Epidermal growth factor receptor activation is essential for kidney fibrosis development.

Fibrosis is the progressive accumulation of excess extracellular matrix and can cause organ failure. Fibrosis can affect nearly every organ including kidney and there is no specific treatment currently. Although Epidermal Growth Factor Receptor (EGFR) signaling pathway has been implicated in development of kidney fibrosis, underlying mechanisms by which EGFR itself mediates kidney fibrosis have not been elucidated. We find that EGFR expression increases in interstitial myofibroblasts in human and mouse fibrotic kidneys. Selective EGFR deletion in the fibroblast/pericyte population inhibits interstitial fibrosis in response to unilateral ureteral obstruction, ischemia or nephrotoxins. In vivo and in vitro studies and single-nucleus RNA sequencing analysis demonstrate that EGFR activation does not induce myofibroblast transformation but is necessary for the initial pericyte/fibroblast migration and proliferation prior to subsequent myofibroblast transformation by TGF-ß or other profibrotic factors. These findings may also provide insight into development of fibrosis in other organs and in other conditions.

Genome Engineering of Human Urine-Derived Stem Cells to Express Lactoferrin and Deoxyribonuclease.

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon modification. USCs from a healthy donor sample were isolated according to established protocols. Within 2 months, 10 clones had been expanded, analyzed, and frozen. Fluorescence-activated cell sorting analysis of individual clones revealed that all 10 clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90, and CD146; negative for CD31, CD34, and CD45). The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN (NPHS1), podocalyxin, and Wilms tumor 1 (WT1). Transfection of USCs with a strongly expressing Green fluorescent protein plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by live animal imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, USCs represent a clinically relevant cell type that can express nonviral transgenes. Impact statement Acute kidney injury (AKI) affects over 13 million people worldwide each year, with hospitalization rates on the rise. There are no therapies that directly regenerate the kidney after AKI. Each human kidney contains approximately one million nephrons that process ∼100 L of urinary filtrate each day. Thousands of kidney cells become detached and are excreted in the urine. A small percentage of these cells can be clonally derived into urine-derived stem cells. We have optimized methods for genome engineering of adult human urine-derived stem cells for future applications in regenerative approaches to treat kidney injury.

Cell and gene therapy for kidney disease.

Kidney disease is a leading cause of morbidity and mortality across the globe. Current interventions for kidney disease include dialysis and renal transplantation, which have limited efficacy or availability and are often associated with complications such as cardiovascular disease and immunosuppression. There is therefore a pressing need for novel therapies for kidney disease. Notably, as many as 30% of kidney disease cases are caused by monogenic disease and are thus potentially amenable to genetic medicine, such as cell and gene therapy. Systemic disease that affects the kidney, such as diabetes and hypertension, might also be targetable by cell and gene therapy. However, although there are now several approved gene and cell therapies for inherited diseases that affect other organs, none targets the kidney. Promising recent advances in cell and gene therapy have been made, including in the kidney research field, suggesting that this form of therapy might represent a potential solution for kidney disease in the future. In this Review, we describe the potential for cell and gene therapy in treating kidney disease, focusing on recent genetic studies, key advances and emerging technologies, and we describe several crucial considerations for renal genetic and cell therapies.

Inhibition of Transforming Growth Factor-ß Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-ß (TGF-ß) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-ß receptors possibly responsible for the improved differentiation, evaluate whether TGF-ß inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-ß signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-ß inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-ß signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-ß by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-ß signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells.

DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

Kidney collecting duct cells make vasopressin in response to NaCl-induced hypertonicity.

Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.

Ligand-independent integrin ß1 signaling supports lung adenocarcinoma development.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin ß1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin ß1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin ß1-mediated adhesion to ECM but are dependent on integrin ß1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin ß1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

Gene therapy for kidney disease: targeting cystinuria.

PURPOSE OF REVIEW: The aim of this study was to summarize recent findings in kidney gene therapy while proposing cystinuria as a model kidney disease target for genome engineering therapeutics. RECENT FINDINGS: Despite the advances of gene therapy for treating diseases of other organs, the kidney lags behind. Kidney-targeted gene delivery remains an obstacle to gene therapy of kidney disease. Nanoparticle and adeno-associated viral vector technologies offer emerging hope for kidney gene therapy. Cystinuria represents a model potential target for kidney gene therapy due to its known genetic and molecular basis, targetability, and capacity for phenotypic rescue. SUMMARY: Although gene therapy for kidney disease remains a major challenge, new and evolving technologies may actualize treatment for cystinuria and other kidney diseases.

Myeloid cyclooxygenase-2/prostaglandin E2/E-type prostanoid receptor 4 promotes transcription factor MafB-dependent inflammatory resolution in acute kidney injury.

Following acute injury to the kidney, macrophages play an important role in recovery of functional and structural integrity, but organ fibrosis and progressive functional decline occur with incomplete recovery. Pro-resolving macrophages are characterized by increased cyclooxygenase 2 (COX-2) expression and this expression was selectively increased in kidney macrophages following injury and myeloid-specific COX-2 deletion inhibited recovery. Deletion of the myeloid prostaglandin E2 (PGE2) receptor, E-type prostanoid receptor 4 (EP4), mimicked effects seen with myeloid COX-2-/- deletion. PGE2-mediated EP4 activation induced expression of the transcription factor MafB in kidney macrophages, which upregulated anti-inflammatory genes and suppressed pro-inflammatory genes. Myeloid Mafb deletion recapitulated the effects seen with either myeloid COX-2 or EP4 deletion following acute kidney injury, with delayed recovery, persistent presence of pro-inflammatory kidney macrophages, and increased kidney fibrosis. Thus, our studies identified a previously unknown mechanism by which prostaglandins modulate macrophage phenotype following acute organ injury and provide new insight into mechanisms underlying detrimental kidney effects of non-steroidal anti-inflammatory drugs that inhibit cyclooxygenase activity.

Cognate restriction of transposition by piggyBac-like proteins.

Mobile genetic elements have been harnessed for gene transfer for a wide variety of applications including generation of stable cell lines, recombinant protein production, creation of transgenic animals, and engineering cell and gene therapy products. The piggyBac transposon family includes transposase or transposase-like proteins from a variety of species including insect, bat and human. Recently, human piggyBac transposable element derived 5 (PGBD5) protein was reported to be able to transpose piggyBac transposons in human cells raising possible safety concerns for piggyBac-mediated gene transfer applications. We evaluated three piggyBac-like proteins across species including piggyBac (insect), piggyBat (bat) and PGBD5 (human) for their ability to mobilize piggyBac transposons in human cells. We observed a lack of cross-species transposition activity. piggyBac and piggyBat activity was restricted to their cognate transposons. PGBD5 was unable to mobilize piggyBac transposons based on excision, colony count and plasmid rescue analysis, and it was unable to bind piggyBac terminal repeats. Within the piggyBac family, we observed a lack of cross-species activity and found that PGBD5 was unable to bind, excise or integrate piggyBac transposons in human cells. Transposition activity appears restricted within species within the piggyBac family of mobile genetic elements.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Functional analysis of the catalytic triad of the hAT-family transposase TcBuster.

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 µm while the E589Q rodlets (N = 33) averaged 1.157 µm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons.

Structural basis of seamless excision and specific targeting by piggyBac transposase.

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.

EGF receptor-mediated FUS phosphorylation promotes its nuclear translocation and fibrotic signaling.

Excessive accumulation of collagen leads to fibrosis. Integrin α1ß1 (Itgα1ß1) prevents kidney fibrosis by reducing collagen production through inhibition of the EGF receptor (EGFR) that phosphorylates cytoplasmic and nuclear proteins. To elucidate how the Itgα1ß1/EGFR axis controls collagen synthesis, we analyzed the levels of nuclear tyrosine phosphorylated proteins in WT and Itgα1-null kidney cells. We show that the phosphorylation of the RNA-DNA binding protein fused in sarcoma (FUS) is higher in Itgα1-null cells. FUS contains EGFR-targeted phosphorylation sites and, in Itgα1-null cells, activated EGFR promotes FUS phosphorylation and nuclear translocation. Nuclear FUS binds to the collagen IV promoter, commencing gene transcription that is reduced by inhibiting EGFR, down-regulating FUS, or expressing FUS mutated in the EGFR-targeted phosphorylation sites. Finally, a cell-penetrating peptide that inhibits FUS nuclear translocation reduces FUS nuclear content and collagen IV transcription. Thus, EGFR-mediated FUS phosphorylation regulates FUS nuclear translocation and transcription of a major profibrotic collagen gene. Targeting FUS nuclear translocation offers a new antifibrotic therapy.

CRISPR/Cas9 engineering of albino cystinuria Type A mice.

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.

Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

INTRODUCTION: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. METHODS: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. RESULTS: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. CONCLUSIONS: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

Metabolic consequences of cystinuria.

BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.