Combination of everolimus and low-dose tacrolimus controls histological liver allograft injury as sufficiently as high-dose tacrolimus

IntroductionThe combination of everolimus (EVR) and low-dose tacrolimus (lowTAC) prevents T cell-mediated rejection of liver grafts as sufficiently as high-dose tacrolimus (highTAC) and mycophenolate, but is associated with a preserved kidney function within the first years after orthotopic liver transplantation (OLT). However, none of the available studies assessed the histological pattern of graft injury or fibrosis in surveillance biopsies (svLbx).MethodsAll svLbx taken under at least one month of stable immunosuppression with either EVR (aim 3-8 ng/ml) combined with lowTAC (aim 3-5 ng/ml) or highTAC (aim 5-8 ng/ml) combined with mycophenolate (500-1500 mg/day) within the first three to four years after OLT at our center were included. Patients who were switched to EVR because of insufficient control of alloreactivity were excluded.ResultsReasons for switches to EVR were mainly malignancies before or after OLT, or chronic kidney injury. We were able to include 20 svLbx with EVR/lowTAC and 49 with highTAC/mycophenolate. Both groups had similar liver enzymes and similar kidney function. The EVR/lowTAC group exhibited lower TAC trough levels at svLbx (4.4 vs. 6.6 ng/ml; p<.001) in comparison to highTAC/mycophenolate. Histological graft injury quantified by the rejection activity index and hepatitis activity index (Ishak), as well as fibrosis were not significantly different between the EVR/lowTAC and highTAC/mycophenolate groups. Likewise, subclinical TCMR, histological criteria justifying immunosuppression minimization, and steatosis had equal prevalence in both regimens. Immunosuppression was adjusted according to the svLbx findings. Immunosuppression regimens had similarly low rates of rejection after immunosuppression reduction, when relevant graft injury was absent in the biopsy.DiscussionIn conclusion, EVR/lowTAC seems to control alloreactivity and histological graft injury as sufficiently as highTAC/mycophenolate within the first 3-4 years after OLT

Albumin-associated free fatty acids induce macropinocytosis in podocytes

Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes actively internalize fluid from the plasma and that the rate of internalization is increased when the filtration barrier is disrupted. In cultured podocytes, the presence of free fatty acids (FFAs) associated with serum albumin stimulated macropinocytosis through a pathway that involves FFA receptors, the Gβ/Gγ complex, and RAC1. Moreover, mice with elevated levels of plasma FFAs as the result of a high-fat diet were more susceptible to Adriamycin-induced proteinuria than were animals on standard chow. Together, these results support a model in which podocytes sense the disruption of the filtration barrier via FFAs bound to albumin and respond by enhancing fluid-phase uptake. The response to FFAs may function in the development of nephrotic syndrome by amplifying the effects of proteinuria

Cholemic nephropathy causes acute kidney injury and is accompanied by loss of aquaporin 2 in collecting ducts

Impairment of renal function often occurs in patients with liver disease. Hepatorenal syndrome is a significant cause of acute kidney injury (AKI) in cirrhotic patients (HRS-AKI, type 1). Causes of non-HRS AKI include cholemic nephropathy (CN), a disease that is characterized by intratubular bile casts and tubular injury. As data on patients with CN is mostly obtained from case reports or autopsy studies, we aimed to investigate the frequency and clinical course of CN. We identified 149 patients who underwent kidney biopsy between 2000 to 2016 at the Department of Gastroenterology, Hepatology and Endocrinology. Of these, 79 had a history of liver disease and deterioration of renal function. When applying recent EASL criteria 45 of the 79 patients (57%) presented with AKI, whereas 34 patients (43%) had chronic kidney disease (CKD) (43%). Renal biopsy revealed the diagnosis of CN in 8 of the 45 patients with AKI (17.8%), whereas none of the patients with CKD was diagnosed with CN. Univariate analysis identified serum bilirubin, alkaline phosphatase and urinary bilirubin and urobilinogen as predictive factors for the diagnosis of CN. Histological analysis of AKI patients with normal bilirubin, elevated bilirubin and the diagnosis of CN revealed loss aquaporin 2 (AQP2) expression in collecting ducts in patients with elevated bilirubin and CN. Biopsy related complications requiring medical intervention occurred in four of 79 patients (5.1%). In conclusion, CN is a common finding in patients with liver disease, AKI and highly elevated bilirubin. Loss of AQP2 in AKI patients with elevated bilirubin and CN might be the result of toxic effects of cholestasis and be in part responsible for the impairment of renal function

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression

P2Y2R Signaling Is Involved in the Onset of Glomerulonephritis

Endogenously released adenosine-5’-triphosphate (ATP) is a key regulator of physiological function and inflammatory responses in the kidney. Genetic or pharmacological inhibition of purinergic receptors has been linked to attenuation of inflammatory disorders and hence constitutes promising new avenues for halting and reverting inflammatory renal diseases. However, the involvement of purinergic receptors in glomerulonephritis (GN) has only been incompletely mapped. Here, we demonstrate that induction of GN in an experimental antibody-mediated GN model results in a significant increase of urinary ATP-levels and an upregulation of P2Y2R expression in resident kidney cells as well as infiltrating leukocytes pointing toward a possible role of the ATP/P2Y2R-axis in glomerular disease initiation. In agreement, decreasing extracellular ATP-levels or inhibition of P2R during induction of antibody-mediated GN leads to a reduction in all cardinal features of GN such as proteinuria, glomerulosclerosis, and renal failure. The specific involvement of P2Y2R could be further substantiated by demonstrating the protective effect of the lack of P2Y2R in antibody-mediated GN. To systematically differentiate between the function of P2Y2R on resident renal cells versus infiltrating leukocytes, we performed bone marrow-chimera experiments revealing that P2Y2R on hematopoietic cells is the main driver of the ATP/P2Y2R-mediated disease progression in antibody-mediated GN. Thus, these data unravel an important pro-inflammatory role for P2Y2R in the pathogenesis of GN

Molecular basis of the functional podocin-nephrin complex:mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains

Hereditary nephrotic syndrome is a heterogeneous disease, characterized by heavy proteinuria and renal failure. Mutations of NPHS1 or NPHS2, the genes encoding for nephrin and podocin, lead to early onset of heavy proteinuria, and rapid progression to end-stage renal disease, suggesting that both proteins are essential for the integrity of the glomerular filter. Podocin is a stomatin protein family member with a predicted hairpin-like structure localizing to the insertion site of the slit diaphragm of podocytes, the visceral glomerular epithelial cells of the kidney. Here we investigate the pathomechanisms of different disease-causing podocin mutations. We show that wild-type podocin is targeted to the plasma membrane, and forms homo-oligomers involving the carboxy and amino terminal cytoplasmic domains. The association of podocin with specialized lipid raft microdomains of the plasma membrane was a prerequisite for recruitment of nephrin into rafts. In contrast, disease-causing mutations of podocin (R138Q and R138X) failed to recruit nephrin into rafts either because these mutants were retained in the endoplasmic reticulum (R138Q), or because they failed to associate with rafts (R138X) despite their presence in the plasma membrane. None of the mutants did augment nephrin signaling, suggesting that lipid raft targeting facilitates nephrin signaling. Our findings demonstrate that the failure of mutant podocin to recruit nephrin into lipid rafts may be essential for the pathogenesis of NPHS2

DSA are associated with more graft injury, more fibrosis and upregulation of rejection associated transcripts in subclinical rejection.

Background: Subclinical T cell-mediated rejection (subTCMR) is commonly found after liver transplantation and has a good short-term prognosis, even when it is left untreated. Donor-specific antibodies (DSA) are putatively associated with a worse prognosis for recipient and graft after liver transplantation. Methods: To assess the immune regulation in subTCMR grafts, gene expression of 93 transcripts for graft injury, tolerance and immune regulation was analyzed in 77 biopsies with “no histological rejection” (NHR; n=25), “clinical TCMR” (cTMCR; n=16) and subTCMR (n=36). In addition, all available subTCMR biopsies (n=71) were tested for DSA with bead assays. Results: SubTCMR showed heterogeneous and intermediate expression profiles of transcripts that were upregulated in cTCMR. Graft gene expression suggested a lower activation of effector lymphocytes and a higher activation of regulatory T cells in grafts with subTCMR compared to cTCMR.DSA positivity in subTCMR was associated with histological evidence of more severe graft inflammation and fibrosis. This more severe DSA+ associated graft injury in subTCMR was converged with an upregulation of cTCMR associated transcripts. In nonsupervised analysis DSA positive subTCMR mostly clustered together with cTCMR, while DSA negative subTCMR clustered together with NHR. Conclusion: T cell-mediated rejection seem to form a continuum of alloimmune activation. Although subTCMR exhibited less expression of TCMR associated transcript, DSA positivity in subTCMR was associated with an upregulation of rejection associated transcripts. The identification of DSA positive subclinical rejection might help to define patients with more inflammation in the graft and development of fibrosis

Datasheet1_Combination of everolimus and low-dose tacrolimus controls histological liver allograft injury as sufficiently as high-dose tacrolimus.docx

IntroductionThe combination of everolimus (EVR) and low-dose tacrolimus (lowTAC) prevents T cell-mediated rejection of liver grafts as sufficiently as high-dose tacrolimus (highTAC) and mycophenolate, but is associated with a preserved kidney function within the first years after orthotopic liver transplantation (OLT). However, none of the available studies assessed the histological pattern of graft injury or fibrosis in surveillance biopsies (svLbx).MethodsAll svLbx taken under at least one month of stable immunosuppression with either EVR (aim 3-8 ng/ml) combined with lowTAC (aim 3-5 ng/ml) or highTAC (aim 5-8 ng/ml) combined with mycophenolate (500-1500 mg/day) within the first three to four years after OLT at our center were included. Patients who were switched to EVR because of insufficient control of alloreactivity were excluded.ResultsReasons for switches to EVR were mainly malignancies before or after OLT, or chronic kidney injury. We were able to include 20 svLbx with EVR/lowTAC and 49 with highTAC/mycophenolate. Both groups had similar liver enzymes and similar kidney function. The EVR/lowTAC group exhibited lower TAC trough levels at svLbx (4.4 vs. 6.6 ng/ml; pDiscussionIn conclusion, EVR/lowTAC seems to control alloreactivity and histological graft injury as sufficiently as highTAC/mycophenolate within the first 3-4 years after OLT.</p

Loss of Podocyte aPKCλ/ι Causes Polarity Defects and Nephrotic Syndrome

Atypical protein kinase C (aPKC) is a central component of the evolutionarily conserved Par3-Par6-aPKC complex, one of the fundamental regulators of cell polarity. We recently demonstrated that these proteins interact with Neph-nephrin molecules at the slit diaphragm of the glomerular filtration barrier. Here, we report that podocyte-specific deletion of aPKCλ/ι in mice results in severe proteinuria, nephrotic syndrome, and death at 4 to 5 wk after birth. Podocyte foot processes of knockout mice developed structural defects, including mislocalization of the slit diaphragm. In the glomerulus, aPKCλ/ι was primarily expressed in developing glomerular epithelial cells and podocyte foot processes. Interestingly, under physiologic conditions, aPKCλ/ι translocated from the apical surface to the basolateral side of developing podocytes, and this translocation preceded the development of foot processes and formation of slit diaphragms. Supporting a critical role for aPKCλ/ι in the maintenance of slit diaphragms and podocyte foot processes, aPKCλ/ι associated with the Neph-nephrin slit diaphragm complex and localized to the tips of filopodia and leading edges of cultured podocytes. These results suggest that aPKC signaling is fundamental to glomerular maintenance and development