Image3_A high-throughput drug discovery pipeline to optimize kidney normothermic machine perfusion.TIFF

Kidney transplantation is the only definitive therapy for end-stage kidney disease. The shortage of organs for transplantation is the main limitation of this life-saving treatment. Normothermic machine perfusion (NMP) is a novel preservation technique with the potential to increase the number of transplantable kidneys through reducing delayed graft function and organ evaluation under physiological conditions. To date, the cellular effects and possible pharmacological interventions during machine perfusion are incompletely understood. A major limitation is the technically complex, time-consuming, and small-scale replication of NMP in rodent models. To overcome this, we developed a 3D-printed, high throughput ex-vivo mouse kidney slice incubator (KSI) mimicking mouse kidney NMP by working under closely resembling conditions. KSI significantly reduced the time per experiment and increased the sample throughput (theoretical: 54 incubations with n = 500/day). The model recapitulated the cellular responses during NMP, namely increased endoplasmic reticulum stress (ER stress). Using KSI, five pharmacological interventions against ER stress taken from the literature were tested. While four were ineffective and excluded, one, β-Nicotinamide-adenine-dinucleotide (NADH), ameliorated ER stress significantly during KSI. The test of NADH in mouse kidney NMP replicated the positive effects against ER stress. This suggests that testing the addition of NADH during clinical kidney NMP might be warranted.</p

MAP1B is highly expressed in kidney podocytes.

<p>Immunofluorescence staining of kidney sections derived from adult human and rat kidney. WT1 served as a marker for podocyte nuclei. Nephrin and synaptopodin served as foot process markers. DAPI was used for staining nuclei. MAP1B is expressed highly specific in podocytes (scale for overview: 50 μm; higher magnification: 25 μm) (A,C). Expression of MAP1B during glomerulogenesis in newborn rat kidney. Glomerular differentiation advances from cortex to medulla (upper right: immature; lower left: mature) (scale for overview: 50 μm; higher magnification: 25 μm) (B). Transmission electron microscopy of kidney sections. Immunolabeling of MAP1B confirms an enrichment in areas with a dense MT cytoskeleton in primary processes and the cell body (primary processes (PP), foot processes (arrowheads), Nucleus (Nu)), glomerular basement membrane (GBM); scale: 200 nm) (D).</p

Constitutive MAP1B Knock out (KO) mice do not exhibit a glomerular phenotype.

<p>Immunofluorescence microscopy of kidney sections from constitutive MAP1B KO mice versus control littermates. Autofluorescence of PFA treated tissue is used to display glomerular anatomy. Total depletion of MAP1B HC in podocytes of KO animals as well as the specificity of our antibody for MAP1B expression is confirmed (scale: 50 μm) (A). We did not observe alterations of glomerular morphology using light microscopy of Periodic-acid Schiff (PAS) stained kidney sections of MAP1B KO animals (scale: 50 μm) (B). MAP1B podocyte foot process morphology examination using transmission electron microscopy of MAP1B KO podocytes (Podocyte (Pd), capillary (C), Bowman’s urinary space (B), podocyte foot processes (arrow heads), scale: 10 μm) (C). Proteinuria in the physiological range in both KO and control littermate by measuring urinary albumin/creatinin ratios (n = 7 for KO and control animals, p = 0,205) (D). 9 week old MAP1B KO animals showed a significantly reduced body weight compared to control littermates (n = 7 for KO and control animals, p = 0,010) (E).</p

Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response

<div><p>The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive <i>Cxadr</i> knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, <i>Cxadr</i> is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of <i>Cxadr</i> for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific <i>Cxadr</i> knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of <i>Cxadr</i> on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of <i>Cxadr</i>.</p></div

<i>Cxadr</i> is highly expressed during kidney development.

<p><b>(A-D)</b> In-Situ-Hybridization showed intense expression of <i>Cxadr</i> in E15.5 embryos, especially in the nervous system, gut, lung and kidney. <b>(E&F)</b> After birth at P1 and P14 <i>Cxadr</i> could be detected in glomerular cells as well as in the nephron (<i>thick ascending limb of Henle</i>). <b>(G)</b> Western blot confirmed CXADR expression in heart, kidney, lung, brain and more specifically in isolated murine podocytes. <b>(H-K)</b> Developmental assessment of <i>Cxadr</i> expression during kidney development started in the S-shaped body phase where podocytes and parietal epithelial cells formed a continuous stretch of cells. <b>(L&M)</b> In both, podocytes (arrows) and parietal epithelial cells (asterisk) CXADR could be robustly detected in the early capillary loop stage, while mature glomeruli only possessed reduced amounts of CXADR in podocytes.</p

Glomerular function, histology and ultrastructure are maintained in podocyte specific <i>Cxadr</i> knock-out animals.

<p><b>(A)</b> Functional assessment only showed a slightly elevated albuminuria in <i>Cxadr</i> deficient animals at P2 which was lost during further maturation of the kidney. <b>(B&C)</b> Histology of glomeruli was normal in podocyte deficient <i>Cxadr</i> animals as was <b>(D&E)</b> ultrastructural assessment by SEM <b>(D–D”</b>, <b>E–E”)</b> and TEM <b>(D”‘</b>, <b>E”‘)</b>.</p

Nephrotoxic serum (NTS) enhances podocyte specific CXADR expression, but lack of CXADR does not influence the course of NTS induced disease.

<p><b>(A-C)</b> On both d4 and d5 after NTS injection CXADR was increased in glomerular podocytes as shown in co-labeling experiments with NEPHRIN (control: white arrows—parietal epithelial cells, NTS: arrow heads—podocytes). As can be easily depicted from the NEPHRIN specific panel <b>(A”, B”, C”)</b> NEPHRIN abundance is greatly reduced during the course of the disease, underlining the severity of the chosen stress model. <b>(D)</b> Schematic of the injection scheme and follow-up using urine collections. <b>(E&F)</b> Western blot using decapsulated glomerular lysates was used to quantify CXADR expression which was increased two fold 5 days after NTS injection. <b>(G&H)</b> As shown with immunofluorescence stainings CXADR induction was absent in podocyte specific <i>Cxadr</i> knock-out animals. <b>(I)</b> Proteinuria developed similarly in wild-type and knock-out animals showing no functional differences.</p

Adriamycin enhances podocyte specific CXADR expression, but lack of CXADR does not influence the course of Adriamycin induced FSGS.

<p><b>(A)</b> In adult wild-type animals CXADR was restricted to parietal epithelial cells (white arrows). <b>(B)</b> Administration of Adriamycin led to an increased glomerular CXADR expression which could be localized to glomerular podocytes using co-labeling experiments with NEPHRIN (white arrow heads). <b>(C)</b> Adriamycin injection scheme and subsequent urine measurements. <b>(D&E)</b> Western blots of decapsulated glomerular lysates were used to quantify CXADR expression in podocytes which indeed was upregulated two-fold. We next assessed abundance and distribution of the slit diaphragm molecules PODOCIN and NEPHRIN after adriamycin injection, and could not discern any differences between the two genotypes <b>(F-F”, G-G”</b> dashed lines-podocyte compartment<b>)</b>. In contrast to NEPHRIN the pattern of ZO-1 expression was largely maintained in both genotypes. <b>(H-H”, I-I”</b> dashed lines—podocyte compartment<b>).</b> We next assessed functional consequences on two different mouse genetic background strains. <b>(J)</b> Wild-type and knock-out animals on a C57Bl6/NCrl were analysed to check for increased susceptibility of knock-out animals while <b>(K)</b> wild-type and knock-out animals on an ICR background were used to test for a protective effect of podocyte specific CXADR deficiency. On both mouse genetic backgrounds we could not discern a difference between wild-type and knock-out mice over a follow-up period of 5 weeks.</p

image_1_P2Y2R Signaling Is Involved in the Onset of Glomerulonephritis.tif

<p>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.</p