Naphthalenephenylalanine-phenylalanine-glycine-arginine-glycine-aspartic promotes self-assembly of nephron progenitor cells in decellularized scaffolds to construct bioengineered kidneys.

The shortage of donor kidneys is an important factor restricting kidney transplantation for patients with end-stage renal disease. To overcome this problem, we used decellularized kidney scaffolds and nephron progenitor cells (NPCs) as seed cells to construct bioengineered kidneys (BEKs). To reduce the effect of extracellular matrix (ECM) loss during the decellularization process on the cell growth microenvironment, we used dextrose to minimize collagen loss in decellularized kidney scaffolds. At the same time, to further improve the growth microenvironment of seed cells in the decellularized scaffolds, we modified the decellularized scaffolds with the self-assembling polypeptide Naphthalenephenylalanine-phenylalanine-glycine-arginine-glycine-aspartic (Nap-FFGRGD) to promote the adhesion and proliferation of seed cells in the scaffolds. NPCs were perfused into the decellularized kidney scaffolds and then the BEKs were cultured in vitro and transplanted in vivo. Markers of podocytes and renal tubules expressed in the glomeruli and renal tubules of the BEKs were detected by immunofluorescence staining, respectively were, suggesting that NPCs can continue to differentiate into renal cells and achieve nephron segment-specific re-population through self-assembly. These results indicate that by relying on the microenvironment provided by Nap-FFGRGD modified decellularized scaffolds, NPCs can be used to construct BEKs for transplantation in the future due to the self-assembly properties of organoids.

Ganab Haploinsufficiency Does Not Cause Polycystic Kidney Disease or Polycystic Liver Disease in Mice.

BACKGROUND: Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) have been described previously, but their roles in ADPKD and PLD are largely unknown. With the increase in polycystic kidney disease caused by GANAB gene mutations in recent years, a suitable animal model is still needed to further explore the pathogenic role of this gene. METHODS: To construct a mouse model of Ganab gene deletion, we analyzed the Ganab gene structure and designed two CRISPR-/Cas9-based targeting strategies. The Cas9/sgRNA we constructed was microinjected into fertilized mouse eggs to obtain chimeric F0 mice. Mice with stable genotypes were selected from offspring born after mating F0 mice with wild-type mice. RESULTS: We found that homozygous mutation of the Ganab gene in C57BL/6 mice resulted in early embryonic lethality, and there were no cysts in the kidneys or livers of Ganab +/- mice. Additionally, Ganab protein expression was reduced by at least 50%, while the expression of ADPKD proteins (PC1 and PC2) and acetylated tubulin was not affected in the Ganab +/- kidney. However, the Ganab +/- mice did not show any abnormal clinical phenotypes after birth and failed to reveal renal tubule dilatation or any abnormalities of the glomeruli in the Ganab +/- kidney. CONCLUSIONS: Homozygous Ganab mutations are lethal in the fetal stage, and Ganab haploinsufficiency does not cause kidney or liver cysts in mice, suggesting that it may not be the causative gene in polycystic kidney disease.

Disruption of Robo2-Baiap2 integrated signaling drives cystic disease.

Hereditary renal cystic diseases are characterized by defects in primary cilia of renal tubular epithelial cells and abnormality of tubular epithelium, which ultimately result in the development of renal cysts. However, the mechanism leading from abnormality of the tubular epithelium to cystogenesis is not well understood. In this report, we demonstrate a critical role for Robo2 in regulating epithelial development, including ciliogenesis, polarization, and differentiation. We found that Robo2 deficiency results in cystic kidneys, and the cyst cells showed defective cilia and polarity defects in tubular epithelium. The cyst cells, less than terminally differentiated, continue to proliferate. We further established that Robo2 works with p53 as well as polarity and ciliary proteins (Par3, PKCς, ZO-2, and Claudin-2) to regulate these processes. Robo2 binds to Baiap2 (also known as IRSp53) through the IRSp53/MIM homology domain in renal epithelial cells. This binding allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2 and maintain p53 homeostasis. Disruption of the Robo2-Baiap2 complex causes MDM2 to be subjected to dephosphorylation, leading to a high level of active p53, and initiated p53-mediated cellular senescence via p21 and decreased the expression of ZO-1, ZO-2, PKCς, Par3, and Claudin-2 proteins, resulting in defects in epithelial development, including ciliogenesis, polarization, and differentiation. Importantly, double knockout of Robo2 and p53 rescued all the epithelial defects in kidneys compared with those in Robo2-knockout kidneys. Taken together, the present results demonstrate that Robo2 deficiency causes renal cystic disease, which is largely dependent on defective Robo2-Baiap2 integrated signaling in kidneys.

Identifying gene mutations of Chinese patients with polycystic kidney disease through targeted next-generation sequencing technology.

BACKGROUND: Polycystic kidney disease (PKD) is the most common hereditary kidney disease. The main mutational genes causing autosomal dominant polycystic kidney disease (ADPKD) are PKD1 and PKD2 as well as some rare pathogenic genes. Unilateral PKD is rare in clinics, and its association with gene mutations is unclear. METHODS: Targeted next-generation sequencing (NGS) was performed to detect the renal ciliopathy-associated genes (targeted NGS panel including 63 genes) in PKD patients. RESULTS: Forty-eight PKD1 and PKD2 mutation sites were detected in 44 bilateral PKD patients, of which 48 were PKD1 mutation sites (87.5%) and six were PKD2 mutation sites (12.5%). All of which exhibited typical ADPKD. Furthermore, we detected HNF1B heterozygous mutations in three families. Although these three patients showed HNF1B heterozygous mutations, their clinical characteristics differed and showed phenotypic heterogeneity. CONCLUSIONS: Targeted NGS panel was helpful in detecting typical ADPKD patients and even in non-typical PKD patients. Macromutation in HNF1B may lead to bilateral PKD. The 16 novel PKD gene mutation sites and two novel PKD2 gene mutation sites discovered in this study have some significance in genetic counseling for ADPKD patients, and increase the number of studied families and expand the mutation database of ADPKD.

Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers.