BackgroundHigh dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in normotensive rats converts renal HS into a pro-inflammatory phenotype, able to bind more sodium and orchestrate inflammation, fibrosis and lymphangiogenesis.MethodsWistar rats received a normal diet for 4 weeks, or 8% NaCl diet for 2 or 4 weeks. Blood pressure was monitored, and plasma, urine and tissue collected. Tissue sodium was measured by flame spectroscopy. Renal HS and tubulo-interstitial remodeling were studied by biochemical, immunohistochemical and qRT-PCR approaches.ResultsHigh sodium rats showed a transient increase in blood pressure (week 1; p<0.01) and increased sodium excretion (p<0.05) at 2 and 4 weeks compared to controls. Tubulo-interstitial T-cells, myofibroblasts and mRNA levels of VCAM1, TGF-β1 and collagen type III significantly increased after 4 weeks (all p<0.05). There was a trend for increased macrophage infiltration and lymphangiogenesis (both p = 0.07). Despite increased dermal sodium over time (p<0.05), renal concentrations remained stable. Renal HS of high sodium rats showed increased sulfation (p = 0.05), increased L-selectin binding to HS (p<0,05), and a reduction of sulfation-sensitive anti-HS mAbs JM403 (p<0.001) and 10E4 (p<0.01). Hyaluronan expression increased under high salt conditions (p<0.01) without significant changes in the chondroitin sulfate proteoglycan versican. Statistical analyses showed that sodium-induced tissue remodeling responses partly correlated with observed HS changes.ConclusionWe show that high salt intake by healthy normotensive rats convert renal HS into high sulfated pro-inflammatory glycans involved in tissue remodeling events, but not in increased sodium storage.

Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not yet clear. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately in a rat model to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in 3-month old male Wistar rats by adriamycin injection. After 6 weeks, when proteinuria has developed, rats were treated for another 6 weeks by anti-VEGFR3 antibody, clodronate liposomes or FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however, without significant effects on inflammatory and fibrotic markers or proteinuria. Clodronate liposomes inhibited macrophage influx and partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation, T-cell influx and interstitial fibrosis, and partially reduced macrophage number and proteinuria; however, it did not significantly influence lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or the influx of macrophages. On the other hand, FTY720 treatment did prevent T-cell influx, myofibroblast accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions.

BackgroundExcess dietary sodium is not only excreted by the kidneys, but can also be stored by non-osmotic binding with glycosaminoglycans in dermal connective tissue. Such storage has been associated with dermal inflammation and lymphangiogenesis. We aim to investigate if skin storage of sodium is increased in kidney patients and if this storage is associated with clinical parameters of sodium homeostasis and dermal tissue remodeling.MethodsAbdominal skin tissue of 12 kidney patients (5 on hemodialysis) and 12 healthy kidney donors was obtained during surgery. Skin biopsies were processed for dermal sodium measurement by atomic absorption spectroscopy, and evaluated for CD68+ macrophages, CD3+ T-cells, collagen I, podoplanin + lymph vessels, and glycosaminoglycans by qRT-PCR and immunohistochemistry.ResultsDermal sodium content of kidney patients did not differ from healthy individuals, but was inversely associated with plasma sodium values (p < 0.05). Compared to controls, kidney patients showed dermal tissue remodeling by increased CD68+ macrophages, CD3+ T-cells and Collagen I expression (all p < 0.05). Also, both N- and O-sulfation of heparan sulfate glycosaminoglycans were increased (all p < 0.05), most outspoken in hemodialysis patients. Plasma and urinary sodium associates with dermal lymph vessel number (both p < 0.05), whereas loss of eGFR, proteinuria and high systolic blood pressure associated with dermal macrophage density (all p < 0.05).ConclusionKidney patients did not show increased skin sodium storage compared to healthy individuals. Results do indicate that kidney failure associates with dermal inflammation, whereas increased sodium excretion and plasma sodium associate with dermal lymph vessel formation and loss of dermal sodium storage capacity.Trial registration The cohort is registered at clinicaltrials.gov as NCT (September 6, 2017). NCT, NCT03272841. Registered 6 September 2017—Retrospectively registered, https://clinicaltrials.gov