Meta-analysis of single-cell and single-nucleus transcriptomics reveals kidney cell type consensus signatures.

While the amount of studies involving single-cell or single-nucleus RNA-sequencing technologies grows exponentially within the biomedical research area, the kidney field requires reference transcriptomic signatures to allocate each cluster its matching cell type. The present meta-analysis of 39 previously published datasets, from 7 independent studies, involving healthy human adult kidney samples, offers a set of 24 distinct consensus kidney cell type signatures. The use of these signatures may help to assure the reliability of cell type identification in future studies involving single-cell and single-nucleus transcriptomics while improving the reproducibility in cell type allocation.

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease.

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1-/- iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale.

Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

The renal inflammatory network of nephronophthisis.

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment.

The primary cilium is a sensory organelle at the cell surface with integral functions in cell signaling. It contains a microtubular axoneme that is rooted in the basal body (BB) and serves as a scaffold for the movement of intraflagellar transport (IFT) particles by Kinesin-2 along the cilium. Ift88, a member of the anterograde moving IFT-B1 complex, as well as the Kinesin-2 subunit Kif3a are required for cilia formation. To facilitate signaling, the cilium restricts the access of molecules to its membrane ("ciliary gate"). This is thought to be mediated by cytoskeletal barriers ("subciliary domains") originating from the BB subdistal/distal appendages, the periciliary membrane compartment (PCMC) as well as the transition fibers and zone (TF/TZ). The PCMC is a poorly characterized membrane domain surrounding the ciliary base with exclusion of certain apical membrane proteins. Here we describe that Ift88, but not Kinesin-2, is required for the establishment of the PCMC in MDCK cells. Likewise, in C. elegans mutants of the Ift88 ortholog osm-5 fail to establish the PCMC, while Kinesin-2 deficient osm-3 mutants form PCMCs normally. Furthermore, disruption of IFT-B1 into two subcomplexes, while disrupting ciliogenesis, does not interfere with PCMC formation. Our findings suggest that cilia are not a prerequisite for the formation of the PCMC, and that separate machineries with partially overlapping functions are required for the establishment of each.

Identification of pathological transcription in autosomal dominant polycystic kidney disease epithelia.

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

Loss of PKD1/polycystin-1 impairs lysosomal activity in a CAPN (calpain)-dependent manner.

Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.

Tubular STAT3 Limits Renal Inflammation in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: The inactivation of the ciliary proteins polycystin 1 or polycystin 2 leads to autosomal dominant polycystic kidney disease (ADPKD). Although signaling by primary cilia and interstitial inflammation both play a critical role in the disease, the reciprocal interactions between immune and tubular cells are not well characterized. The transcription factor STAT3, a component of the cilia proteome that is involved in crosstalk between immune and nonimmune cells in various tissues, has been suggested as a factor fueling ADPKD progression. METHOD: To explore how STAT3 intersects with cilia signaling, renal inflammation, and cyst growth, we used conditional murine models involving postdevelopmental ablation of Pkd1, Stat3, and cilia, as well as cultures of cilia-deficient or STAT3-deficient tubular cell lines. RESULTS: Our findings indicate that, although primary cilia directly modulate STAT3 activation in vitro, the bulk of STAT3 activation in polycystic kidneys occurs through an indirect mechanism in which primary cilia trigger macrophage recruitment to the kidney, which in turn promotes Stat3 activation. Surprisingly, although inactivating Stat3 in Pkd1-deficient tubules slightly reduced cyst burden, it resulted in a massive infiltration of the cystic kidneys by macrophages and T cells, precluding any improvement of kidney function. We also found that Stat3 inactivation led to increased expression of the inflammatory chemokines CCL5 and CXCL10 in polycystic kidneys and cultured tubular cells. CONCLUSIONS: STAT3 appears to repress the expression of proinflammatory cytokines and restrict immune cell infiltration in ADPKD. Our findings suggest that STAT3 is not a critical driver of cyst growth in ADPKD but rather plays a major role in the crosstalk between immune and tubular cells that shapes disease expression.

Divergent function of polycystin 1 and polycystin 2 in cell size regulation.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC1 and PC2 localize to the primary cilium and form a protein complex, which is thought to regulate signaling events. PKD1 mutations are associated with a stronger phenotype than PKD2, suggesting the existence of PC1 specific functions in renal tubular cells. However, the evidence for diverging molecular functions is scant. The bending of cilia by fluid flow induces a reduction in cell size through a mechanism that involves the kinase LKB1 but not PC2. Here, using different in vitro approaches, we show that contrary to PC2, PC1 regulates cell size under flow and thus phenocopies the loss of cilia. PC1 is required to couple mechanical deflection of cilia to mTOR in tubular cells. This study pinpoints divergent functions of the polycystins in renal tubular cells that may be relevant to disease severity in ADPKD.

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

Hepatic Production of Fibroblast Growth Factor 23 in Autosomal Dominant Polycystic Kidney Disease.

Context: The bone-derived hormone fibroblast growth factor (FGF) 23 controls phosphate homeostasis and urinary phosphate excretion. FGF23 plasma levels increase in the early stage of renal insufficiency to prevent hyperphosphatemia. Recent evidence suggests that this increase has effects on cardiac and immune cells that compromise patients' health. Patients with autosomal dominant polycystic kidney disease (ADPKD) have been reported to have higher FGF23 concentrations than other patients with similar renal function. The significance of this finding has remained unknown. Methods and Results: Analyzing the FGF23 plasma levels in 434 patients with ADPKD and 355 control subjects with a measured glomerular filtration rate (mGFR) between 60 and 120 mL/min per 1.73 m2, we confirmed that patients with ADPKD had higher FGF23 plasma concentrations than controls. Remarkably, this difference did not translate into renal phosphate leakage. Using different assays for FGF23, we found that this discrepancy was explained by a predominant increase in the cleaved C-terminal fragment of FGF23, which lacks phosphaturic activity. We found that FGF23 plasma concentration independently correlated with the severity of cystic liver disease in ADPKD. We observed that, in contrast to control liver tissues, the cystic liver from patients with ADPKD markedly expressed FGF23 messenger RNA and protein. In line with this finding, the surgical reduction of polycystic liver mass was associated with a decrease in FGF23 plasma levels independently of any modification in mGFR, phosphate, or iron status. Conclusion: Our findings demonstrate that severely polycystic livers produce FGF23 and increase levels of circulating FGF23 in patients with ADPKD.

Spindle pole cohesion requires glycosylation-mediated localization of NuMA.

Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress.

Targeting mTOR Signaling Can Prevent the Progression of FSGS.

Mammalian target of rapamycin (mTOR) signaling is involved in a variety of kidney diseases. Clinical trials administering mTOR inhibitors to patients with FSGS, a prototypic podocyte disease, led to conflicting results, ranging from remission to deterioration of kidney function. Here, we combined complex genetic titration of mTOR complex 1 (mTORC1) levels in murine glomerular disease models, pharmacologic studies, and human studies to precisely delineate the role of mTOR in FSGS. mTORC1 target genes were significantly induced in microdissected glomeruli from both patients with FSGS and a murine FSGS model. Furthermore, a mouse model with constitutive mTORC1 activation closely recapitulated human FSGS. Notably, the complete knockout of mTORC1 by induced deletion of both Raptor alleles accelerated the progression of murine FSGS models. However, lowering mTORC1 signaling by deleting just one Raptor allele ameliorated the progression of glomerulosclerosis. Similarly, low-dose treatment with the mTORC1 inhibitor rapamycin efficiently diminished disease progression. Mechanistically, complete pharmacologic inhibition of mTOR in immortalized podocytes shifted the cellular energy metabolism toward reduced rates of oxidative phosphorylation and anaerobic glycolysis, which correlated with increased production of reactive oxygen species. Together, these data suggest that podocyte injury and loss is commonly followed by adaptive mTOR activation. Prolonged mTOR activation, however, results in a metabolic podocyte reprogramming leading to increased cellular stress and dedifferentiation, thus offering a treatment rationale for incomplete mTOR inhibition.

Efficient genome editing of differentiated renal epithelial cells.

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

Stat3 Controls Tubulointerstitial Communication during CKD.

In CKD, tubular cells may be involved in the induction of interstitial fibrosis, which in turn, leads to loss of renal function. However, the molecular mechanisms that link tubular cells to the interstitial compartment are not clear. Activation of the Stat3 transcription factor has been reported in tubular cells after renal damage, and Stat3 has been implicated in CKD progression. Here, we combined an experimental model of nephron reduction in mice from different genetic backgrounds and genetically modified animals with in silico and in vitro experiments to determine whether the selective activation of Stat3 in tubular cells is involved in the development of interstitial fibrosis. Nephron reduction caused Stat3 phosphorylation in tubular cells of lesion-prone mice but not in resistant mice. Furthermore, specific deletion of Stat3 in tubular cells significantly reduced the extent of interstitial fibrosis, which correlated with reduced fibroblast proliferation and matrix synthesis, after nephron reduction. Mechanistically, in vitro tubular Stat3 activation triggered the expression of a specific subset of paracrine profibrotic factors, including Lcn2, Pdgfb, and Timp1. Together, our results provide a molecular link between tubular and interstitial cells during CKD progression and identify Stat3 as a central regulator of this link and a promising therapeutic target.

Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow.

Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo. PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1-AMPK-mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases.

Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2.

In chronic kidney disease (CKD), proteinuria results in severe tubulointerstitial lesions, which ultimately lead to end-stage renal disease. Here we identify 4-phenylbutyric acid (PBA), a chemical chaperone already used in humans, as a novel therapeutic strategy capable to counteract the toxic effect of proteinuria. Mechanistically, we show that albumin induces tubular unfolded protein response via cytosolic calcium rise, which leads to tubular apoptosis by Lipocalin 2 (LCN2) modulation through ATF4. Consistent with the key role of LCN2 in CKD progression, Lcn2 gene inactivation decreases ER stress-induced apoptosis, tubulointerstitial lesions and mortality in proteinuric mice. More importantly, the inhibition of this pathway by PBA protects kidneys from morphological and functional degradation in proteinuric mice. These results are relevant to human CKD, as LCN2 is increased in proteinuric patients. In conclusion, our study identifies a therapeutic strategy susceptible to improve the benefit of RAS inhibitors in proteinuria-induced CKD progression.

Cystic gene dosage influences kidney lesions after nephron reduction.

Cystic kidney disease is characterized by the progressive development of multiple fluid-filled cysts. Cysts can be acquired, or they may appear during development or in postnatal life due to specific gene defects and lead to renal failure. The most frequent form of this disease is the inherited polycystic kidney disease (PKD). Experimental models of PKD showed that an increase of cellular proliferation and apoptosis as well as defects in apico-basal and planar cell polarity or cilia play a critical role in cyst development. However, little is known about the mechanisms and the mediators involved in acquired cystic kidney diseases (ACKD). In this study, we used the nephron reduction as a model to study the mechanisms underlying cyst development in ACKD. We found that tubular dilations after nephron reduction recapitulated most of the morphological features of ACKD. The development of tubular dilations was associated with a dramatic increase of cell proliferation. In contrast, the apico-basal polarity and cilia did not seem to be affected. Interestingly, polycystin 1 and fibrocystin were markedly increased and polycystin 2 was decreased in cells lining the dilated tubules, whereas the expression of several other cystic genes did not change. More importantly, Pkd1 haploinsufficiency accelerated the development of tubular dilations after nephron reduction, a phenotype that was associated to a further increase of cell proliferation. These data were relevant to humans ACKD, as cystic genes expression and the rate of cell proliferation were also increased. In conclusion, our study suggests that the nephron reduction can be considered a suitable model to study ACKD and that dosage of genes involved in PKD is also important in ACKD.

AKT2 is essential to maintain podocyte viability and function during chronic kidney disease.

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2(-/-) but not in Akt1(-/-) mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.

Lipocalin 2 is essential for chronic kidney disease progression in mice and humans.

Mechanisms of progression of chronic kidney disease (CKD), a major health care burden, are poorly understood. EGFR stimulates CKD progression, but the molecular networks that mediate its biological effects remain unknown. We recently showed that the severity of renal lesions after nephron reduction varied substantially among mouse strains and required activation of EGFR. Here, we utilized two mouse strains that react differently to nephron reduction--FVB/N mice, which develop severe renal lesions, and B6D2F1 mice, which are resistant to early deterioration--coupled with genome-wide expression to elucidate the molecular nature of CKD progression. Our results showed that lipocalin 2 (Lcn2, also known as neutrophil gelatinase-associated lipocalin [NGAL]), the most highly upregulated gene in the FVB/N strain, was not simply a marker of renal lesions, but an active player in disease progression. In fact, the severity of renal lesions was dramatically reduced in Lcn2-/- mice. We discovered that Lcn2 expression increased upon EGFR activation and that Lcn2 mediated its mitogenic effect during renal deterioration. EGFR inhibition prevented Lcn2 upregulation and lesion development in mice expressing a dominant negative EGFR isoform, and hypoxia-inducible factor 1α (Hif-1α) was crucially required for EGFR-induced Lcn2 overexpression. Consistent with this, cell proliferation was dramatically reduced in Lcn2-/- mice. These data are relevant to human CKD, as we found that LCN2 was increased particularly in patients who rapidly progressed to end-stage renal failure. Together our results uncover what we believe to be a novel function for Lcn2 and a critical pathway leading to progressive renal failure and cystogenesis.