum IL-6 and improved outcomes with prophylactic PD in children, we believe that our results are highly clinically relevant.