Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we ...examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.
The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively ...investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).
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•Anaerobic glycolysis represents the predominant energy source of podocytes•Neither mitochondrial turnover nor mtDNA transcription impairs podocyte function•These findings elucidate podocyte metabolism, function, and glomerular integrity
Glomerular podocytes form the third and most outer layer of the kidney filtration barrier responsible for restricting the passage of proteins into the urine. Brinkkoetter et al. show that podocyte metabolism primarily relies on anaerobic glycolysis and the fermentation of glucose to lactate.
Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular ...level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR-dependent regulatory network for nutrient transport in renal proximal tubular cells.
The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into ...specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair.
Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source.
Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome.
Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.
The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide ...3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.
Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to ...the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear.
We used mouse models, including DNA methyltransferase (
and
) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development.
We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction
and
and that DNA methyltransferases
and
are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of
in nephron progenitor cells (in contrast to deletion of
or
) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In
-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis,
and its target
Derepression of germline genes, protocadherins,
genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression.
These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.
Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; ...as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2–/– mice, which lack mature and functioning B and T lymphocytes, developed neither anti-PLA2R1 antibodies nor proteinuria. Thus, our work demonstrates that podocyte expression of human PLA2R1 can induce membranous nephropathy with an underlying antibody-mediated pathogenesis in mice. Importantly, this antibody-mediated model enables proof-of-concept evaluations of antigen-specific treatment strategies, e.g., targeting autoantibodies or autoantibody-producing cells, and may further help understand the autoimmune pathogenesis of membranous nephropathy.
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Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. However, both the initiation pathways and the ...pathogenic significance of complement activation in MN are poorly understood. Here, we show that components from all three complement pathways (alternative, classical and lectin) are found in renal biopsies from patients with MN. Proximity ligation assays to directly visualize complement assembly in the tissue reveal dominant activation via the classical pathway, with a close correlation to the degree of glomerular C1q-binding IgG subclasses. In an antigen-specific autoimmune mouse model of MN, glomerular damage and proteinuria are reduced in complement-deficient mice compared with wild-type littermates. Severe disease with progressive ascites, accompanied by extensive loss of the integral podocyte slit diaphragm proteins, nephrin and neph1, only occur in wild-type animals. Finally, targeted silencing of C3 using RNA interference after the onset of proteinuria significantly attenuates disease. Our study shows that, in MN, complement is primarily activated via the classical pathway and targeting complement components such as C3 may represent a promising therapeutic strategy.
Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a ...comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking.
We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped
, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting
. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy;
proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated
knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics.
We demonstrated SRGAP1 localization to podocyte foot processes
and to cellular protrusions
.
but not
knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of
by
resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore,
-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery
. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS.
SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how ...parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
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