Decellularized Human Kidney Cortex Hydrogels Enhance Kidney Microvascular Endothelial Cell Maturation and Quiescence

Abstract

The kidney peritubular microvasculature is highly susceptible to injury from drugs and toxins, often resulting in acute kidney injury and progressive chronic kidney disease. Little is known about the process of injury and regeneration of human kidney microvasculature, resulting from the lack of appropriate kidney microvascular models that can incorporate the proper cells, extracellular matrices (ECMs), and architectures needed to understand the response and contribution of individual vascular components in these processes. In this study, we present methods to recreate the human kidney ECM (kECM) microenvironment by fabricating kECM hydrogels derived from decellularized human kidney cortex. The majority of native matrix proteins, such as collagen-IV, laminin, and heparan sulfate proteoglycan, and their isoforms were preserved in similar proportions as found in normal kidneys. Human kidney peritubular microvascular endothelial cells (HKMECs) became more quiescent when cultured on this kECM gel compared with culture on collagen-I-assessed using phenotypic, genotypic, and functional assays; whereas human umbilical vein endothelial cells became stimulated on kECM gels. We demonstrate for the first time that human kidney cortex can form a hydrogel suitable for use in flow-directed microphysiological systems. Our findings strongly suggest that selecting the proper ECM is a critical consideration in the development of vascularized organs on a chip and carries important implications for tissue engineering of all vascularized organs.

FIG. 1.

Decellularization and matrix characterization of human kidney cortex. (A) Overview of the decellularization process beginning with whole tissue (A.1), sectioning (A.2), and chemical processing at 1 h (A.3), 24 h (A.4), and 120 h (A.5), then rinsing with water for 24 h (A.6) and 120 h (A.7). (B, C) Histological evaluation of untreated and decellularized kidney sections. (B) Hematoxylin and eosin staining reveals the removal of nuclei, but preservation of structural integrity of the ECM following decellularization, including glomerular structures (asterisks). Scale bar, 100 μm. (C) Matrix protein expression of FN, HSPG, LAM, Col-IV, and VN. Scale bar, 50 μm. Col-IV, collagen-IV; ECM, extracellular matrix; FN, fibronectin; HSPG, heparan sulfate proteoglycan; LAM, laminin; VN, versican. Color images available online at www.liebertpub.com/tea

FIG. 2.

Quantitative assessment of decellularization by LC-MS/MS. (A) Relative quantification of ECM proteins in decellularized kidney tissue with LC-MS/MS. (B) Relative percentage of subdomains for collagen-IV (B.1), laminin (B.2), and collagen-I (B.3). LC-MS/MS, liquid chromatography–tandem mass spectrometry. Color images available online at www.liebertpub.com/tea

FIG. 3.

Fabrication and characterization of kECM gels. (A) Overview of the gelation process from the decellularized product (A.1), lyophilization (A.2), and digestion and homogenization over ice (A.3) to a final kECM gel at 15 mg/mL (A.4). (B) Hydrogel formation and immunofluorescence images of ECM proteins of 7.5 mg/mL kECM (B.1) and kECM/collagen-I mixture (B.2) gels. Scale bar, 100 μm. (C) Ultrastructural characterization of 7.5 mg/mL collagen (C.1), kECM (C.2), and mixture gel (C.3) using scanning electron microscopy. Scale bar, 10 μm. (D) Rheology testing of different gel compositions showing the complex modulus as a function of strain. kECM, kidney ECM. Color images available online at www.liebertpub.com/tea

FIG. 4.

Morphological, functional, and molecular differences of HKMECs on different ECM gels. (A) Immunofluorescence images of HKMECs cultured on 7.5 mg/mL collagen-I (A.1, A.4), 7.5 mg/mL kECM (A.2, A.5), and 7.5 mg/mL mixture gel (A.3, A.6). (A.1–A.3): red, CD31; green, VWF; blue, nuclei. Scale bar, 50 μm. (A.4–A.6): red, F-actin; green, PV1; blue, nuclei. Scale bar, 50 μm. (B) Real-time q-PCR for Human kidney peritubular microvascular endothelial cells (HKMECs) cultured on three different hydrogel conditions showing selective genes implicated in proliferation (PCNA), quiescence (VWF, DLL4, NOS3, VCAM1), proteolytic activity (MMP10), and vessel integrity and permeability (VEGFR2, JAM2, OCLN, PECAM, PLVALP, ANGPT2). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ******p < 0.000001. HKMECs, Human kidney peritubular microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; q-PCR, quantitative polymerase chain reaction; VWF, von Willebrand factor. Color images available online at www.liebertpub.com/tea

FIG. 5.

Morphological, functional, and molecular differences of HUVECs on different gels. (A) Immunofluorescence image of HUVECs cultured on 7.5 mg/mL collagen-I (A.1), 7.5 mg/mL kECM (A.2), and 7.5 mg/mL mixture gel (A.3). Red, CD31; green, VWF; blue, nuclei. Scale bar, 50 μm. (B) Cell density comparing HUVEC and HKMEC was quantified at different hydrogel culture conditions of collagen-I, kECM, and 1:1 mixture at mass fraction of 7.5 mg/mL. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ******p < 0.000001. (C) Real time q-PCR for HKMECs cultured on three different hydrogel conditions showing selective genes. (D) Angiogenesis assay for HUVECs cultured on ECM gels comprising 4 mg/mL collagen-I (D.1) and 7.5 mg/mL kECM mixture (D.2) gels. (D.1–2) Confocal image of HUVEC monolayer on the surface of hydrogels in the xy plane (upper panel) and yz cross sections at dash lines (bottom panel) after 72 h of culture. Scale bar, 50 μm. (D.3) Quantification of the number of sprouts per area for different culture conditions showing significant increment of sprouts on mixture gels, p < 0.05. Error bars: ±2 SEM. SEM, standard error of the mean. Color images available online at www.liebertpub.com/tea

FIG. 6.

kECM mixture gels support the formation of engineered microvessels. (A) Microvessel fabrication. Schematic of fabricated microvessel network and housing (A.1). Depiction of kidney microvessels embedded within any ECM gel (A.2). (B–C) z-stack projection of confocal image of engineered human microvessels from HKMECs (B) and HUVECs (C) in kECM mixture gel in xy plane (left panel) and cross sectional yz plane at the dash line (right and bottom panels). Scale bar, 100 (B.1 and C.1) and 50 μm (B.2 and C.2). Color images available online at www.liebertpub.com/tea