Priya Ravikumar

Renal Production, Uptake, and Handling of Circulating αKlotho

αKlotho is a multifunctional protein highly expressed in the kidney. Soluble αKlotho is released through cleavage of the extracellular domain from membrane αKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating αKlotho production and handling is incompletely understood, however. Here, we found higher αKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum αKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of αKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous αKlotho in anephric rats was four- to five-fold longer than that in normal rats, and exogenously injected labeled recombinant αKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that αKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in αKlotho homeostasis, producing and releasing αKlotho into the circulation and clearing αKlotho from the blood into the urinary lumen.

α-Klotho protects against oxidative damage in pulmonary epithelia

α-Klotho exerts pleiotropic biological actions. Heterozygous α-Klotho haplo-insufficient mice (kl/+) appear normal at baseline except for age-related changes in the lung, suggesting heightened pulmonary susceptibility to α-Klotho deficiency. We used in vivo and in vitro models to test whether α-Klotho protects lung epithelia against injury. Normally, α-Klotho is not expressed in the lung, but circulating α-Klotho levels are reduced -40% in kl/+ mice and undetectable in homozygous α-Klotho-deficient mice (kl/kl). kl/+ mice show distal air space enlargement at a given airway pressure, with elevated lung oxidative damage marker (8-hydroxydeoxyguanosine; 8-OHdG); these abnormalities are exacerbated in kl/kl mice. Studies were performed in A549 lung epithelial cells and/or primary culture of alveolar epithelial cells. Hyperoxia (95% O2) and high inorganic phosphate concentrations (Pi, 3-5 mM) additively caused cell injury (lactate dehydrogenase release), oxidative DNA damage (8-OHdG), lipid oxidation (8-isoprostane), protein oxidation (carbonyl), and apoptosis (caspase-8 activity and TUNEL stain). Transfection of transmembrane or soluble α-Klotho, or addition of soluble α-Klotho-containing conditioned media, increased cellular antioxidant capacity (Cu- and Fe-based assays) via increased nuclear factor erythroid-derived 2-related factors 1 and 2 (Nrf1/2) transcriptional activity and ameliorated hyperoxic and phosphotoxic injury. To validate the findings in vivo, we injected α-Klotho-containing conditioned media into rat peritoneum before and during hyperoxia exposure and found reduced alveolar interstitial edema and oxidative damage. We conclude that circulating α-Klotho protects the lung against oxidative damage and apoptosis partly via increasing endogenous antioxidative capacity in pulmonary epithelia. Cytoprotection by α-Klotho may play an important role in degenerative diseases of the lung.

Fatty diabetic lung: altered alveolar structure and surfactant protein expression.

Pulmonary dysfunction develops in type 2 diabetes mellitus (T2DM) in direct correlation with glycemia and is exacerbated by obesity; however, the associated structural derangement has not been quantified. We studied lungs from obese diabetic (fa/fa) male Zucker diabetic fatty (ZDF) rats at 4, 12, and 36 wk of age, before and after onset of T2DM, compared with lean nondiabetic (+/+) rats. Surfactant proteins A and C (SP-A and SP-C) immunoexpression in lung tissue was quantified at ages 14 and 18 wk, after the onset of T2DM. In fa/fa animals, lung volume was normal despite obesity. Numerous lipid droplets were visible within alveolar interstitium, lipofibroblasts, and macrophages, particularly in subpleural regions. Total triglyceride content was 136% higher. By 12 wk, septum volume was 21% higher, and alveolar duct volume was 36% lower. Capillary basement membrane was 29% thicker. Volume of lamellar bodies was 45% higher. By age 36 wk, volumes of interstitial collagen fibers, cells, and matrix were respectively 32, 25, and 80% higher, and capillary blood volume was 18% lower. ZDF rats exhibited a strain-specific increase in resistance of the air-blood diffusion barrier with age, which was exaggerated in fa/fa lungs compared with +/+ lungs. In fa/fa lungs, SP-A and SP-C expression were elevated at age 14-18 wk; the normal age-related increase in SP-A expression was accelerated, whereas SP-C expression declined with age. Thus lungs from obese T2DM animals develop many qualitatively similar changes as in type 1 diabetes mellitus but with extensive lipid deposition, altered alveolar type 2 cell ultrastructure, and surfactant protein expression patterns that suggest additive effects of hyperglycemia and lipotoxicity.

Polymeric nanoparticles for pulmonary protein and DNA delivery

Polymeric nanoparticles (NPs) are promising carriers of biological agents to the lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vitro. We screened six polymeric NPs: gelatin, chitosan, alginate, poly(lactic-co-glycolic) acid (PLGA), PLGA-chitosan and PLGA-poly(ethylene glycol) (PEG), for inhalational protein/DNA delivery. All NPs except PLGA-PEG and alginate were <300nm in size with a bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA-PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gambles solution) over 5days. PLGA-based NPs and natural polymer NPs exhibited the highest cytocompatibility and dose-dependent in vitro uptake, respectively, by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate plasmid DNA encoding yellow fluorescent protein (YFP) or rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10days while increasing YFP expression was observed for at least 7days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery.

Noninvasive quantification of heterogeneous lung growth following extensive lung resection by high-resolution computed tomography

To quantify the in vivo magnitude and distribution of regional compensatory lung growth following extensive lung resection, we performed high-resolution computed tomography at 15- and 30-cmH(2)O transpulmonary pressures and measured air and tissue (including microvascular blood) volumes within and among lobes in six adult male foxhounds, before and after balanced 65% lung resection ( approximately 32% removed from each side). Each lobe was identified from lobar fissures. Intralobar gradients in air and tissue volumes were expressed along standardized x,y,z-coordinate axes. Fractional tissue volume (FTV) was calculated as the volume ratio of tissue/(tissue + air). Following resection compared with before, lobar air and tissue volumes increased 1.8- to 3.5-fold, and whole lung air and tissue volumes were 67 and 90% of normal, respectively. Lobar-specific compliance doubled post-resection, and whole lung-specific compliance normalized. These results are consistent with vigorous compensatory growth in all remaining lobes. Compared with pre-resection, post-resection interlobar heterogeneity of FTV, assessed from the coefficient of variation, decreased at submaximal inflation, but was unchanged at maximal inflation. The coefficient of variation of intralobar FTV gradients changed variably due to the patchy development of thickened pleura and alveolar septa, with elevated alveolar septal density and connective tissue content in posterior-caudal and peripheral regions of the remaining lobes; these areas likely experienced disproportional mechanical stress. We conclude that HRCT can noninvasively and quantitatively assess the magnitude and spatial distribution of compensatory lung growth. Following extensive resection, heterogeneous regional mechanical lung strain may exceed the level that could be sustained solely by existing connective tissue elements.

Permanent alveolar remodeling in canine lung induced by high-altitude residence during maturation.

Young canines born at sea level (SL) and raised for 5 mo at high altitude (HA, 3,800 m), followed by return to SL before somatic maturation, showed enhanced alveolar gas exchange and diffusing capacity at rest and exercise that persisted into adulthood (McDonough P, Dane DM, Hsia CC, Yilmaz C, Johnson RL Jr. J Appl Physiol 100: 474-81, 2006; Hsia CCW, Johnson RL Jr, McDonough P, Dane DM, Hurst MD, Fehmel JL, Wagner HE, Wagner PD. J Appl Physiol 102: 1448-55, 2007). To examine the associated structural response, we quantified lung ultrastructure in male foxhounds raised at 3,800 m HA or their littermates raised at SL (n = 6 each) from 2.5 to 7.5 mo of age. Three years following return to SL, lungs were fixed for morphometric analysis. In HA-exposed animals compared with SL controls, lung volume at a given inflation pressure was higher with enlargement of alveolar ducts and sacs without significant differences in the volumes of alveolar cell components, septal tissue, or in alveolar-capillary surface areas. There was a shift toward a significantly lower harmonic mean thickness of the blood-gas diffusion barrier in HA-raised animals. As a control organ, muscle capillary length density of costal diaphragm was significantly higher in HA-raised animals, indicating parallel adaptation in oxygen transport organs. We conclude that, in actively growing animals, 5 mo of HA exposure that was discontinued before somatic maturation induced acinar remodeling that increased lung compliance and reduced the resistance of blood-gas diffusion barrier to diffusion that persisted into adulthood, but without permanent enhancement of alveolar tissue growth.

Long-term post-pneumonectomy pulmonary adaptation following all-trans-retinoic acid supplementation

In adult dogs following right pneumonectomy (PNX) and receiving all-trans-retinoic acid (RA) supplementation for 4 mo, we found modestly enhanced alveolar-capillary growth in the remaining lung without enhanced resting lung function (J Appl Physiol 96: 1080-1089 and 96: 1090-1096, 2004). Since alveolar remodeling progresses beyond this period and the lipid-soluble RA continues to be released from tissue stores, we hypothesized that RA supplementation may exert additional long-term effects. To examine this issue, adult male litter-matched foxhounds underwent right PNX followed by RA supplementation (2 mg/kg po 4 days/wk, n = 6) or placebo (n = 4) for 4 mo. Cardiopulmonary function was measured at rest and during exercise at 4 and 20 mo post-PNX. The remaining lung was fixed under a constant airway pressure for morphometric analysis. Comparing RA treatment to placebo controls, there were no differences in aerobic capacity, cardiopulmonary function, or lung volume at rest or exercise. Alveolar-capillary basal lamina thickness and mean harmonic thickness of air-blood diffusion barrier were 23-29% higher. The prevalence of double-capillary profiles remained 82% higher. Absolute volumes of septal interstitium, collagen fibers, cells, and matrix were 32% higher; the relative volumes of other septal components and alveolar-capillary surface areas expressed as ratios to control values were up to 24% higher. Thus RA supplementation following right PNX modestly and persistently enhanced long-term alveolar-capillary structural dimensions, especially the deposition of interstitial and connective tissue elements, in such a way that caused a net increase in barrier resistance to diffusion without improving lung mechanics or gas exchange.

Separating in vivo mechanical stimuli for postpneumonectomy compensation: imaging and ultrastructural assessment

Following right pneumonectomy (PNX), the remaining lung expands and its perfusion more than doubles. Tissue and microvascular mechanical stresses are putative stimuli for compensatory lung growth and remodeling, but their relative contribution remains uncertain. To temporally separate expansion- and perfusion-related stimuli, we replaced the right lung of adult dogs with a customized inflated prosthesis. Four months later, the prosthesis was either acutely deflated (DEF) or kept inflated (INF). Thoracic high-resolution computed tomography (HRCT) was performed pre- and post-PNX before and after prosthesis deflation. Lungs were fixed for morphometric analysis ∼12 mo post-PNX. The INF prosthesis prevented mediastinal shift and lateral lung expansion while allowing the remaining lung to expand 27-38% via caudal elongation, associated with reversible capillary congestion in dependent regions at low inflation and 40-60% increases in the volumes of alveolar sepal cells, matrix, and fibers. Delayed prosthesis deflation led to further significant increases in lung volume, alveolar tissue volumes, and alveolar-capillary surface areas. At postmortem, alveolar tissue volumes were 33% higher in the DEF than the INF group. Lateral expansion explains ∼65% of the total post-PNX increase in left lung volume assessed in vivo or ex vivo, ∼36% of the increase in HRCT-derived (tissue + microvascular blood) volume, ∼45% of the increase in ex vivo septal extravascular tissue volume, and 60% of the increase in gas exchange surface areas. This partition agrees with independent physiological measurements obtained in these animals. We conclude that in vivo signals related to lung expansion and perfusion contribute separately and nearly equally to post-PNX growth and remodeling.

Progressive adaptation in regional parenchyma mechanics following extensive lung resection assessed by functional computed tomography

In adult canines following major lung resection, the remaining lobes expand asymmetrically, associated with alveolar tissue regrowth, remodeling, and progressive functional compensation over many months. To permit noninvasive longitudinal assessment of regional growth and function, we performed serial high-resolution computed tomography (HRCT) on six male dogs (∼9 mo old, 25.0 ± 4.5 kg, ±SD) at 15 and 30 cmH(2)O transpulmonary pressure (Ptp) before resection (PRE) and 3 and 15 mo postresection (POST3 and POST15, respectively) of 65-70% of lung units. At POST3, lobar air volume increased 83-148% and tissue (including microvascular blood) volume 120-234% above PRE values without further changes at POST15. Lobar-specific compliance (Cs) increased 52-137% from PRE to POST3 and 28-79% from POST3 to POST15. Inflation-related parenchyma strain and shear were estimated by detailed registration of corresponding anatomical features at each Ptp. Within each lobe, regional displacement was most pronounced at the caudal region, whereas strain was pronounced in the periphery. Regional three-dimensional strain magnitudes increased heterogeneously from PRE to POST3, with further medial-lateral increases from POST3 to POST15. Lobar principal strains (PSs) were unchanged or modestly elevated postresection; changes in lobar maximum PS correlated inversely with changes in lobar air and tissue volumes. Lobar shear distortion increased in coronal and transverse planes at POST3 without further changes thereafter. These results establish a novel use of functional HRCT to map heterogeneous regional deformation during compensatory lung growth and illustrate a stimulus-response feedback loop whereby postresection mechanical stress initiates differential lobar regrowth and sustained remodeling, which in turn, relieves parenchyma stress and strain, resulting in progressive increases in lobar Cs and a delayed increase in whole lung Cs.

Defining a stimuli-response relationship in compensatory lung growth following major resection

Major lung resection is a robust model that mimics the consequences of loss-of-functioning lung units. We previously observed in adult canines, following 42% and 58% lung resection, a critical threshold of stimuli intensity for the initiation of compensatory lung growth. To define the range and limits of this stimuli-response relationship, we performed morphometric analysis on the remaining lobes of adult dogs, 2-3 years after surgical removal of ∼ 70% of lung units in the presence or absence of mediastinal shift. Results were expressed as ratios to that in corresponding control lobes. Lobar expansion and extravascular tissue growth (∼ 3.8- and ∼ 2.0-fold of normal, respectively) were heterogeneous; the lobes remaining next to the diaphragm exhibited a greater response. Tissue growth and capillary formation, indexed by double-capillary profiles, increased, regardless of mediastinal shift. Septal collagen fibers increased up to 2.7-fold, suggesting a greater need for structural support. Compared with previous cohorts following less-extensive resection, tissue volume and gas-exchange surface areas increased significantly only in the infracardiac lobe following 42% resection, exceeded two- to threefold in all lobes following 58% resection, and then exhibited diminished gains following ∼ 70% resection. In contrast, alveolar-capillary formation increased with incremental resection without reaching an upper limit. Overall structural regrowth was most vigorous and uniform following 58% resection. The diminishment of gains in tissue growth, following ∼ 70% resection, could reflect excessive or maldistributed mechanical stress that threatens septal integrity. Results also suggest additional independent stimuli of alveolar-capillary formation, possibly related to the postresection augmentation of regional perfusion.