Validation of eGFR for Detecting Associations Between Serum Protein Biomarkers and Subsequent GFR Decline.

SIGNIFICANCE STATEMENT: eGFR from creatinine, cystatin C, or both has been primarily used in search of biomarkers for GFR decline. Whether the relationships between biomarkers and eGFR decline are similar to associations with measured GFR (mGFR) decline has not been investigated. This study revealed that some biomarkers showed statistically significant different associations with eGFR decline compared with mGFR decline, particularly for eGFR from cystatin C. The findings indicate that non-GFR-related factors, such as age, sex, and body mass index, influence the relationship between biomarkers and eGFR decline. Therefore, the results of biomarker studies using eGFR, particularly eGFRcys, should be interpreted with caution and perhaps validated with mGFR. BACKGROUND: Several serum protein biomarkers have been proposed as risk factors for GFR decline using eGFR from creatinine or cystatin C. We investigated whether eGFR can be used as a surrogate end point for measured GFR (mGFR) when searching for biomarkers associated with GFR decline. METHODS: In the Renal Iohexol Clearance Survey, GFR was measured with plasma iohexol clearance in 1627 individuals without diabetes, kidney, or cardiovascular disease at baseline. After 11 years of follow-up, 1409 participants had one or more follow-up GFR measurements. Using logistic regression and interval-censored Cox regression, we analyzed the association between baseline levels of 12 serum protein biomarkers with the risk of accelerated GFR decline and incident CKD for both mGFR and eGFR. RESULTS: Several biomarkers exhibited different associations with eGFR decline compared with their association with mGFR decline. More biomarkers showed different associations with eGFRcys decline than with eGFRcre decline. Most of the different associations of eGFR decline versus mGFR decline remained statistically significant after adjustment for age, sex, and body mass index, but several were attenuated and not significant after adjusting for the corresponding baseline mGFR or eGFR. CONCLUSIONS: In studies of some serum protein biomarkers, eGFR decline may not be an appropriate surrogate outcome for mGFR decline. Although the differences from mGFR decline are attenuated by adjustment for confounding factors in most cases, some persist. Therefore, proposed biomarkers from studies using eGFR should preferably be validated with mGFR.

Serum matrix metalloproteinase 7 and accelerated glomerular filtration rate decline in a general non-diabetic population.

BACKGROUND: Age-related reduction of glomerular filtration rate (GFR) is a major contributor to the global chronic kidney disease (CKD) epidemic. We investigated whether baseline serum levels of the pro-fibrotic matrix metalloproteinase 2 (MMP2), MMP7 and their inhibitor, tissue inhibitor of metalloproteinase 1 (TIMP1), which mediates fibrosis development in aging animals, were associated with GFR decline in a general non-diabetic population. METHODS: In the Renal Iohexol Clearance Survey, we measured GFR using iohexol clearance in 1627 subjects aged 50-64 years without self-reported diabetes, kidney or cardiovascular disease. After a median of 5.6 years, 1324 had follow-up GFR measurements. Using linear mixed models and logistic regression analyses, we evaluated the association of MMP7, MMP2 and TIMP1 with the mean GFR decline rate, risk of accelerated GFR decline (defined as subjects with the 10% steepest GFR slopes: ≥1.8 mL/min/1.73 m2/year) and incident CKD [GFR <60 mL/min/1.73 m2 and/or urinary albumin to creatinine ratio (ACR) ≥3.0 mg/mmol]. RESULTS: Higher MMP7 levels (per standard deviation increase of MMP7) were associated with steeper GFR decline rates [-0.23 mL/min/1.73 m2/year (95% confidence interval -0.34 to -0.12)] and increased risk of accelerated GFR decline and incident CKD [odds ratios 1.58 (1.30-1.93) and 1.45 (1.05-2.01), respectively, in a model adjusted for age, sex, baseline GFR, ACR and cardiovascular risk factors]. MMP2 and TIMP1 showed no association with GFR decline or incident CKD. CONCLUSIONS: The pro-fibrotic biomarker MMP7, but not MMP2 or TIMP1, is associated with increased risk of accelerated GFR decline and incident CKD in middle-aged persons from the general population.

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

Dietary macronutrients and the aging liver sinusoidal endothelial cell.

Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology.

Highly oxidized albumin is cleared by liver sinusoidal endothelial cells via the receptors stabilin-1 and -2.

Oxidized albumin (oxHSA) is elevated in several pathological conditions, such as decompensated cirrhosis, acute on chronic liver failure and liver mediated renal failure. Patient derived oxidized albumin was previously shown to be an inflammatory mediator, and in normal serum levels of oxHSA are low. The removal from circulation of oxidized albumins is therefore likely required for maintenance of homeostasis. Liver sinusoidal endothelial cells (LSEC) are prominent scavenger cells specialized in removal of macromolecular waste. Given that oxidized albumin is mainly cleared by the liver, we hypothesized the LSEC are the site of uptake in the liver. In vivo oxHSA was cleared rapidly by the liver and distributed to mainly the LSEC. In in vitro studies LSEC endocytosed oxHSA much more than other cell populations isolated from the liver. Furthermore, it was shown that the uptake was mediated by the stabilins, by affinity chromatography-mass spectrometry, inhibiting uptake in LSEC with other stabilin ligands and showing uptake in HEK cells overexpressing stabilin-1 or -2. oxHSA also inhibited the uptake of other stabilin ligands, and a 2-h challenge with 100 µg/mL oxHSA reduced LSEC endocytosis by 60% up to 12 h after. Thus the LSEC and their stabilins mediate clearance of highly oxidized albumin, and oxidized albumin can downregulate their endocytic capacity in turn.

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins.

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.

Crystallized but not soluble uric acid elicits pro-inflammatory response in short-term whole blood cultures from healthy men.

Several epidemiological studies have pointed at serum uric acid (SUA) as an independent risk factor for mortality, diabetes, hypertension, cardiovascular and kidney disease; however, no clear pathogenic pathway is established. Uric acid (UA) crystals show pro-inflammatory properties and can thus create or contribute to the state of chronic low-grade inflammation, a widely accepted pathogenic mechanism in several of the above-mentioned pathologies. On the other hand, soluble uric acid possesses antioxidant properties that might attenuate inflammatory responses. We aimed to explore the net effects of experimentally rising SUA in human whole blood cultures on several mediators of inflammation. Production of TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8 was assessed upon addition of 200 µM UA, 500 µM UA or monosodium urate (MSU) crystals in the presence or absence of 5 ng/ml lipopolysaccharide (LPS). RT-qPCR and multiplex bead based immunoassay were used to measure mRNA expression and cytokine release at 2 and 4 h of culture, respectively. 14C labeled UA was used to assess intracellular uptake of UA. We show that crystallized, but not soluble, UA induces production of pro-inflammatory mediators in human whole blood. Soluble UA is internalized in blood cells but does not potentiate or reduce LPS-induced release of cytokines.

Age-related changes in the hepatic microcirculation in mice.

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

Liver aging and pseudocapillarization in a Werner syndrome mouse model.

Werner syndrome is a progeric syndrome characterized by premature atherosclerosis, diabetes, cancer, and death in humans. The knockout mouse model created by deletion of the RecQ helicase domain of the mouse Wrn homologue gene (Wrn(∆hel/∆hel)) is of great interest because it develops atherosclerosis and hypertriglyceridemia, conditions associated with aging liver and sinusoidal changes. Here, we show that Wrn(∆hel/∆hel) mice exhibit increased extracellular matrix, defenestration, decreased fenestration diameter, and changes in markers of liver sinusoidal endothelial cell inflammation, consistent with age-related pseudocapilliarization. In addition, hepatocytes are larger, have increased lipofuscin deposition, more frequent nuclear morphological anomalies, decreased mitochondria number, and increased mitochondrial diameter compared to wild-type mice. The Wrn(∆hel/∆hel) mice also have altered mitochondrial function and altered nuclei. Microarray data revealed that the Wrn(∆hel/∆hel) genotype does not affect the expression of many genes within the isolated hepatocytes or liver sinusoidal endothelial cells. This study reveals that Wrn(∆hel/∆hel) mice have accelerated typical age-related liver changes including pseudocapillarization. This confirms that pseudocapillarization of the liver sinusoid is a consistent feature of various aging models. Moreover, it implies that DNA repair may be implicated in normal aging changes in the liver.

Hepatic disposal of advanced glycation end products during maturation and aging.

UNLABELLED: Aging is characterized by progressive loss of metabolic and biochemical functions and accumulation of metabolic by-products, including advanced glycation end products (AGEs), which are observed in several pathological conditions. A number of waste macromolecules, including AGEs are taken up from the circulation by endocytosis mainly into liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). However, AGEs still accumulate in different tissues with aging, despite the presence of this clearance mechanism. The aim of the present study was to determine whether the efficiency of LSECs and KCs for disposal of AGEs changes through aging. RESULTS: After intravenous administration of (14)C-AGE-albumin in pre-pubertal, young adult, middle aged and old mice, more than 90% of total recovered (14)C-AGE was liver associated, irrespective of age. LSECs and KCs represented the main site of uptake. A fraction of the (14)C-AGE degradation products ((14)C-AGE-DPs) was stored for months in the lysosomes of these cells after uptake. The overall rate of elimination of (14)C-AGE-DPs from the liver was markedly faster in pre-pubertal than in all post-pubertal age groups. The ability to eliminate (14)C-AGE-DPs decreased to similar extents after puberty in LSECs and KCs. A rapid early removal phase was characteristic for all age groups except the old group, where this phase was absent. CONCLUSIONS: Removal of AGE-DPs from the liver scavenger cells is a very slow process that changes with age. The ability of these cells to dispose of AGEs declines after puberty. Decreased AGE removal efficiency early in life may lead to AGE accumulation.

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism.

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

The Relationship between fenestrations, sieve plates and rafts in liver sinusoidal endothelial cells.

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.

Lack of recognition of Nepsilon-(carboxymethyl)lysine by the mouse liver reticulo-endothelial system: implications for pathophysiology.

Advanced glycation end products (AGEs) are known to be associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer's disease, uremia, as well as with normal aging. This study was undertaken to investigate whether Nepsilon-(carboxymethyl)lysine (CML), a major structure among numerous AGEs, engenders hepatic AGE clearance. For this purpose uptake of BSA substituted with heterogeneous AGEs or with CML only was monitored in vivo and in cultured hepatic scavenger cells. Here, we show that following intravenous administration of 125I-AGE-BSA and 125I-CML-BSA, blood radioactivity was reduced by 50% after 50s and >100 min, respectively. Recoveries from the circulation at 6 min after injection were: 5% for AGE-BSA, 95% for CML-BSA. More than 80% of the injected AGE-BSA was recovered from the liver. AGE-BSA, but not CML-BSA, was avidly endocytosed by cultured liver scavenger cells. Our results suggest that CML does not engender AGE-BSA clearance. Macromolecules substituted with CML only may escape elimination and cause pathological effects.