SGLT2 inhibition promotes glomerular repopulation by cells of renin lineage in experimental kidney disease.

AIM: Sodium glucose co-transporter-2 (SGLT2) inhibitors stimulate renal excretion of sodium and glucose and exert renal protective effects in patients with (non-)diabetic chronic kidney disease (CKD) and may as well protect against acute kidney injury (AKI). The mechanism behind this kidney protective effect remains unclear. Juxtaglomerular cells of renin lineage (CoRL) have been demonstrated to function as progenitors for multiple adult glomerular cell types in kidney disease. This study assesses the impact of SGLT2 inhibition on the repopulation of glomerular cells by CoRL and examines their phenotypic commitment. METHODS: Experiments were performed in Ren1cre-tdTomato lineage-trace mice. Either 5/6 nephrectomy (5/6NX) modeling CKD or bilateral ischaemia reperfusion injury (bIRI) mimicking AKI was applied, while the SGLT2 inhibitor empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days. RESULTS: Both 5/6NX and bIRI-induced kidney injury increased the number of glomerular CoRL-derived cells. SGLT2 inhibition improved kidney function after 5/6NX, indicated by decreased blood creatinine and urea levels, but not after bIRI. In line with this, empagliflozin in 5/6NX animals resulted in less glomerulosclerosis, while it did not affect histopathological features in bIRI. Treatment with empagliflozin resulted in an increase in the number of CoRL-derived glomerular cells in both 5/6NX and bIRI conditions. Interestingly, SGLT2 inhibition led to more CoRL-derived podocytes in 5/6NX animals, whereas empagliflozin-treated bIRI mice presented with increased levels of parietal epithelial and mesangial cells derived from CoRL. CONCLUSION: We conclude that SGLT2 inhibition by empagliflozin promotes CoRL-mediated glomerular repopulation with selective CoRL-derived cell types depending on the type of experimental kidney injury. These findings suggest a previously unidentified mechanism that could contribute to the renoprotective effect of SGLT2 inhibitors.

Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis.

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking.

Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

Tsc2 mutation induces renal tubular cell nonautonomous disease.

TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.

Tsc Gene Locus Disruption and Differences in Renal Epithelial Extracellular Vesicles.

In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and -16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and -19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and -20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and ß-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.

Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content.

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100-250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.

Multifaceted highly targeted sequential multidrug treatment of early ambulatory high-risk SARS-CoV-2 infection (COVID-19).

The SARS-CoV-2 virus spreading across the world has led to surges of COVID-19 illness, hospitalizations, and death. The complex and multifaceted pathophysiology of life-threatening COVID-19 illness including viral mediated organ damage, cytokine storm, and thrombosis warrants early interventions to address all components of the devastating illness. In countries where therapeutic nihilism is prevalent, patients endure escalating symptoms and without early treatment can succumb to delayed in-hospital care and death. Prompt early initiation of sequenced multidrug therapy (SMDT) is a widely and currently available solution to stem the tide of hospitalizations and death. A multipronged therapeutic approach includes 1) adjuvant nutraceuticals, 2) combination intracellular anti-infective therapy, 3) inhaled/oral corticosteroids, 4) antiplatelet agents/anticoagulants, 5) supportive care including supplemental oxygen, monitoring, and telemedicine. Randomized trials of individual, novel oral therapies have not delivered tools for physicians to combat the pandemic in practice. No single therapeutic option thus far has been entirely effective and therefore a combination is required at this time. An urgent immediate pivot from single drug to SMDT regimens should be employed as a critical strategy to deal with the large numbers of acute COVID-19 patients with the aim of reducing the intensity and duration of symptoms and avoiding hospitalization and death.

Tuberous sclerosis complex exhibits a new renal cystogenic mechanism.

Tuberous sclerosis complex (TSC) is a tumor predisposition syndrome with significant renal cystic and solid tumor disease. While the most common renal tumor in TSC, the angiomyolipoma, exhibits a loss of heterozygosity associated with disease, we have discovered that the renal cystic epithelium is composed of type A intercalated cells that have an intact Tsc gene that have been induced to exhibit Tsc-mutant disease phenotype. This mechanism appears to be different than that for ADPKD. The murine models described here closely resemble the human disease and both appear to be mTORC1 inhibitor responsive. The induction signaling driving cystogenesis may be mediated by extracellular vesicle trafficking.

Suppressive effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation.

Increasing data implicate iron accumulation in tumorigenesis of the kidney, particularly the clear cell renal cell carcinoma (ccRCC) subtype. The von Hippel Lindau (VHL)/hypoxia inducible factor-α (HIF-α) axis is uniquely dysregulated in ccRCC and is a major regulator and regulatory target of iron metabolism, yet the role of iron in ccRCC tumorigenesis and its potential interplay with VHL inactivation remains unclear. We investigated whether ccRCC iron accumulation occurs due to increased cell dependency on iron for growth and survival as a result of VHL inactivation. Free iron levels were compared between four VHL-mutant ccRCC cell lines (786-0, A704, 769-P, RCC4) and two benign renal tubule epithelial cell lines (RPTEC, HRCEp) using the Phen Green SK fluorescent iron stain. Intracellular iron deprivation was achieved using two clinical iron chelator drugs, deferasirox (DFX) and deferoxamine (DFO), and chelator effects were measured on cell line growth, cell cycle phase, apoptosis, HIF-1α and HIF-2α protein levels and HIF-α transcriptional activity based on expression of target genes CA9, OCT4/POU5F1 and PDGFß/PDGFB. Similar assays were performed in VHL-mutant ccRCC cells with and without ectopic wild-type VHL expression. Baseline free iron levels were significantly higher in ccRCC cell lines than benign renal cell lines. DFX depleted cellular free iron more rapidly than DFO and led to greater growth suppression of ccRCC cell lines (>90% at ~30-150 µM) than benign renal cell lines (~10-50% at up to 250 µM). Similar growth responses were observed using DFO, with the exception that a prolonged treatment duration was necessary to deplete cellular iron adequately for differential growth suppression of the less susceptible A704 ccRCC cell line relative to benign renal cell lines. Apoptosis and G1-phase cell cycle arrest were identified as potential mechanisms of chelator growth suppression based on their induction in ccRCC cell lines but not benign renal cell lines. Iron chelation in ccRCC cells but not benign renal cells suppressed HIF-1α and HIF-2α protein levels and transcriptional activity, and the degree and timing of HIF-2α suppression correlated with the onset of apoptosis. Restoration of wild-type VHL function in ccRCC cells was sufficient to prevent chelator-induced apoptosis and G1 cell cycle arrest, indicating that ccRCC susceptibility to iron deprivation is VHL inactivation-dependent. In conclusion, ccRCC cells are characterized by high free iron levels and a cancer-specific dependency on iron for HIF-α overexpression, cell cycle progression and apoptotic escape. This iron dependency is introduced by VHL inactivation, revealing a novel interplay between VHL/HIF-α dysregulation and ccRCC iron metabolism. Future study is warranted to determine if iron deprivation using chelator drugs provides an effective therapeutic strategy for targeting HIF-2α and suppressing tumor progression in ccRCC patients.

Angiotensin II Short-Loop Feedback: Is There a Role of Ang II for the Regulation of the Renin System In Vivo?

The activity of the renin-angiotensin-aldosterone system is triggered by the release of the protease renin from the kidneys, which in turn is controlled in the sense of negative feedback loops. It is widely assumed that Ang II (angiotensin II) directly inhibits renin expression and secretion via a short-loop feedback by an effect on renin-producing cells (RPCs) mediated by AT1 (Ang II type 1) receptors. Because the concept of such a direct short-loop negative feedback control, which originates mostly from in vitro experiments, has not yet been systematically proven in vivo, we aimed to test the validity of this concept by studying the regulation of renin synthesis and secretion in mice lacking Ang II-AT1 receptors on RPCs. We found that RPCs of the kidney express Ang II-AT1 receptors. Mice with conditional deletion of Ang II-AT1 receptors in RPCs were normal with regard to the number of renin cells, renal renin mRNA, and plasma renin concentrations. Renin expression and secretion of these mice responded to Ang I (angiotensin I)-converting enzyme inhibition and to Ang II infusion like in wild-type (WT) controls. In summary, we did not obtain evidence that Ang II-AT1 receptors on RPCs are of major relevance for the normal regulation of renin expression and secretion in mice. Therefore, we doubt the existence of a direct negative feedback function of Ang II on RPCs.

Sex differences in transcriptomic profiles in aged kidney cells of renin lineage.

Renin expressing cells in the kidney's juxta-glomeruluar compartment likely also serve as progenitors for adult glomerular cells in disease. Although these cells of renin lineage (CoRL) decrease in number with advancing kidney age, accompanied by less responsiveness to typical stimuli such as ACE-inhibition, mechanisms and the impact of sex as a biological variable with age are not known. Accordingly, labeled CoRL were sorted from individual young (2m) and aged (27m) male and female Ren1cCre|ZsGreen reporter mice, and their transcriptomic profiles analyzed by RNA seq. When both aged female and male mice were combined, there were 48 differentially expressed genes (DEG) compared to young mice. However, when compared to their young sex-matched mice, aged female and male mice had 159 and 503 DEGs respectively. In addition to marked differences in individual genes between aged female and male mice, gene ontology analysis showed major pathway differences by sex. The majority of DEGs in one sex did not significantly change or changed in the opposite direction in the other sex. These results show that in CoRL of advanced age, individual genes and gene ontologies change, but differ between female and male mice, highlighting sex related differences the aging process.

Detection of renin lineage cell transdifferentiation to podocytes in the kidney glomerulus with dual lineage tracing.

Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow-colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.

Lack of connexin 40 decreases the calcium sensitivity of renin-secreting juxtaglomerular cells.

The so-called calcium paradoxon of renin describes the phenomenon that exocytosis of renin from juxtaglomerular cells of the kidney is stimulated by lowering of the extracellular calcium concentration. The yet poorly understood effect of extracellular calcium on renin secretion appears to depend on the function of the gap junction protein connexin 40 (Cx40) in renin-producing cells. This study aimed to elucidate the role of Cx40 for the calcium dependency of renin secretion in more detail by investigating if Cx40 function is really essential for the influence of extracellular calcium on renin secretion, if and how Cx40 affects intracellular calcium dynamics in renin-secreting cells and if Cx40-mediated gap junctional coupling of renin-secreting cells with the mesangial cell area is relevant for the influence of extracellular calcium on renin secretion. Renin secretion was studied in isolated perfused mouse kidneys. Calcium measurements were performed in renin-producing cells of microdissected glomeruli. The ultrastructure of renin-secreting cells was examined by electron microscopy. We found that Cx40 was not essential for stimulation of renin secretion by lowering of the extracellular calcium concentration. Instead, Cx40 increased the sensitivity of renin secretion response towards lowering of the extracellular calcium concentration. In line, the sensitivity and dynamics of intracellular calcium in response to lowering of extracellular calcium were dampened when renin-secreting cells lacked Cx40. Disruption of gap junctional coupling of renin-secreting cells by selective deletion of Cx40 from mesangial cells, however, did not change the stimulation of renin secretion by lowering of the extracellular calcium concentration. Deletion of Cx40 from renin cells but not from mesangial cells was associated with a shift of renin expression from perivascular cells of afferent arterioles to extraglomerular mesangial cells. Our findings suggest that Cx40 is not directly involved in the regulation of renin secretion by extracellular calcium. Instead, it appears that in renin-secreting cells of the kidney lacking Cx40, intracellular calcium dynamics and therefore also renin secretion are desensitized towards changes of extracellular calcium. Whether the dampened calcium response of renin-secreting cells lacking Cx40 function results from a direct involvement of Cx40 in intracellular calcium regulation or from the cell type shift of renin expression from perivascular to mesangial cells remains to be clarified. In any case, Cx40-mediated gap junctional coupling between renin and mesangial cells is not relevant for the calcium paradoxon of renin secretion.

Lineage tracing aged mouse kidneys shows lower number of cells of renin lineage and reduced responsiveness to RAAS inhibition.

Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.

Charting the transcriptional landscape of cells of renin lineage following podocyte depletion.

Renin producing cells of the juxtaglomerulus, herein called cells of renin lineage (CoRL), have garnered recent interest for their propensity to act as a progenitor source for various kidney cell types including podocytes. Despite recent advances, the process of transdifferentiation of CoRL to podocytes is poorly understood. In this study, we employed a transgenic reporter mouse line which permanently labels CoRL with ZsGreen fluorescent protein, allowing for isolation by fluorescence-activated cell sorting. At 5 days following induction of abrupt podocyte ablation via anti-podocyte sheep IgG, mice were sacrificed and CoRL were isolated by FACS. RNA was subsequently analyzed by microarray. Gene set enrichment analysis (GSEA) was performed and revealed that CoRL display a distinct phenotype following podocyte ablation, primarily consisting of downregulation of metabolic processes and upregulation of immuno-modulatory processes. Additionally, RNA-biology and cell cycle-related processes were also upregulated. Changes in gene expression or activity of a core set of transcription factors including HNF1 and E2F were identified through changes in enrichment of their respective target genes. However, integration of results from transcription factor and canonical pathway analysis indicated that ERR1 and PU-box family members may be the major contributors to the post-podocyte ablation phenotype of CoRL. Finally, top ranking genes were selected from the microarray-based analysis and confirmed by qPCR. Collectively, our results provide valuable insights into the transcriptional regulation of CoRL following abrupt podocyte ablation.

WT1 Is Necessary for the Proliferation and Migration of Cells of Renin Lineage Following Kidney Podocyte Depletion.

Wilms' tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.

Utility of a Noninvasive 2-Gene Molecular Assay for Cutaneous Melanoma and Effect on the Decision to Biopsy.

Importance: Expression of long intergenic non-protein coding RNA 518 (LINC00518) and preferentially expressed antigen in melanoma (PRAME) genes, obtained via noninvasive adhesive patch biopsy, is a sensitive and specific method for detection of cutaneous melanoma. However, the utility of this test in biopsy decisions made by dermatologists has not been evaluated. Objective: To determine the utility of the pigmented lesion assay (PLA) for LINC00518/PRAME expression in decisions to biopsy a series of pigmented skin lesions. Design, Setting, and Participants: In this secure web-based, multiple-reader-multiple-case study, 45 board-certified dermatologists each evaluated 60 clinical and dermoscopic images of clinically atypical pigmented lesions, first without and then with PLA gene expression information and were asked whether the lesions should be biopsied. Data were collected from March 24, 2014, through November 13, 2015. Interventions: Participants were given a report for each lesion, which included the results of an assay for expression of LINC00518/PRAME and a PLA score with data on the predictive values of the information provided. Main Outcomes and Measures: Biopsy sensitivity and specificity with vs without PLA data. Results: Forty-five dermatologists (29 male and 16 female) performed the evaluation. After incorporating the PLA into their decision as to whether to biopsy a pigmented lesion suggestive of melanoma, dermatologists improved their mean biopsy sensitivity from 95.0% to 98.6% (P = .01); specificity increased from 32.1% to 56.9% (P < .001) with PLA data. Conclusions and Relevance: The noninvasive PLA enables dermatologists to significantly improve biopsy specificity while maintaining or improving sensitivity. This result may increase the number of early melanomas biopsied and reduce the number of benign lesions biopsied, thereby improving patient outcomes and reducing health care costs.

Transcriptome Analysis of Human Reninomas as an Approach to Understanding Juxtaglomerular Cell Biology.

Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular cells of the kidney. Chronic stimulation of renin release results in a recruitment of new juxtaglomerular cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because juxtaglomerular cells rapidly dedifferentiate when removed from the kidney, their developmental origin and the mechanism that explains their phenotypic plasticity remain unclear. To overcome this limitation, we have performed RNA expression analysis on 4 human renin-producing tumors. The most highly expressed genes that were common between the reninomas were subsequently used for in situ hybridization in kidneys of 5-day-old mice, adult mice, and adult mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of human embryonic kidney 293 cells transfected with the mouse cDNA encoding these ligands was applied to (pro)renin-synthesizing As4.1 cells. Among the ligands, only platelet-derived growth factor B reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. In addition, platelet-derived growth factor B-exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was accompanied by a downregulated expression of α-smooth muscle actin and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myoendocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for platelet-derived growth factor B as a regulator of renin-synthesizing cells.

Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging.

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.