Integrated multiomics implicates dysregulation of ECM and cell adhesion pathways as drivers of severe COVID-associated kidney injury.

COVID-19 has been a significant public health concern for the last four years; however, little is known about the mechanisms that lead to severe COVID-associated kidney injury. In this multicenter study, we combined quantitative deep urinary proteomics and machine learning to predict severe acute outcomes in hospitalized COVID-19 patients. Using a 10-fold cross-validated random forest algorithm, we identified a set of urinary proteins that demonstrated predictive power for both discovery and validation set with 87% and 79% accuracy, respectively. These predictive urinary biomarkers were recapitulated in non-COVID acute kidney injury revealing overlapping injury mechanisms. We further combined orthogonal multiomics datasets to understand the mechanisms that drive severe COVID-associated kidney injury. Functional overlap and network analysis of urinary proteomics, plasma proteomics and urine sediment single-cell RNA sequencing showed that extracellular matrix and autophagy-associated pathways were uniquely impacted in severe COVID-19. Differentially abundant proteins associated with these pathways exhibited high expression in cells in the juxtamedullary nephron, endothelial cells, and podocytes, indicating that these kidney cell types could be potential targets. Further, single-cell transcriptomic analysis of kidney organoids infected with SARS-CoV-2 revealed dysregulation of extracellular matrix organization in multiple nephron segments, recapitulating the clinically observed fibrotic response across multiomics datasets. Ligand-receptor interaction analysis of the podocyte and tubule organoid clusters showed significant reduction and loss of interaction between integrins and basement membrane receptors in the infected kidney organoids. Collectively, these data suggest that extracellular matrix degradation and adhesion-associated mechanisms could be a main driver of COVID-associated kidney injury and severe outcomes.

cGAS Activation Accelerates the Progression of Autosomal Dominant Polycystic Kidney Disease.

SIGNIFICANCE STATEMENT: The renal immune infiltrate observed in autosomal polycystic kidney disease contributes to the evolution of the disease. Elucidating the cellular mechanisms underlying the inflammatory response could help devise new therapeutic strategies. Here, we provide evidence for a mechanistic link between the deficiency polycystin-1 and mitochondrial homeostasis and the activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of the interferon genes (STING) pathway. Our data identify cGAS as an important mediator of renal cystogenesis and suggest that its inhibition may be useful to slow down the disease progression. BACKGROUND: Immune cells significantly contribute to the progression of autosomal dominant polycystic kidney disease (ADPKD), the most common genetic disorder of the kidney caused by the dysregulation of the Pkd1 or Pkd2 genes. However, the mechanisms triggering the immune cells recruitment and activation are undefined. METHODS: Immortalized murine collecting duct cell lines were used to dissect the molecular mechanism of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) activation in the context of genotoxic stress induced by Pkd1 ablation. We used conditional Pkd1 and knockout cGas-/- genetic mouse models to confirm the role of cGAS/stimulator of the interferon genes (STING) pathway activation on the course of renal cystogenesis. RESULTS: We show that Pkd1 -deficient renal tubular cells express high levels of cGAS, the main cellular sensor of cytosolic nucleic acid and a potent stimulator of proinflammatory cytokines. Loss of Pkd1 directly affects cGAS expression and nuclear translocation, as well as activation of the cGAS/STING pathway, which is reversed by cGAS knockdown or functional pharmacological inhibition. These events are tightly linked to the loss of mitochondrial structure integrity and genotoxic stress caused by Pkd1 depletion because they can be reverted by the potent antioxidant mitoquinone or by the re-expression of the polycystin-1 carboxyl terminal tail. The genetic inactivation of cGAS in a rapidly progressing ADPKD mouse model significantly reduces cystogenesis and preserves normal organ function. CONCLUSIONS: Our findings indicate that the activation of the cGAS/STING pathway contributes to ADPKD cystogenesis through the control of the immune response associated with the loss of Pkd1 and suggest that targeting this pathway may slow disease progression.

Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

Computational study of biomechanical drivers of renal cystogenesis.

Renal cystogenesis is the pathological hallmark of autosomal dominant polycystic kidney disease, caused by PKD1 and PKD2 mutations. The formation of renal cysts is a common manifestation in ciliopathies, a group of syndromic disorders caused by mutation of proteins involved in the assembly and function of the primary cilium. Cystogenesis is caused by the derailment of the renal tubular architecture and tissue deformation that eventually leads to the impairment of kidney function. However, the biomechanical imbalance of cytoskeletal forces that are altered in cells with Pkd1 mutations has never been investigated, and its nature and extent remain unknown. In this computational study, we explored the feasibility of various biomechanical drivers of renal cystogenesis by examining several hypothetical mechanisms that may promote morphogenetic markers of cystogenesis. Our objective was to provide physics-based guidance for our formulation of hypotheses and our design of experimental studies investigating the role of biomechanical disequilibrium in cystogenesis. We employed the finite element method to explore the role of (1) wild-type versus mutant cell elastic modulus; (2) contractile stress magnitude in mutant cells; (3) localization and orientation of contractile stress in mutant cells; and (4) sequence of cell contraction and cell proliferation. Our objective was to identify the factors that produce the characteristic tubular cystic growth. Results showed that cystogenesis occurred only when mutant cells contracted along the apical-basal axis, followed or accompanied by cell proliferation, as long as mutant cells had comparable or lower elastic modulus than wild-type cells, with their contractile stresses being significantly greater than their modulus. Results of these simulations allow us to focus future in vitro and in vivo experimental studies on these factors, helping us formulate physics-based hypotheses for renal tubule cystogenesis.

SARS-CoV-2 viral protein ORF3A injures renal tubules by interacting with TRIM59 to induce STAT3 activation.

Acute kidney injury occurs frequently in COVID-19 patients infected by the coronavirus SARS-CoV-2, and infection of kidney cells by this virus has been reported. However, little is known about the direct impact of the SARS-CoV-2 infection upon the renal tubular cells. We report that SARS-CoV-2 activated signal transducer and activator of transcription 3 (STAT3) signaling and caused cellular injury in the human renal tubular cell line. Mechanistically, the viral protein ORF3A of SARS-CoV-2 augmented both NF-κB and STAT3 signaling and increased the expression of kidney injury molecule 1. SARS-CoV-2 infection or expression of ORF3A alone elevated the protein level of tripartite motif-containing protein 59 (TRIM59), an E3 ubiquitin ligase, which interacts with both ORF3A and STAT3. The excessive TRIM59 in turn dissociated the phosphatase TCPTP from binding to STAT3 and hence inhibited the dephosphorylation of STAT3, leading to persistent STAT3 activation. Consistently, ORF3A induced renal injury in zebrafish and mice. In addition, expression of TRIM59 was elevated in the kidney autopsies of COVID-19 patients with acute kidney injury. Thus, the aberrant activation of STAT3 signaling by TRIM59 plays a significant role in the renal tubular cell injury caused by SARS-CoV-2, which suggests a potential targeted therapy for the renal complications of COVID-19.

Epithelial proliferation and cell cycle dysregulation in kidney injury and disease.

Various cellular insults and injury to renal epithelial cells stimulate repair mechanisms to adapt and restore the organ homeostasis. Renal tubular epithelial cells are endowed with regenerative capacity, which allows for a restoration of nephron function after acute kidney injury. However, recent evidence indicates that the repair is often incomplete, leading to maladaptive responses that promote the progression to chronic kidney disease. The dysregulated cell cycle and proliferation is also a key feature of renal tubular epithelial cells in polycystic kidney disease and HIV-associated nephropathy. Therefore, in this review, we provide an overview of cell cycle regulation and the consequences of dysregulated cell proliferation in acute kidney injury, polycystic kidney disease, and HIV-associated nephropathy. An increased understanding of these processes may help define better targets for kidney repair and combat chronic kidney disease progression.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

Integrin-ß1 is required for the renal cystogenesis caused by ciliary defects.

Defects in the function of primary cilia are commonly associated with the development of renal cysts. On the other hand, the intact cilium appears to contribute a cystogenic signal whose effectors remain unclear. As integrin-ß1 is required for the cystogenesis caused by the deletion of the polycystin 1 gene, we asked whether it would be similarly important in the cystogenetic process caused by other ciliary defects. We addressed this question by investigating the effect of integrin-ß1 deletion in a ciliopathy genetic model in which the Ift88 gene, a component of complex B of intraflagellar transport that is required for the proper assembly of cilia, is specifically ablated in principal cells of the collecting ducts. We showed that the renal cystogenesis caused by loss of Ift88 is prevented when integrin-ß1 is simultaneously depleted. In parallel, pathogenetic manifestations of the disease, such as increased inflammatory infiltrate and fibrosis, were also significantly reduced. Overall, our data indicate that integrin-ß1 is also required for the renal cystogenesis caused by ciliary defects and point to integrin-ß1-controlled pathways as common drivers of the disease and as possible targets to interfere with the cystogenesis caused by ciliary defects.

The Joubert syndrome protein ARL13B binds tubulin to maintain uniform distribution of proteins along the ciliary membrane.

Cilia-mediated signal transduction involves precise targeting and localization of selected molecules along the ciliary membrane. However, the molecular mechanism underlying these events is unclear. The Joubert syndrome protein ARL13B is a membrane-associated G-protein that localizes along the cilium and functions in protein transport and signaling. We identify tubulin as a direct interactor of ARL13B and demonstrate that the association occurs via the G-domain and independently from the GTPase activity of ARL13B. The G-domain is necessary for the interaction of ARL13B with the axoneme both in vitro and in vivo We further show that exogenously expressed mutants lacking the tubulin-binding G-domain (ARL13B-ΔGD) or whose GTPase domain is inactivated (ARL13B-T35N) retain ciliary localization, but fail to rescue ciliogenesis defects of null Arl13bhnn mouse embryonic fibroblasts (MEFs). However, while ARL13B-ΔGD and the membrane proteins Smoothened (SMO) and Somatostatin receptor-3 (SSTR3) distribute unevenly along the cilium of Arl13bhnn MEFs, ARL13B-T35N distributes evenly along the cilium and enables the uniform distribution of SMO and SSTR3. Thus, we propose a so far unknown function of ARL13B in anchoring ciliary membrane proteins to the axoneme through the direct interaction of its G-domain with tubulin.

Prothymosin-α Variants Elicit Anti-HIV-1 Response via TLR4 Dependent and Independent Pathways.

BACKGROUND: Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intracellular and extracellular-associated functions. Recently, we identified two new ProTα variants with potent anti-HIV activity from CD8+ T cells and cervicovaginal lavage. The first is a splice variant of the ProTα gene known as isoB and the second is the product of ProTα pseudogene 7 (p7). Similarly to iso2, the anti-HIV activity of both variants is mediated by type I IFN. Here we tested whether the immunomodulatory activity of isoB and p7 are also TLR4 dependent and determined their kinetic of release in response to HIV-1 infection. METHODS: Type I, type III, TNF-α and IL-6 mRNA inducing activity was determined in macrophages from wild type and TLR4 knockout mice treated with recombinant ProTα variants. Supernatants from mock and HIV infected cells were analyzed by mass spectrometry in positive and negative modes for the presence of ProTα variants. In silico structural and functional analysis of ProTα variants were performed. RESULTS: We show that both isoB and p7 upregulate IFN-ß, IFN-λ1, IL-6, TNF-α and RANTES mRNAs in primary human macrophages. The potent stimulation of IFN-ß by the recombinant ProTα variants in human macrophages is dependent on the TLR4 pathway, whereas the induction of TNF-α and IL-6 may also occur independently of TLR4, suggesting the interaction of ProTα variants with other signaling molecules/receptors. In silico analyses confirmed that the novel isoB and p7 variants are intrinsically disordered proteins, which lack the NLS and mass spectrometry showed release of ProTα variants within minutes post HIV-1 infection. These features are consistent with the function of ProTα variants as damage associate molecular patterns (DAMPs). CONCLUSIONS: Our findings indicate that ProTα variants strongly inhibit viral replication mainly, but not exclusively, through TLR4 signaling and that they are released within minutes of viral infection suggesting that they may function as DAMPs.

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

Inactivation of integrin-ß1 prevents the development of polycystic kidney disease after the loss of polycystin-1.

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-ß1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-ß1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-ß1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-ß1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.

Prostaglandin E(2) mediates proliferation and chloride secretion in ADPKD cystic renal epithelia.

Prostaglandin E(2) (PGE(2)) contributes to cystogenesis in genetically nonorthologous models of autosomal dominant polycystic kidney disease (ADPKD). However, it remains unknown whether PGE(2) induces the classic features of cystic epithelia in genetically orthologous models of ADPKD. We hypothesized that, in ADPKD epithelia, PGE(2) induces proliferation and chloride (Cl(-)) secretion, two archetypal phenotypic features of ADPKD. To test this hypothesis, proliferation and Cl(-) secretion were measured in renal epithelial cells deficient in polycystin-1 (PC-1). PC-1-deficient cells increased in cell number (proliferated) faster than PC-1-replete cells, and this proliferative advantage was abrogated by cyclooxygenase inhibition, indicating a role for PGE(2) in cell proliferation. Exogenous administration of PGE(2) increased proliferation of PC-1-deficient cells by 38.8 ± 5.2% (P < 0.05) but inhibited the growth of PC-1-replete control cells by 49.4 ± 1.9% (P < 0.05). Next, we tested whether PGE(2)-specific E prostanoid (EP) receptor agonists induce intracellular cAMP and downstream ß-catenin activation. PGE(2) and EP4 receptor agonism (TCS 2510) increased intracellular cAMP concentration and the abundance of active ß-catenin in PC-1-deficient cells, suggesting a mechanism for PGE(2)-mediated proliferation. Consistent with this hypothesis, antagonizing EP4 receptors reverted the growth advantage of PC-1-deficient cells, implicating a central role for the EP4 receptor in proliferation. To test whether PGE(2)-dependent Cl(-) secretion is also enhanced in PC-1-deficient cells, we used an Ussing chamber to measure short-circuit current (I(sc)). Addition of PGE(2) induced a fivefold higher increase in I(sc) in PC-1-deficient cells compared with PC-1-replete cells. This PGE(2)-induced increase in I(sc) in PC-1-deficient cells was blocked by CFTR-172 and flufenamic acid, indicating that PGE(2) activates CFTR and calcium-activated Cl(-) channels. In conclusion, PGE(2) activates aberrant signaling pathways in PC-1-deficient epithelia that contribute to the proliferative and secretory phenotype characteristic of ADPKD and suggests a therapeutic role for PGE(2) inhibition and EP4 receptor antagonism.

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons.

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

Cilium, centrosome and cell cycle regulation in polycystic kidney disease.

Polycystic kidney disease is the defining condition of a group of common life-threatening genetic disorders characterized by the bilateral formation and progressive expansion of renal cysts that lead to end stage kidney disease. Although a large body of information has been acquired in the past years about the cellular functions that characterize the cystic cells, the mechanisms triggering the cystogenic conversion are just starting to emerge. Recent findings link defects in ciliary functions, planar cell polarity pathway, and centrosome integrity in early cystic development. Many of the signals dysregulated during cystogenesis may converge on the centrosome for its central function as a structural support for cilia formation and a coordinator of protein trafficking, polarity, and cell division. Here, we will discuss the contribution of proliferation, cilium and planar cell polarity to the cystic signal and will analyze in particular the possible role that the basal bodies/centrosome may play in the cystogenetic mechanisms. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Primary cilia dynamics instruct tissue patterning and repair of corneal endothelium.

Primary cilia are required for several signaling pathways, but their function in cellular morphogenesis is poorly understood. Here we show that emergence of an hexagonal cellular pattern during development of the corneal endothelium (CE), a monolayer of neural crest-derived cells that maintains corneal transparency, depends on a precise temporal control of assembly of primary cilia that subsequently disassemble in adult corneal endothelial cells (CECs). However, cilia reassembly occurs rapidly in response to an in vivo mechanical injury and precedes basal body polarization and cellular elongation in mature CECs neighboring the wound. In contrast, CE from hypomorphic IFT88 mutants (Tg737(orpk)) or following in vivo lentiviral-mediated IFT88 knockdown display dysfunctional cilia and show disorganized patterning, mislocalization of junctional markers, and accumulation of cytoplasmic acetylated tubulin. Our results indicate an active role of cilia in orchestrating coordinated morphogenesis of CECs during development and repair and define the murine CE as a powerful in vivo system to study ciliary-based cellular dynamics.

Fluid shear stress induces renal epithelial gene expression through polycystin-2-dependent trafficking of extracellular regulated kinase.

BACKGROUND: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1). METHODS: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells. RESULTS: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not. CONCLUSIONS: PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.

Prothymosin-alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction.

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-alpha (ProTalpha), a small acidic protein produced and released by CD8(+) T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTalpha acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTalpha, retained by an acidic peptide derived from ProTalpha, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTalpha accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8(+) cells. Thus, a protein produced by CD8(+) T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTalpha may provide therapeutic leads for IFN-sensitive viruses.

Loss of polycystin-1 causes centrosome amplification and genomic instability.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.

Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells.

Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in renal epithelial cell lines derived from murine models of autosomal recessive polycystic kidney disease (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule [human fetal collecting tubule (HFCT) pool and clone 2C] cell lines were grown in culture, loaded with a Ca(2+) indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca(2+)](i) were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca(2+)](i) was greater in ARPKD 5E (700 +/- 87 nM, n = 21) than in HFCT 2C (315 +/- 58 nM, n = 12; P < 0.01) cells. ARPKD 5E cells treated with Gd(3+), an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [Ca(2+)](i) transient. Cilia were approximately 20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd(3+)-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca(2+) stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated Gd(3+)-sensitive mechano-induced Ca(2+) response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.