PART Ⅰ: Effects Of Environmental Conditions On Nephron Number
Mar 20, 2022
for more information:ali.ma@wecistanche.com
PART Ⅰ: Effects of Environmental Conditions on Nephron Number: Modeling Maternal Disease and Epigenetic Regulation in Renal Development
Lars Fuhrmann, Saskia Lindner, Alexander-Thomas Hauser, Clemens Höse & et al.
1. Introduction
Fetal development is affected by the in utero environment, and an adverse milieu can predispose to diseases such as hypertension, cardiovascular disease, and chronic kidney disease later in life [1-4]. A range of intrauterine disturbances can result in a reduction in nephron endowment and compromised renal function in the offspring. In rodents, conditions leading to reduced nephron numbers at birth include intrauterine growth restriction (IUGR), maternal low protein diet, medications (including corticosteroids or nonsteroidal anti-inflammatory drugs), monogenetic mutations, and low vitamin A levels, as well as maternal diabetes and iron deficiency [5-10]. In humans, there is currently no non-invasive method of measuring nephron numbers. However, postmortal studies have demonstrated a negative correlation between nephron numbers and blood pressure [4,11]. Additionally, intrauterine conditions associated with reduced nephron numbers such as IUGR are known to be are associated with increased rates of hypertension and CKD[12].
In vivo experiments where adverse intrauterine conditions are artificially induced in pregnant animals have proven invaluable for the detection and description of the renal alterations induced in the offspring. However, any experimental intervention during gestation results in complex alterations in the maternal, placental and fetal physiology which may themselves affect the environment of the developing metanephroi. Ex vivo modeling techniques circumvent this by enabling the investigation of kidney development completely separated from the influence of the mother animal, the placenta, or other organs of the fetus. Isolated cultures of metanephroi on a medium-air interface were initially performed by Trowell in 1950 [13] and have subsequently been refined by culturing of kidneys on filter membranes[14], providing a basis for single-variable culture conditions.
In summary, there is an increasing body of evidence suggesting that prenatal insults associated with low nephron numbers are relevant to risk factors for hypertension and CKD. In order to develop preventative strategies to ensure adequate nephron endowment at birth, a mechanistic understanding of the factors influencing renal development and nephrogenesis is necessary. In the present work, metanephric organ culture was used to model different aspects of environmental regulation to study their effects on renal growth and possible implications for long-term renal function and used to screen for epigenetic regulators using FDA-approved small compounds.
Click to Cistanche herba products for kidney function
2. Results
2.1. Use of Metanephric Organ Cultures to Study the Effect of Environmental Conditions on Renal Development
To model adverse environmental conditions, kidneys from embryonic day (E)12.5 embryos were cultured at the medium-air interface (Figure 1A). To facilitate monitoring of nephron and glomerular development, Six2. Cre and Pod. Cre dual-fluorescent reporter mice were used, respectively(Figure 1B, C). A common condition during pregnancy is fever, which affects more than 10% of pregnancies during the first 16 weeks of gestation [15]Heat is a well-characterized teratogen, and hyperthermia during pregnancy has been shown to lead to fetal abortion, growth retardation, and developmental defects, such as renal agenesis, hypoplasia and low birth weight in several species [16-21]. To assess the impact of prolonged, fever-range hyperthermic conditions on kidney growth and nephron formation, kidneys were isolated and maintained at either 37 or 40°C(Figure 1D). After 7 days of culture, kidneys cultured at 40C were, on average, 18.36%smaller than their counterparts cultured under physiological conditions (Figure 1E). However, no significant difference in the number of glomeruli per kidney was found between the groups(Figure 1F). Nevertheless, the decreased overall growth of the kidneys demonstrates a negative effect of increased temperature on metanephric growth.
With an estimated 19% of pregnant women suffering from iron-deficiency anemia [22], iron deficiency is one of the most widespread conditions with the potential to disturb renal development [23]. In vivo data have shown that renal growth, glomerular numbers, and renal iron uptake are reduced during pregnancies affected by maternal iron deficiency [24]. In order to assess the impact of reduced transferrin-bound iron supply on kidney growth and nephrogenesis, explants were cultured in a medium containing 50 ug/mL of iron-saturated holo-transferrin or 50 ug/mL of iron-depleted apo-transferrin, respectively. Iron-restricted kidneys remained much smaller than their iron-sufficient counterparts and showed increased apoptosis in the ureteric buds and reduced ureteric bud branching and proliferation (Figure 1G, Supplementary Figure S1A-F). While the nephron population was morphologically unaffected, a decrease in the developing distal part of the nephron, as well as distal tubules, could be seen(Figure 1H,I, Supplementary Figure S1G-J). The iron-deficient kidneys were, on average, 47.9% of the size of their holo-transferrin cultured counterparts (Figure 1) and showed a reduction in the overall number of glomeruli per kidney of 69.9% after 7 days in culture (Figure 1K). Thus, iron depletion by apo-transferrin showed severe effects on kidney growth with an all-proximal nephrogenesis phenotype.
Figure 1. Use of metanephric organ cultures to study the effect of environmental conditions on renal development. (A) The urogenital ridge from E12.5 mouse embryos(left panel) was microsurgically extracted (second panel), and the kidneys were isolated（third panel） and placed on Transwell inserts （right panel）. Scale bars∶ 1 mm （left panel）， 500 μm（third panel）. (B)Transgenic mice with dual Tomato/EGFP expression were used for conditional labeling of Six2-positive cells and their offspring using Six2. Cre or(C) podocin-positive cells using Pod. Cre mice. (D)Explants from the same embryo were cultured for 7 days at 37 or 40°C. Scale bars∶ 500 μm. （E） Surface areas of explants grown for7 days at 37 or 40°C.n=40 pairs， paired t-test, mean±SD.(F) The number of glomeruli in the explant groups after7 days.n=21 pairs, paired t-test, mean ±SD. （G） Explants from the same embryo were cultured for 7 days in a medium containing holo-Tf or apo-Tf. Scale bars∶ 500 μm. (H)Widefield images of holo-Tf and apo-Tf cultured explant pair stained against SIX2 and E-cadherin after 48 h of culture show normal progenitor cell pool and defects in early nephron morphology. Scale bar∶100 μm.（I） Widefield images of holo-Tf and apo-Tfcultured explant pair stained against WT1 and JAG1. Scale bars∶100 μm. （） Surface areas of holo-Tf and apo-Tf cultured explants after 7 days.n=30 pairs, paired t-test, mean ± SD.(K) Number of glomeruli in the explant groups after 7 days.n =17 pairs, paired t-test, mean ± SD.
2.2. Ex Vio High Glucose Exposure Leads to Diabetic Nephropathy-Associated Changes in the Developing Kidney
Maternal diabetes is another common condition during pregnancy, with a global prevalence of hyperglycemia in pregnancy of~17% and over 20 million live births each year [25]. Diabetes induced in mouse and rat models has been shown to lead to offspring with a lower nephron number[10,26,27]. Metanephric organ culture has been used before to study the effect of high glucose on renal development [27-29]. Previously, we reported a reduction in size, nephron number, and DNA methylation under high glucose conditions of 55 mM[30]. In contrast to published data [28], no effect on explant size or glomerular number could be seen in our samples when cultured in different 30 mM glucose media compared to 5 mM control conditions after 7 days (Supplementary Figure S2A, B). Furthermore, no decrease in DNA methylation at LINE-1 and major satellite sites could be detected(Supplementary Figure S2C, D). The effect of 55 mM high glucose on renal development after a 7-day period culture was further analyzed, showing a decrease in the growth rate starting at day 3 in culture (Figure 2A, B). Immunofluorescence stainings showed no morphological defects of the SIX2-positive progenitor cell pool (Figure 2C)but showed reduced staining of podocyte marker podocalyxin (PODXL, Figure 2D). The glomeruli were found to contain a thickened glomerular basement matrix visible in histological stainings (Figure 2E). Similar findings were made in electron microscopy, showing an increase in glomerular basement membrane thickness(Figure 2F), one of the earliest markers of pre-diabetes and diabetic nephropathy (DN)[31,32], in five out of six kidneys and none of seven littermate control kidneys. To further unravel changes in the transcriptome, pair-wise differential gene expression (DGE) analysis of kidney cultures from three litters was performed, with one kidney from each embryo cultured with high glucose and the other with control medium and the kidneys pooled for analysis (n=3).DGE confirmed the upregulation of extracellular matrix components as the primary upregulated biological process (Supplementary Table S1). Downregulated genes were mainly involved in (immune)cell activation and exocytosis/secretion (Supplementary Table S1). Mammalian phenotype ontology indicated abnormal kidney cortex and renal corpuscle morphology due to downregulated genes such as Pdgfb, Podxl, Ren, Ptpro, Mafb, and Vegfa(Supplementary Table S1). Renal expression of several genes, such as Angptl4, Spon2 (Mindin), Pappa, and Txnip, which have been shown to be upregulated in diabetic nephropathy [33-36], was found to be increased under hyperglycemic conditions. To compare the high glucose kidney culture gene expression profile to human DN, the DGE data were matched to human data from microdissected glomeruli and tubules from diabetic nephropathy patient biopsies from the European Renal cDNA Bank(ERCB). From the 216 differentially regulated genes matched after batch analysis, 94 genes were correspondingly differentially regulated in the glomerular and/or tubular fractions (Figure 2G). The overlap of our model and human DNgenes showed 40 out of 95 genes upregulated in the glomeruli and 34 genes in tubules (25 genes in common)(Figure 2H). The genes were mostly involved in an extracellular matrix organization (COL4A5, COL4A6, COL8A2, LAMB3, LAMC3) and cell adhesion (ITGBL1, CLDN15). Additionally, diabetes-associated genes such as TXNIP, SPON2, and PAPPA were upregulated. Out of 120 downregulated genes from the kidney cultures,22 were also downregulated in the glomeruli and 39 were also downregulated in the tubules (16 in com-mon (Figure 2I). These genes were involved in response to endogenous stimulus (BMP2, KLF15, JUNB, CTSB), nephron epithelium development (PTPRO, PODXL, VEGFA), and positive regulation of endothelial cell chemotaxis (LGMN, P2RX4, VEGFA). Additionally, diabetes-associated genes such as RASGRP3, SIRPA, GATM, and ESM1 were downregulated [37-39]. Differentially regulated genes not overlapping with ERCB data also reflected diabetes-associated changes, such as extracellular matrix(ANGPTL4, TNN, DPT, COL9A2)or gestational diabetes(LAT2, HP, CXCL10, CD86, CD68, REN, SLC2A3, VCAM1). Thus, renal development under high glucose conditions displayed remarkable similarities to human adult diabetic nephropathy.
Figure 2. Ex vivo high glucose exposure leads to diabetic nephropathy-associated changes in the developing kidney. (A) Embryonic kidneys from Pod.Cre; Tomato/EGFP animals cultured for 7 days in low glucose (LG,5.5 mMα-D-glucose, 55 mM mannitol) or high glucose (HG, 55 mM α-D-glucose)conditions. Scale bar: 500 um.(B)Kidney surface area of HG and LG conditions.*,p=0.0474;*,p=0.0052;*,p<0.0001.Paired t-test, mean ± SD.(C) Confocal immunofluorescent stainings of day 7 kidney cultures against Six2 and (D) podocalyxin with pan-cytokeratin and Hoechst.Scale bar: 100 um. (E) Stainings of 6 um sections from day 7 kidney cultures. Scale bar: 20 um. (F)Transmission electron microscopy of sections from day 7 kidney cultures. Glomerular basement membranes are thickened in kidneys exposed to high glucose conditions. Left column: magnification showing podocyte foot processes. Scale bars: 500 nm (left panels),100 nm (right panels).(G) Fold change of RNA-seq data from HG compared to LG kidneys and ERCB diabetic nephropathy (DN)patient microarray data from microdissected glomeruli and tubules showing differentially expressed genes. (H) Genes upregulated in the kidney cultures(KC)overlapping with ERCB DN patient data and selected genes highlighted. (I)Genes downregulated in the kidney cultures (KC) overlapping with ERCB DN patient data and selected genes highlighted.
2.3. Ex Vivo High Glucose Exposure Influences to Long-Term Memory Formation via DNA Methylation
To further understand the molecular changes mediated by a hyperglycemic environment, kidney cultures were grown at high glucose conditions for 3.5 days and then changed to low glucose conditions for the same amount of time (Figure 3A). Remarkably, incubation under physiological conditions after the shorter incubation period in high glucose medium did not reverse growth reduction after 7 days in culture with the cultures growing at the same rate as under continuous high glucose treatment (Figure 3B). Furthermore, DNA methylation showed hypomethylation of LINE-1 element and major satellite loci (Figure 2C, D), as well as sustained DNA hypermethylation of the Ppargcla promoter, under both high glucose and reversed conditions (Figure 3E), indicating the formation of metabolic memory via DNA methylation due to the earlier adverse environmental conditions as a means of fetal programming.
Figure 3. Ex vivo high glucose exposure influences long-term memory formation via DNA methylation. (A)Imaging of E12.5embryonic kidneys from day 0 to day 7 in low glucose medium (5.5 mM), high glucose medium (55 mM), or high glucose medium for3.5 days and reversal to low glucose medium for the remaining days.Scale bar:500 um. (B) Kidney surface area over 7 days. Mean ± SD. n=36 kidneys. LG, low glucose treatment; HG, high glucose treatment; HG to LG,3.5 days high and 3.5 days low glucose treatment.***,LG-HG (unpaired t-test):p=0.0004;**,LG-HG to LG (paired t-test):p<0.0001.(C) Analysis of the DNA methylation at LINE-1 and(D)major satellite loci shows continuous DNA hypomethylation in high glucose treated conditions.**,p-value=0.0022.*,p-value=0.0357.(E) Analysis of the DNA methylation at the Ppargcla locus shows continuous DNA hypermethylation in high glucose conditions. Mean ± SD. *,p-value = 0.0130.
2.4.Ex Vivo Small Compound Screen identifies Epigenetic Regulators of Renal Development
The results of this work as well as previous works suggest that epigenetic mechanisms play a role in kidney development [30, A40-45]. Therefore, we wanted to systematically evaluate the effect of epigenetic modulators of the different enzyme classes on renal development. For this, we selected a library of 22 FDA-approved small compounds with demonstrated inhibitory activity [46-56](Figure 4A). Using Six2.Cre-reporter mice to evaluate nephron development, the renal structures were cultured for 3 days with the inhibitors in the medium. Size increase over time as compared to the littermate control organs, and the morphology was checked for abnormalities in development (Figure 4B). Several inhibitors could be shown to interfere with normal ex vivo renal development. HDAC inhibitor entinostat, a benzamide histone deacetylase inhibitor with a high affinity for HDAC1,2 and 3 [57], showed consistent growth reduction and lack of differentiation and proliferation after 3 days (Figure 4C).TH39 developed as a selective HDAC8 inhibitor (IC50 HDAC8 88 nM, 26-fold selective against HDAC1,28-fold selective against HDAC6[56]), showed a similarly severe inhibition of growth compared to littermate control organs. Furthermore, iron chelators and inhibitors of JmJC deferasirox and deferoxamine showed growth reduction analogous to iron-deficient medium. Additionally, SET7/9 inhibitor cyproheptadine [58,59] showed growth reduction and lack of differentiation and proliferation compared to control kidneys (Figure 4D)but also seemed to interfere with Wnt signaling (Supplementary Figure S3). Other HDAC, HAT, HDM, and HMT inhibitors, and DNMT inhibitor 5-azacytidine did not show growth reduction or developmental anomalies within the measured time frame (Figure 4A).
Figure 4. Ex vivo small compound screen identifies modulators of renal development. (A)List of small compounds, their epigenetic targets, and concentration used shows renal growth reduction with entinostat, TH39, deferasirox, deferoxamine, and cyproheptadine in ≥3 independent experiments after 3 days in culture. Control cultures were treated with DMSO. ***, p-value<0.0001. (B)Examples of two sets of embryonic kidney cultures with pictures taken from day 0 until day 3 showing growth reduction and morphological differences in kidneys treated with TH39, deferasirox, and deferoxamine compared to littermate control kidneys. Scale bar∶ 500 μm. (C）Entinostat showed growth reduction at 5 and 10 μM concentrations, no nephron differentiation, and lack of proliferation after 3 days (D)Cvproheptadine showed growth reduction at 100 and 50 μM concentrations， lack differentiation， ureter dilation, and lack of proliferation after 3 days in culture. Scale bar: 500 um. PanCK, pan-cytokeratin. CC3, cleaved caspase-3. PCNA, proliferating cell nuclear antigen.
