A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-ß (TGF-ß). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-ß-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Meis1 is specifically upregulated in kidney myofibroblasts during aging and injury but is not required for kidney homeostasis or fibrotic response.

The homeobox transcription factor Meis1 is required for mammalian development, and its overexpression plays a role in tumorigenesis, especially leukemia. Meis1 is known to be expressed in kidney stroma, but its function in kidney is undefined. We hypothesized that Meis1 may regulate stromal cell proliferation in kidney development and disease and tested the hypothesis using cell lineage tracing and cell-specific Meis1 deletion in development, aging, and fibrotic disease. We observed strong expression of Meis1 in platelet-derived growth factor receptor-ß-positive pericytes and perivascular fibroblasts, both in adult mouse kidney and to a lesser degree in human kidney. Either bilateral ischemia-reperfusion injury or aging itself led to strong upregulation of Meis1 protein and mRNA in kidney myofibroblasts, and genetic lineage analysis confirmed that Meis1-positive cells proliferate as they differentiate into myofibroblasts after injury. Conditional deletion of Meis1 in all kidney stroma with two separate tamoxifen-inducible Cre recombinase drivers had no phenotype with the exception of consistent induction of the tubular injury marker kidney injury molecule-1 (Kim-1) only in Meis1 mutants. Further examination of Kim-1 expression revealed linkage disequilibrium of Kim-1 and Meis1, such that Meis1 mutants carried the longer BALB/c Kim-1 allele. Unexpectedly, we report that this Kim-1 allele is expressed at baseline in wild-type BALB/c mice, without any associated abnormalities, including long-term fibrosis, as predicted from the literature. We conclude that Meis1 is specifically expressed in stroma and myofibroblasts of mouse and human kidney, that it is not required for kidney development, disease, or aging, and that BALB/c mice unexpectedly express Kim-1 protein at baseline without other kidney abnormality.

Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model.

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1ß and NLRP3 were individually assessed. Tlr4, Tlr5, Lbp, Nlrp3, Casp1, Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2, Tlr7, Tlr9, Nod2, Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.

Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury.

Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean±SEM: 3.3±0.1 µm before injury versus 12.5±0.2 µm after injury; P<0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean±SEM capillaries/high-power field: 67.6±4.7 in control versus 44.1±4.8 at 56 days; P<0.05) and increased cross-sectional area (mean±SEM: 21.9±0.4 µm2 in control versus 24.1±0.6 µm2 at 10 days; P<0.01 and 24.6±0.6 µm2 at 56 days; P<0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction.

Adventitial MSC-like Cells Are Progenitors of Vascular Smooth Muscle Cells and Drive Vascular Calcification in Chronic Kidney Disease.

Mesenchymal stem cell (MSC)-like cells reside in the vascular wall, but their role in vascular regeneration and disease is poorly understood. Here, we show that Gli1+ cells located in the arterial adventitia are progenitors of vascular smooth muscle cells and contribute to neointima formation and repair after acute injury to the femoral artery. Genetic fate tracing indicates that adventitial Gli1+ MSC-like cells migrate into the media and neointima during athero- and arteriosclerosis in ApoE-/- mice with chronic kidney disease. Our data indicate that Gli1+ cells are a major source of osteoblast-like cells during calcification in the media and intima. Genetic ablation of Gli1+ cells before induction of kidney injury dramatically reduced the severity of vascular calcification. These findings implicate Gli1+ cells as critical adventitial progenitors in vascular remodeling after acute and during chronic injury and suggest that they may be relevant therapeutic targets for mitigation of vascular calcification.

Chronic VEGF blockade worsens glomerular injury in the remnant kidney model.

VEGF inhibition can promote renal vascular and parenchymal injury, causing proteinuria, hypertension and thrombotic microangiopathy. The mechanisms underlying these side effects are unclear. We investigated the renal effects of the administration, during 45 days, of sunitinib (Su), a VEGF receptor inhibitor, to rats with 5/6 renal ablation (Nx). Adult male Munich-Wistar rats were distributed among groups S+V, sham-operated rats receiving vehicle only; S+Su, S rats given Su, 4 mg/kg/day; Nx+V, Nx rats receiving V; and Nx+Su, Nx rats receiving Su. Su caused no change in Group S. Seven and 45 days after renal ablation, renal cortical interstitium was expanded, in association with rarefaction of peritubular capillaries. Su did not worsen hypertension, proteinuria or interstitial expansion, nor did it affect capillary rarefaction, suggesting little angiogenic activity in this model. Nx animals exhibited glomerulosclerosis (GS), which was aggravated by Su. This effect could not be explained by podocyte damage, nor could it be ascribed to tuft hypertrophy or hyperplasia. GS may have derived from organization of capillary microthrombi, frequently observed in Group Nx+Su. Treatment with Su did not reduce the fractional glomerular endothelial area, suggesting functional rather than structural cell injury. Chronic VEGF inhibition has little effect on normal rats, but can affect glomerular endothelium when renal damage is already present.

Programmed hypertension in rats treated with a NF-κB inhibitor during nephrogenesis: renal mechanisms.

Suppression of the renin-angiotensin system (RAS) during murine lactation causes progressive renal injury, indicating a physiological action of angiotensin II on nephrogenesis. The nuclear factor NF-κB system is one of the main intracellular mediators of angiotensin II. We investigated whether inhibition of this system with pyrrolidine dithiocarbamate (PDTC) during rat nephrogenesis would lead to similar hypertension and renal injury as observed with RAS suppressors. Immediately after delivery, 32 Munich-Wistar dams, each nursing 6 male pups, were divided into 2 groups: C, untreated, and PDTC, receiving PDTC, 280 mg kg(-1) day(-1) orally, during 21 days. After weaning, the offspring were followed until 10 months of age without treatment. Adult rats that received neonatal PDTC exhibited stable hypertension and myocardial injury, without albuminuria. To gain additional insight into this process, the renal expression of RAS components and sodium transporters were determined by quantitative real-time PCR (qRT-PCR) at 3 and 10 months of life. Renal renin and angiotensinogen were upregulated at 3 and downregulated at 10 months of age, suggesting a role for early local RAS activation. Likewise, there was early upregulation of the proximal sodium/glucose and sodium/bicarbonate transporters, which abated later in life, suggesting that additional factors sustained hypertension in the long run. The conclusions drawn from the findings were as follows: (1) an intact NF-κB system during nephrogenesis may be essential to normal renal and cardiovascular function in adult life; (2) neonatal PDTC represents a new model of hypertension, lacking overt structural injury or functional impairment of the kidneys; and (3) hypertension in this model seems associated with early temporary activation of renal RAS and sodium transporters.

Regression of glomerular injury by losartan in experimental diabetic nephropathy.

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowman's capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.