Autophagy Protects From Uremic Vascular Media Calcification

Chronic kidney disease and diabetes mellitus are associated with extensive media calcification, which leads to increased cardiovascular morbidity and mortality. Here, we investigated the role of autophagy in the pathogenesis of uremic vascular media calcification. DBA/2 mice were fed with high-phosphate diet (HPD) in order to cause vascular calcification. DBA/2 mice on standard chow diet were used as control. In parallel, autophagy and its response to rapamycin, 3-methyladenine (3-MA), and bafilomycin were studied in an in vitro model using mouse vascular smooth muscle cells (MOVAS). DBA/2 mice on HPD developed severe vascular media calcification, which is mirrored in vitro by culturing MOVAS under calcifying conditions. Both, in vitro and in vivo, autophagy significantly increased in MOVAS under calcifying conditions and in aortas of HPD mice, respectively. Histologically, autophagy was located to the aortic Tunica media, but also vascular endothelial cells, and was found to continuously increase during HPD treatment. 3-MA as well as bafilomycin blocked autophagy in MOVAS and increased calcification. Vice versa, rapamycin treatment further increased autophagy and resulted in a significant decrease of vascular calcification in vitro and in vivo. Rapamycin reduced Runx2 transcription levels in aortas and MOVAS to control levels, whereas it increased α-smooth muscle actin and Sm22α transcription in MOVAS to control levels. Furthermore, rapamycin-treated HPD mice survived significantly longer compared to HPD controls. These findings indicate that autophagy is an endogenous response of vascular smooth muscle cells (VSMC) to protect from calcification in uremia. Induction of autophagy by rapamycin protects cells and mice from uremic media calcification possibly by inhibiting osteogenic transdifferentiation of VSMC

Low-Dose rIL-15 Protects from Nephrotoxic Serum Nephritis via CD8+ T Cells

Rapid progressive glomerulonephritis (GN) often leads to end-stage kidney disease, driving the need for renal replacement therapy and posing a global health burden. Low-dose cytokine-based immunotherapies provide a new strategy to treat GN. IL-15 is a strong candidate for the therapy of immune-mediated kidney disease since it has proven to be tubular-protective before. Therefore, we set out to test the potential of low-dose rIL-15 treatment in a mouse model of nephrotoxic serum nephritis (NTS), mimicking immune complex-driven GN in humans. A single low-dose treatment with rIL-15 ameliorated NTS, reflected by reduced albuminuria, less tissue scarring, fewer myeloid cells in the kidney, and improved tubular epithelial cell survival. In addition, CD8+ T cells, a primary target of IL-15, showed altered gene expression and function corresponding with less cytotoxicity mediated by rIL-15. With the use of transgenic knock-out mice, antibody depletion, and adoptive cell transfer studies, we here show that the beneficial effects of rIL-15 treatment in NTS depended on CD8+ T cells, suggesting a pivotal role for them in the underlying mechanism. Our findings add to existing evidence of the association of IL-15 with kidney health and imply a potential for low-dose rIL-15 immunotherapies in GN

Agonism of Prostaglandin E2 Receptor 4 Ameliorates Tubulointerstitial Injury in Nephrotoxic Serum Nephritis in Mice

Selectively targeting the E-type prostanoid receptor 4 (EP4) might be a new therapeutic option in the treatment of glomerulonephritis (GN), since the EP4 receptor is expressed on different immune cells, resident kidney cells, and endothelial cells, which are all involved in the pathogenesis of immune-complex GN. This study aimed to evaluate the therapeutic potential and to understand the mode of action of EP4 agonist in immune-complex GN using the murine model of nephrotoxic serum nephritis (NTS). In vivo, NTS mice were treated two times daily with two different doses of an EP4 agonist ONO AE1-329 or vehicle for 14 days total. The effect of PGE2 and EP4 agonism and antagonism was tested on murine distal convoluted tubular epithelial cells (DCT) in vitro. In vivo, the higher dose of the EP4 agonist led to an improved NTS phenotype, including a reduced tubular injury score and reduced neutrophil gelatinase-associated lipocalin (NGAL) and blood urea nitrogen (BUN) levels. EP4 agonist treatment caused decreased CD4+ T cell infiltration into the kidney and increased proliferative capacity of tubular cells. Injection of the EP4 agonist resulted in dose-dependent vasodilation and hypotensive episodes. The low-dose EP4 agonist treatment resulted in less pronounced episodes of hypotension. In vitro, EP4 agonism resulted in cAMP production and increased distal convoluted tubular (DCT) proliferation. Taken together, EP4 agonism improved the NTS phenotype by various mechanisms, including reduced blood pressure, decreased CD4+ T cell infiltration, and a direct effect on tubular cells leading to increased proliferation probably by increasing cAMP levels

The Spleen Plays No Role in Nephrotoxic Serum Nephritis, but Constitutes a Place of Compensatory Haematopoiesis

<div><p>Background</p><p>The spleen has been implicated in the pathogenesis of immune-complex glomerulonephritis by initiating and resolving adaptive immune responses. Thus, we aimed to evaluate the role of the spleen in experimental nephrotoxic serum nephritis (NTS).</p><p>Methods</p><p>In order to accelerate the disease, animals were subjected to NTS by preimmunizing male C57BL/6J mice with rabbit IgG three days before injecting the rabbit anti-glomerular basement antiserum, or were immunized only. A group underwent splenectomy before NTS induction.</p><p>Results</p><p>We observed enlargement of the spleen with a maximum at 14 days after NTS induction or immunization only. Splenectomized mice were found to develop albuminuria and renal histological changes comparable to sham-operated controls. Nevertheless, anaemia was aggravated in mice after splenectomy. During the course of NTS, we detected CD41⁺ megakaryocytes and Ter119⁺ erythroid precursor cells in the spleen of mice with NTS and of immunized mice. Ter119⁺Cxcr4⁺ cells and the binding partner Cxcl12 increased in the spleen, and decreased in the bone marrow. This was accompanied by a significant systemic increase of interferon-gamma in the serum.</p><p>Conclusions</p><p>In summary, splenectomy does not influence the course of NTS <i>per se</i>, but is involved in concomitant anaemia. Extramedullary haematopoiesis in the spleen is probably facilitated through the migration of Cxcr4⁺ erythroid precursor cells from the bone marrow to the spleen via a Cxcl12 gradient and likely arises from the suppressive capacity of chronic inflammation on the bone marrow.</p></div

Enlargement and increase in spleen weight in immunized mice and in NTS is not attributable to leukocytes.

<p>CD4⁺CD69⁺ T cells (A, B) and CD8⁺CD69⁺ T cells (C, D) were analysed in spleens of healthy mice (black bars, n = 4), immunized mice on day 14 (lined bars, n = 4) and of mice 14 days (grey bars, n = 4) after NTS induction (A, B) by relative (A, C) and quantitative (B, D) flow cytometry. (E, F) Monocytes (CD11b⁺SSC^low) and neutrophil granulocytes (CD11b⁺SSC^hi) were evaluated relatively (E) and quantitatively (F) in spleens of healthy (n = 4), immunized (n = 4) and nephritic mice on day 14 (n = 4). All data are given as means ± SEM. *p<0.05; **p<0.01; ***p<0.001.</p

Autophagy protects from uremic vascular media calcification

Chronic kidney disease and diabetes mellitus are associated with extensive media calcification, which leads to increased cardiovascular morbidity and mortality. Here, we investigated the role of autophagy in the pathogenesis of uremic vascular media calcification. DBA/2 mice were fed with high-phosphate diet (HPD) in order to cause vascular calcification. DBA/2 mice on standard chow diet were used as control. In parallel, autophagy and its response to rapamycin, 3-methyladenine (3-MA), and bafilomycin were studied in an in vitro model using mouse vascular smooth muscle cells (MOVAS). DBA/2 mice on HPD developed severe vascular media calcification, which is mirrored in vitro by culturing MOVAS under calcifying conditions. Both, in vitro and in vivo, autophagy significantly increased in MOVAS under calcifying conditions and in aortas of HPD mice, respectively. Histologically, autophagy was located to the aortic Tunica media, but also vascular endothelial cells, and was found to continuously increase during HPD treatment. 3-MA as well as bafilomycin blocked autophagy in MOVAS and increased calcification. Vice versa, rapamycin treatment further increased autophagy and resulted in a significant decrease of vascular calcification in vitro and in vivo. Rapamycin reduced Runx2 transcription levels in aortas and MOVAS to control levels, whereas it increased α-smooth muscle actin and Sm22α transcription in MOVAS to control levels. Furthermore, rapamycin-treated HPD mice survived significantly longer compared to HPD controls. These findings indicate that autophagy is an endogenous response of vascular smooth muscle cells (VSMC) to protect from calcification in uremia. Induction of autophagy by rapamycin protects cells and mice from uremic media calcification possibly by inhibiting osteogenic transdifferentiation of VSMC

Immunized and nephritic mice display increased levels of serum, Il-6, TNF-α and IFN-γ.

<p>Serum Il-6, TNF-α and IFN-γ were measured in healthy (n = 4), immunized mice on day 14 (n = 4) and nephritic mice on day 14 (n = 4). Data are given as means ± SEM. *p<0.05; **p<0.01; ***p<0.001.</p

The red pulp increases in NTS.

<p>(A) Spleens from healthy (black bar, n = 5), immunized (lined bar, n = 5) and nephritic mice (grey bar, n = 5) on day 14 were stained for F4/80, indicating the red pulp. Representative pictures are shown. Magnification x40. (B) Red and white pulp were quantified for F4/80 positive cells. Data are given as means ± SEM. *p<0.05.</p

NTS and immunisation increase erythroid progenitor cells in the spleen and repress the bone marrow haematopoiesis.

<p>Spleens (A, C) and bone marrow (B, C) of healthy (black bars, n = 5) and immunized (lined bars, n = 5) mice on day 14 as well as mice that either underwent splenectomy (SplX, squared bar, n = 5) or sham-operation (Sham, white bars, n = 5) prior to the induction of NTS were evaluated for erythroid precursors in quantitative flow cytometry on day 14. (A, B) Data are given as means ± SEM. (C) Representative dot plots are shown. *p<0.05, ***p<0.001.</p