Bernd Hohenstein

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

The SGLT2 inhibitor empagliflozin ameliorates early features of diabetic nephropathy in BTBR ob/ob type 2 diabetic mice with and without hypertension

Diabetic nephropathy is the leading cause of end-stage renal disease in humans in the Western world. The recent development of Na+-glucose cotransporter 2 (SGLT2) inhibitors offers a new antidiabetic therapy via enhanced glucose excretion. Whether this strategy exerts beneficial effects on the development of type 2 diabetic nephropathy is still largely unclear. We investigated the effects of the specific SGLT2 inhibitor empagliflozin in BTBR.Cg-Lep/WiscJ (BTBR ob/ob) mice, which spontaneously develop type 2 diabetic nephropathy. In the first experiment, BTBR ob/ob mice received either a diet containing 300 ppm empagliflozin or equicaloric placebo chow for 12 wk. In the second experiment, BTBR ob/ob mice received 1 μg·kg body wt(-1)·day(-1) ANG II to induce arterial hypertension and were separated into the same two diet groups for 6 wk. In both experiments, empagliflozin treatment enhanced glucosuria, thereby lowering blood glucose. Independently of hypertension, empagliflozin reduced albuminuria in diabetic mice. However, empagliflozin treatment affected diabetes-related glomerular hypertrophy, markers of renal inflammation, and mesangial matrix expansion only in BTBR ob/ob mice without hypertension. In summary, empagliflozin demonstrated significant antihyperglycemic effects, differentially ameliorating early features of diabetic nephropathy in BTBR ob/ob mice with and without hypertension.

Alirocumab in patients with heterozygous familial hypercholesterolaemia undergoing lipoprotein apheresis: the ODYSSEY ESCAPE trial

Aim To evaluate the effect of alirocumab on frequency of standard apheresis treatments [weekly or every 2 weeks (Q2W)] in heterozygous familial hypercholesterolaemia (HeFH). Methods and results ODYSSEY ESCAPE (NCT02326220) was a double-blind study in 62 HeFH patients undergoing regular weekly or Q2W lipoprotein apheresis. Patients were randomly assigned (2:1, respectively) to receive alirocumab 150 mg (n = 41) or placebo (n = 21) Q2W subcutaneously for 18 weeks. From day 1 to week 6, apheresis rate was fixed according to the patient’s established schedule; from weeks 7 to 18, apheresis rate was adjusted based on the patient’s low-density lipoprotein cholesterol (LDL-C) response in a blinded fashion. Apheresis was not performed when the LDL-C value was ≥30% lower than the baseline (pre-apheresis) value. The primary efficacy endpoint was the rate of apheresis treatments over 12 weeks (weeks 7–18), standardized to number of planned treatments. In the alirocumab group the least square (LS) mean ± SE (95% confidence interval [CI]) per cent change in pre-apheresis LDL-C from baseline at week 6 was −53.7 ± 2.3 (−58.2 to − 49.2) compared with 1.6 ± 3.1 (–4.7 to 7.9) in the placebo group. The primary efficacy endpoint showed statistically significant benefit in favour of alirocumab (Hodges–Lehmann median estimate of treatment difference: 0.75; 95% CI 0.67–0.83; P < 0.0001). Therefore, alirocumab-treated patients had a 0.75 (75%) additional reduction in the standardized rate of apheresis treatments vs. placebo-treated patients. During this period, 63.4% of patients on alirocumab avoided all and 92.7% avoided at least half of the apheresis treatments. Adverse event rates were similar (75.6% of patients on alirocumab vs. 76.2% on placebo). Conclusions Lipoprotein apheresis was discontinued in 63.4% of patients on alirocumab who were previously undergoing regular apheresis, and the rate was at least halved in 92.7% of patients. Alirocumab was generally safe and well tolerated.

Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.

Renin Lineage Cells Repopulate the Glomerular Mesangium after Injury

Mesangial cell injury has a major role in many CKDs. Because renin-positive precursor cells give rise to mesangial cells during nephrogenesis, this study tested the hypothesis that the same phenomenon contributes to glomerular regeneration after murine experimental mesangial injury. Mesangiolysis was induced by administration of an anti-mesangial cell serum in combination with LPS. In enhanced green fluorescent protein-reporter mice with constitutively labeled renin lineage cells, the size of the enhanced green fluorescent protein-positive area in the glomerular tufts increased after mesangial injury. Furthermore, we generated a novel Tet-on inducible triple-transgenic LacZ reporter line that allowed selective labeling of renin cells along renal afferent arterioles of adult mice. Although no intraglomerular LacZ expression was detected in healthy mice, about two-thirds of the glomerular tufts became LacZ positive during the regenerative phase after severe mesangial injury. Intraglomerular renin descendant LacZ-expressing cells colocalized with mesangial cell markers α8-integrin and PDGF receptor-β but not with endothelial, podocyte, or parietal epithelial cell markers. In contrast with LacZ-positive cells in the afferent arterioles, LacZ-positive cells in the glomerular tuft did not express renin. These data demonstrate that extraglomerular renin lineage cells represent a major source of repopulating cells for reconstitution of the intraglomerular mesangium after injury.

PDGF-C mediates glomerular capillary repair.

Glomerular endothelial cell injury is a key component of a variety of diseases. Factors involved in glomerular endothelial cell repair are promising therapeutic agents for such diseases. Platelet-derived growth factor (PDGF)-C has pro-angiogenic properties; however, nothing is known about such functions in the kidney. We therefore investigated the consequences of either PDGF-C infusion or inhibition in rats with mesangioproliferative glomerulonephritis, which is accompanied by widespread glomerular endothelial cell damage. We also assessed the role of PDGF-C in a mouse model of thrombotic microangiopathy as well as in cultured glomerular endothelial cells. PDGF-C infusion in nephritic rats significantly reduced mesangiolysis and microaneurysm formation, whereas glomerular endothelial cell area and proliferation increased. PDGF-C infusion specifically up-regulated glomerular fibroblast growth factor-2 expression. In contrast, antagonism of PDGF-C in glomerulonephritis specifically reduced glomerular endothelial cell area and proliferation and increased mesangiolysis. Similarly, PDGF-C antagonism in murine thrombotic microangiopathy aggravated the disease and reduced glomerular endothelial area. In conditionally immortalized glomerular endothelial cells, PDGF-C was mitogenic and induced a 27-fold up-regulation of fibroblast growth factor-2 mRNA. PDGF-C also exerted indirect pro-angiogenic effects, since it induced endothelial cell mitogens and pro-angiogenic factors in mesangial cells and macrophages. These results identify PDGF-C as a novel, potent pro-angiogenic factor in the kidney that can accelerate capillary healing in experimental glomerulonephritis and thrombotic microangiopathy.

Enhanced progenitor cell recruitment and endothelial repair after selective endothelial injury of the mouse kidney.

Primary and/or secondary injury of the renal microvascular endothelium is a common finding in various renal diseases. Besides well-known endothelial repair mechanisms, including endothelial cell (EC) proliferation and migration, homing of extrinsic cells such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC) has been shown in various organs and may contribute to microvascular repair. However, these mechanisms have so far not been studied after selective microvascular injury in the kidney. The present study investigated the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation. Erythropoietin was used to stimulate progenitor cells in a therapeutic approach. We found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors to orchestrate microvascular repair. Angiogenic factors started to increase as early as 30 min after disease induction. Progenitor cells could be first detected in the circulation and the spleen before they selectively homed to the diseased kidney. Injection of a high dose of erythropoietin 2 h after disease induction markedly attenuated vascular injury through nonhemodynamic mechanisms, possibly involving vascular endothelial growth factor release.

Thrombospondin 2 Functions as an Endogenous Regulator of Angiogenesis and Inflammation in Experimental Glomerulonephritis in Mice

The role of thrombospondin 2 (TSP2) was investigated in an anti-glomerular basement membrane (GBM) nephritis model that compared TSP2-null mice with wild-type (WT) controls. TSP2-null mice were analyzed for kidney function, renal cortical matrix expansion, influx of inflammatory cells, proliferation, and apoptosis, as well as for capillary rarefaction after induction of anti-GBM disease. Whereas the renal cortex of normal control WT mice did not show any detectable TSP2 staining above background, TSP2 protein expression was clearly upregulated in anti-GBM disease. TSP2 deficiency led to an accelerated and enhanced inflammatory response, as indicated by the influx of CD4(+) and CD8a(+) cells and monocytes/macrophages. Glomerular fibrin deposition and a matrix-remodeling response were also observed, as indicated by collagens I and IV staining and a proliferative response within the renal interstitium. These changes were accompanied by increased matrix metalloproteinase 2 activity and enhanced alpha-smooth muscle actin staining in the TSP2-null mice. Neither a compensatory increase in TSP1 nor increased phosphorylation of Smad 2/3, an indicator for TGF-beta activity, was observed. The proliferative response of the peritubular endothelium was accelerated and enhanced, leading to a reversal of capillary rarefaction in TSP2-null mice, whereas interstitial cell death was equivalent to that in WT mice. In conclusion, the lack of the matricellular protein TSP2 in mice accelerates and enhances several responses to renal injury and reveals an important role for TSP2 as a major endogenous antiangiogenic and matrix metalloproteinase 2-regulating factor in renal disease.

P2Y1 Gene Deficiency Protects from Renal Disease Progression and Capillary Rarefaction during Passive Crescentic Glomerulonephritis

The metabotropic receptor P2Y1 is necessary for full ADP-induced platelet activation and is localized on various intrinsic renal cells, including mesangial cells, podocytes, and endothelial cells. To date, nothing is known about the role of the P2Y1 receptor during inflammatory renal disease. The role of the P2Y1 receptor was investigated using 22 P2Y1 gene-deficient (-/-) and 27 wild-type (wt) mice during the time course of passive crescentic nephrotoxic glomerulonephritis. Six P2Y1 -/- and six wt mice served as undiseased controls. Renal tissues were harvested on days 1, 10, and 28 after disease induction. No renal phenotype was found in P2Y1 -/- versus wt mice. In contrast, during crescentic glomerulonephritis, approximately 50% of all wt mice died, whereas all P2Y1 -/- mice survived. Renal function as assessed by creatinine clearance measurements, glomerulosclerosis, and tubulointerstitial injury indices as well as glomerular and interstitial matrix expansion were improved significantly in P2Y1 -/- compared with wt mice. These changes were preceded by reduced glomerular and peritubular capillary rarefaction indices in P2Y1 -/- compared with wt mice. The alteration of the rates of both peritubular apoptosis and endothelial cell proliferation suggests improved capillary preservation in P2Y1 -/- mice early in disease (day 10) and an additional enhanced repair reaction in P2Y1 -/- mice at the late time point (day 28), whereas injury on day 1 seemed to be equivalent in both groups. It is concluded that loss of P2Y1 receptor function safeguards against capillary loss, fibrosis, and death by renal failure during experimental crescentic glomerulonephritis.