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Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo., (© 2023. Springer Nature Limited.)

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that has been linked to high-fructose corn syrup consumption with induction of hepatic de novo lipogenesis (DNL) as the suggested central mechanism. Feeding diets very high in fructose (> 60%) rapidly induce several features of NAFLD in rodents, but similar diets have not yet been applied in larger animals, such as pigs. With the aim to develop a large animal NAFLD model, we analysed the effects of feeding a high-fructose (HF, 60% w/w) diet for four weeks to castrated male Danish Landrace-York-Duroc pigs. HF feeding upregulated expression of hepatic DNL proteins, but levels were low compared with adipose tissue. No steatosis or hepatocellular ballooning was seen on histopathological examination, and plasma levels of transaminases were similar between groups. Inflammatory infiltrates and the amount of connective tissue was slightly elevated in liver sections from fructose-fed pigs, which was corroborated by up-regulation of macrophage marker expression in liver homogenates. Supported by RNA-profiling, quantitative protein analysis, histopathological examination, and biochemistry, our data suggest that pigs, contrary to rodents and humans, are protected against fructose-induced steatosis by relying on adipose tissue rather than liver for DNL.

The scavenger receptor CD163 is highly expressed in macrophages in sites of chronic inflammation where it has a not yet defined role. Here we have investigated development of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in CD163-deficient C57BL/6 mice. Compared to wild-type mice, the CIA in CD163-deficient mice had a several-fold higher arthritis score with early onset, prolonged disease and strongly enhanced progression. Further, the serum anti-collagen antibody isotypes as well as the cytokine profiles and T cell markers in the inflamed joints revealed that CD163-deficient mice after 52 days had a predominant Th2 response in opposition to a predominant Th1 response in CD163+/+ mice. Less difference in disease severity between the CD163+/+ and CD163-/- mice was seen in the CAIA model that to a large extent induces arthritis independently of T-cell response and endogenous Th1/Th2 balance. In conclusion, the present set of data points on a novel strong anti-inflammatory role of CD163.

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE -/- mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors.

CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma 'soluble CD163' (sCD163) has become a biomarker for macrophage activity and inflammation. The present study disclosed that 10% of sCD163 in healthy persons is actually extracellular vesicle (EV)-associated CD163 not being cleaved and shed. Endotoxin injection of human volunteers caused a selective increase in the ectodomain CD163, while septic patients exhibited high levels of both soluble ectodomain CD163 and extracellular vesicle (EV) CD163, the latter representing up 60% of total plasma CD163. A poor prognosis of septic patients measured as the sequential organ failure assessment (SOFA) score correlated with the increase in membrane-associated CD163. Our results show that soluble ectodomain CD163 and EV CD163 in plasma are part of separate macrophage response in the context of systemic inflammation. While that soluble ectodomain CD163 is released during the acute systemic inflammatory response, this is not the case for EV CD163 that instead may be released during a later phase of the inflammatory response. A separate measurement of the two forms of CD163 constituting 'soluble CD163' in plasma may therefore add to the diagnostic and prognostic value.