Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified

Megalin Orchestrates FcRn Endocytosis and Trafficking

The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis

Tubular <i>IKKβ</i> Deletion Alleviates Acute Ischemic Kidney Injury and Facilitates Tissue Regeneration

Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate immune response converging to NF-ĸB pathway induction. Despite the clear role of NF-ĸB in inflammation, it has recently been acknowledged that NF-ĸB may impact other cell functions. To identify NF-ĸB function with respect to metabolism, vascular function and oxidative stress after I/R injury and to decipher in detail the underlying mechanism, we generated a transgenic mouse model with targeted deletion of IKKβ along the tubule and applied I/R injury followed by its analysis after 2 and 14 days after I/R injury. Tubular IKKβ deletion ameliorated renal function and reduced tissue damage. RNAseq data together with immunohistochemical, biochemical and morphometric analysis demonstrated an ameliorated vascular organization and mRNA expression profile for increased angiogenesis in mice with tubular IKKβ deletion at 2 days after I/R injury. RNAseq and protein analysis indicate an ameliorated metabolism, oxidative species handling and timely-adapted cell proliferation and apoptosis as well as reduced fibrosis in mice with tubular IKKβ deletion at 14 days after I/R injury. In conclusion, mice with tubular IKKβ deletion upon I/R injury display improved renal function and reduced tissue damage and fibrosis in association with improved vascularization, metabolism, reactive species disposal and fine-tuned cell proliferation

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage following ischemia/reperfusion injury in mouse

Aim: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. Methods: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30min/48h). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischemic vs. non-ischemic kidneys in WT versus Dusp3-/- . Results: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs. WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs. WT ischemic kidneys. Dusp3-/- ischemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1β and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs. WT kidneys post I/R. Conclusion: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.Rôle de DUSP3 dans l'ischémie rénale chez la souri

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage following ischemia/reperfusion injury in mouse

BACKGROUND AND AIMS: Dual Specificity Phosphatase 3 (DUSP3) is a positive regulator of the innate immune response in case of sepsis, but its role in the ischemic damage is unknown. Here, we study (i) whether and where DUSP3 is expressed in the renal parenchyma, and (ii) whether its genetic deletion in Dusp3 systemic knock-out (Dusp3-/-) mice attenuates the I/R-associated inflammation and injury. METHOD: Experiment 1: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal ischemia for 30 minutes followed by a reperfusion of 48 hours. Blood and kidneys were collected. Renal function was assessed upon I/R biomarkers, i.e. blood urea nitrogen (BUN) and creatinine (SCr). Expressions of inflammatory and immune markers were comparatively quantified at both mRNA (real-time qPCR) and protein (immuno-blotting and –staining) levels in ischemic vs. non-ischemic kidneys in Dusp3 WT vs. KO mice. Experiment 2: Ten C57BL/6 male WT and Dusp3-/- mice were anesthetized. Renal Doppler ultrasound was performed to assess the renal resistivity index (RRI). The expression of CD31 and VEGF vascular markers was quantified by the means of real time qPCR and and immuno-staining (FiJi software). RESULTS: Experiment 1: An immuno-reactive signal for DUSP3 was detected in the glomeruli (in co-localization with nephrin) and in Meca-32-positive endothelial cells of both outer and inner medulla of mouse non-ischemic WT kidneys. No significant immunoreactivity for DUSP3 was detected in Dusp3-/- kidneys. Following renal I/R, the mRNA level of Dusp3 was increased 1.8-fold compared to baseline (p<0.001). Immunoblot quantifications showed a 77-fold increased expression of DUSP3 post renal I/R. Serum levels of I/R biomarkers were significantly lower in Dusp3-/- compared to WT mice following renal I/R (BUN: 78.4633.7 vs. 258.96162.9mg/dL; SCr: 0.160.07 vs. 0.860.9 mg/dL; p<0.01). At mRNA levels, Dusp3-/- ischemic kidneys showed a significantly decreased expression level of CD11b, TNF-a, KIM-1, IL-6, IL-1b and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b positive cells were significantly reduced in Dusp3-/- vs WT renal parenchyma post I/R. Experiment 2: The RRI non-invasively measured by ultrasound was lower in Dusp3-/- group compared to controls (0.566 0.03 vs. 0.6660.02; p<0.001). The Dusp3-/- non ischemic kidneys were characterized by a 1.8-fold increased surface of CD31-positive cells compared to WT kidneys (p<0.001). At mRNA levels, the Dusp3-/- kidneys showed significantly increased basal levels of CD31 and VEGF compared to controls. CONCLUSION: The genetic deletion of DUSP3 is associated with (i) increased renal vascular density, (ii) decreased RRI and (iii) nephroprotection against renal I/R injury

Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

peer reviewedDiabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development

Expression of proteases and Pearson correlation.

(A) Comparison of the relative amounts of ADAM (a disintegrin and metalloprotease domain) proteases as well as BACE (beta-secretase) and MMP2 (matrix metallopeptidase 2) in the SLGC lines indicated (data from proteome array). Based on the assumption that the antibodies spotted on the filters of the proteome array possessed similar KDs, the relative expression of the proteases was calculated. The sum of the signals of all proteases was arbitrary set at 100%. The bars indicate to what percentage the individual proteases contribute to this expression. (B) Similar assay as in (A). The relative Cathepsin D levels were compared to the relative expression of all other proteases, depicted in panel (A). (C) Similar analysis as in (A) comparing the relative expression of the protease inhibitors TIMP (tissue inhibitor of metalloproteinases) 1, 2, 3 and 4.–Parts D and E graphically summarize some of the correlation data shown in Figs 8 and 9. In (B) the coefficients are indicated on the y-axis, highlighting expression levels with a positive correlation to the Sox2 (blue) or CD133 (violet) dots. In (C) the Pearson correlation coefficients were calculated relative to the levels of the neural proteins Tau and GFAP, as well as the hyaluronan receptor CD44, the integrin αv, and the N- and E-cadherin, respectively. In all cases, the expression levels that entered the calculations were determined with the same whole cell extracts. The calculations were confirmed with biological replicates. (TIF)</p

Pearson correlation coefficients (k): SLGC markers.

In (A) cell lines were ranked according to their Sox2 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. Group 1 displayed significantly higher Sox2 levels than Group 2. Group E comprises the established cell lines U87 and CaCo2, both of which lack Sox2 expression. The ranking was as follows: [T1452, T1371, T1495-SC, T1586, T1440, T1587, T1447-SC, T1495, T1447, T1338], [T1522, T1389, T1467, T1442, T1464, T1454, T1600, T1439], [CaCo2, U87]. In (B) cell lines were ranked according to their CD133 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. The CD133 levels were highest in the CaCo2 cell line, followed by the SLGCs assigned to groups with decreasing CD133-levels. The non-SLGC line CaCo2, which encompasses >90% of CD133 positive cells, is indicated on the very left. The ranking was as follows: [CaCo2], [T1452, T1333, T1495-Sc], [T1447-SC], [T1586, T1442, T1587, U87, T1600, T1464, T1447, T1495, T1440], [T1522, T1371, T1389, T1467, T1439, T1454].–For abbreviations, see the legends to Figs 5 and 6.</p

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, <i>Phosphatase and tensin homolog;</i> +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E^§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, Phosphatase and tensin homolog; +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.</p

Proteins characterizing the metaprofiles.

For all proteins that are significant (>95% percentile) for at least one metaprofile (MP1-5), their decoding along all MPs is shown. Solid dots indicate those above 95%; the shallow dots are those above 90% (see also Fig 4A). Furthermore, the proteins are sorted to match their principal MPs. All proteins that can be assigned to exactly one MP are clustered, and these clusters are color coded (MP1: blue, MP2: red, MP3: orange, MP4: green, and MP5: violet). As no unique protein could be linked to MP1, we used for clustering those that have expression above 90% but below 95% for the other MPs. All other proteins are assigned to a minimum of two metaprofiles (e.g., Galectin 3, NCAM L1) or all metaprofiles (CD147).—For abbreviations, see the legend to Fig 3.</p