Effect of Hypoxia on Branching Characteristics and Cell Subpopulations during Kidney Organ Culture

Abstract

During early developmental stages, embryonic kidneys are not fully vascularized and are potentially exposed to hypoxic conditions, which is known to influence cell proliferation and survival, ureteric bud branching, and vascularization of the developing kidney. To optimize the culture conditions of in vitro cultured kidneys and gain further insight into the effect of hypoxia on kidney development, we exposed mouse embryonic kidneys isolated at E11.5, E12.5, and E13.5 to hypoxic and normal culture conditions and compared ureteric bud branching patterns, the growth of the progenitor subpopulation hoxb7+, and the expression patterns of progenitor and differentiation markers. Branching patterns were quantified using whole organ confocal imaging and gradient-vector-based analysis. In our model, hypoxia causes an earlier expression of UB tip cell markers, and a delay in stalk cell marker gene expression. The metanephric mesenchyme (MM) exhibited a later expression of differentiation marker FGF8, marking a delay in nephron formation. Hypoxia further delayed the expression of stroma cell progenitor markers, a delay in cortical differentiation markers, as well as an earlier expression of medullary and ureteral differentiation markers. We conclude that standard conditions do not apply universally and that tissue engineering strategies need to optimize suitable culture conditions for each application. We also conclude that adapting culture conditions to specific aspects of organ development in tissue engineering can help to improve individual stages of tissue generation.

Keywords: developmental biology; kidney; physiology; tissue engineering.

Figure 1

Kidney growth in normoxia and hypoxia. Embryonic kidneys exposed to hypoxic conditions during in vitro culture (E–H) exhibit different growth pattern compared to in vitro culture under normal conditions (A–D). Kidneys in hypoxic condition show increased branching from 24 h (E), but with a reduced overall and lower tip volume. Organs at 48, 72 and 120 h (F–H) present with stunted growth, reduced total volume and thinner collecting duct branches.

Figure 2

Branching generation and kidney volume. The upper panel shows the number of branching generations of embryonic kidneys harvested at E11.5, E12.5, and E13.5 after culture in normal (N) and hypoxic (H) condition. Branching generations are expressed in box plots as [y] = N−H at time points 0 h (fresh), 24 h, 48 h and 72 h. *: p < 0.05 Kidney volume (y-axis) is expressed as voxel.

Figure 3

Diameter, length, and volume of ureteric bud branches. Analysis and comparison of ureteric bud branch diameter, length, and volume of kidneys E11.5, E12.5, and E13.5 after 24 h, 48 h, and 72 h of in vitro culture in normal or hypoxic conditions.

Figure 4

Percentage of Hoxb7+ kidney cells in normoxia vs. hypoxia. Hypoxic conditions exert influence on the growth of hoxb7+ cells in embryonic kidneys in in vitro culture. During the early stages of in vitro culture, hypoxia seems to increase the hoxb7+ cell population in the kidneys (time points 24 h and 48 h). At later time points, 96 h and 120 h, hypoxia seems to have the opposite effect, and hoxb7+ cells represent a smaller portion of the overall cell composition. In addition, hypoxia seems to affect kidneys isolated at E12.5 and E13.5 stronger than kidneys isolated at E11.5. Error bars represent SE.

Figure 5

HIF1α mRNA in response to hypoxia. Quantitative analysis of hypoxia-inducible factor 1 subunit alpha (HIF1α) by real-time polymerase chain reaction (qRT-PCR) shows a gradual up-regulation of HIF1α mRNA in response to hypoxia. ∆Ct is the difference in amplification cycles (Ct) between the gene of interest and the endogenous control (β-actin) of a sample. ∆∆Ct refers to the Ct difference in an experimental sample (hypoxia) and the same gene in the control condition (normoxia). Error bars represent an estimate of the SE of a log2 fold-change.

Figure 6

Gene expression of progenitor and differentiation markers of embryonic kidneys. Heatmap showing hierarchically clustered gene expression data of progenitor and differentiation markers of embryonic kidneys in hypoxic culture, compared to normal condition. Gene expression is presented for 24 h, 48, 72 h, 96 h, and 120 h. Genes that show higher expression in hypoxic condition than in normal control are in red, green is associated with a reduction in mRNA level.

Figure 7

Chart of the expressional shift of developmental and the differentiation marker genes of the renal progenitor subpopulations in hypoxia vs. normal. Information is based on the qRT-PCR analysis above. Hypoxia causes UB tip branching markers Ret and Wnt11 to be expressed earlier, while a UB stalk marker Wntb7 is found delayed. MM differentiation marker FGF8 and Podx1 are delayed. Stroma progenitor cell marker Raldh2 is expressed later and the expression of stroma cell differentiation marker Snai2 is delayed, while stroma medullary marker Bmp4 and urethra marker Tbx18 are expressed earlier. Black arrow indicates an earlier gene expression in hypoxia compared to normoxia. White arrow indicates a later gene expression in hypoxia compared to normoxia.