Over time, memory retrieval is thought to transfer from the hippocampus to a distributed network of neocortical sites. Of these sites, the retrosplenial cortex (RSC) is robustly activated during retrieval of remotely acquired, emotionally-valenced memories. It is unclear, however, whether RSC is specifically involved in memory storage or retrieval, and which neurotransmitter receptor mechanisms serve its function. We addressed these questions by inhibiting N-methyl-d-aspartate receptors (NMDAR) in RSC via infusions of APV prior to tests for context fear in male mice. Anterior cingulate cortex (ACC) and dorsal hippocampus (DH), which have been implicated in the retrieval of remote and recent memory, respectively, served as neuroanatomical controls. Surprisingly, infusion of APV only into RSC, but not ACC or DH, abolished retrieval of remote memory, as revealed by lack of freezing to the conditioning context. APV infused into RSC also impaired retrieval of recent memory, but had no effect on conditioning or memory storage. Within-subject experiments confirmed that the role of RSC in memory retrieval is not time-limited. RSC-dependent context fear memory retrieval was mediated by NR2A, but not NR2B, subunit-containing NMDAR. Collectively, these data are the first demonstration that NMDAR in RSC are necessary for the retrieval of remote and recent memories of fear-evoking contexts. Dysfunction of RSC may thereby contribute significantly to the re-experiencing of traumatic memories in patients with post-traumatic stress disorder (PTSD).

No abstract

Background DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. Methods We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Results Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Conclusions Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4, and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal inter-endothelial cell junctions, and poor attachment between the glomerular endothelial cells and basement membrane. Additionally, we demonstrated that expression of the VEGFR-3 ligand, VEGF-C, by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, our studies support a VEGF-C independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels which has implications for clinical therapies that target this receptor.

Nephrotic syndrome is one of the most common glomerular diseases in children and can be classified on the basis of steroid responsiveness. While multiple genetic causes have been discovered for steroid resistant nephrotic syndrome, the genetics of steroid sensitive nephrotic syndrome remains elusive. Mutations in Epithelial Membrane Protein 2 ( EMP2 ), a member of the GAS3/PMP22 tetraspan family of proteins, were recently implicated as putative monogenic cause of steroid sensitive nephrotic syndrome. We investigated this hypothesis by developing Emp2 reporter and knockout mouse models. In lacZ reporter mice (engineered to drive expression of the enzyme β-galactosidase under the control of the endogenous murine Emp2 promoter), Emp2 promoter activity was not observed in podocytes but was particularly prominent in medium- and large-caliber arterial vessels in the kidney and other tissues where it localizes specifically in vascular smooth muscle cells (vSMCs) but not in the endothelium. Strong Emp2 expression was also found in non-vascular smooth muscle cells found in other organs like the stomach, bladder, and uterus. Global and podocyte-specific Emp2 knockout mice were viable and did not develop nephrotic syndrome showing no evidence of abnormal glomerular histology or ultrastructure. Altogether, our results do not support that loss of function of EMP2 represent a monogenic cause of proteinuric kidney disease. However, the expression pattern of Emp2 indicates that it may be relevant in smooth muscle function in various organs and tissues including the vasculature.

BackgroundLymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease.As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined.MethodsWe generated a new mouse model expressingVegf-Cunder regulation of the nephron progenitor Six2Cre driver strain (Six2Vegf-C). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging.ResultsSix2Vegf-Cmice had reduced body weight and kidney function compared to littermate controls.Six2Vegf-Ckidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density.ConclusionsKidney lymphangiogenesis was robustly induced in theSix2Vegf-Cmice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.

Purpose of reviewThis review will highlight recent advances in our understanding of the kidney lymphatics regarding their development, physiologic function, and their potential role in the progression of kidney disease.Recent findingsAlthough sparse in comparison to the blood vasculature, lymphatic vessels within the healthy kidney perform an important role in maintaining homeostasis. Additionally, in response to kidney injury, lymphatic vessels undergo substantial expansion, termed lymphangiogenesis, which shows a direct correlation to the extent of tubulointerstitial fibrosis. Kidney lymphatics expand through both the proliferation of lymphatic endothelial cells from existing lymphatic vessels, as well as from direct contribution by other cell types of nonvenous origin. The primary driver of lymphatic growth is vascular endothelial growth factor C, both in development and in response to injury. The clinical implications of lymphangiogenesis in the setting of kidney diseases remains debated, however growing evidence suggests lymphatic vessels may perform a protective role in clearing away accumulating interstitial fluid, inflammatory cytokines, and cellular infiltrates that occur with injury.SummaryThere is increasing evidence the kidney lymphatics perform an active role in the response to kidney injury and the development of fibrosis. Recent advances in our understanding of these vessels raise the possibility of targeting kidney lymphatics for the treatment of kidney disease.

Running Title: APOL1 Variant Organoid scRNA-seq Word Count: 205 (Abstract), 1736 (Main Text, excluding Methods)ABSTRACT Background DNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNAseq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls. ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells.Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1mediated kidney disease.