Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing.

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Protocol for multimodal analysis of human kidney tissue by imaging mass spectrometry and CODEX multiplexed immunofluorescence.

Here, we describe the preservation and preparation of human kidney tissue for interrogation by histopathology, imaging mass spectrometry, and multiplexed immunofluorescence. Custom image registration and integration techniques are used to create cellular and molecular atlases of this organ system. Through careful optimization, we ensure high-quality and reproducible datasets suitable for cross-patient comparisons that are essential to understanding human health and disease. Moreover, each of these steps can be adapted to other organ systems or diseases, enabling additional atlas efforts.

Tubular ß-catenin and FoxO3 interactions protect in chronic kidney disease.

The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.

Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA.

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/-) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.

ß-catenin activation in a novel liver progenitor cell type is sufficient to cause hepatocellular carcinoma and hepatoblastoma.

Hepatocellular carcinoma (HCC) was thought historically to arise from hepatocytes, but gene expression studies have suggested that it can also arise from fetal progenitor cells or their adult progenitor progeny. Here, we report the identification of a unique population of fetal liver progenitor cells in mice that can serve as a cell of origin in HCC development. In the transgenic model used, mice carry the Cited1-CreER(TM)-GFP BAC transgene in which a tamoxifen-inducible Cre (CreER(TM)) and GFP are controlled by a 190-kb 5' genomic region of Cited1, a transcriptional coactivator protein for CBP/p300. Wnt signaling is critical for regulating self-renewal of progenitor/stem cells and has been implicated in the etiology of cancers of rapidly self-renewing tissues, so we hypothesized that Wnt pathway activation in CreER(TM)-GFP(+) progenitors would result in HCC. In livers from the mouse model, transgene-expressing cells represented 4% of liver cells at E11.5 when other markers were expressed, characteristic of the hepatic stem/progenitor cells that give rise to adult hepatocytes, cholangiocytes, and SOX9(+) periductal cells. By 26 weeks of age, more than 90% of Cited1-CreER(TM)-GFP;Ctnnb1(ex3(fl)) mice with Wnt pathway activation developed HCC and, in some cases, hepatoblastomas and lung metastases. HCC and hepatoblastomas resembled their human counterparts histologically, showing activation of Wnt, Ras/Raf/MAPK, and PI3K/AKT/mTOR pathways and expressing relevant stem/progenitor cell markers. Our results show that Wnt pathway activation is sufficient for malignant transformation of these unique liver progenitor cells, offering functional support for a fetal/adult progenitor origin of some human HCC. We believe this model may offer a valuable new tool to improve understanding of the cellular etiology and biology of HCC and hepatoblastomas and the development of improved therapeutics for these diseases.

Assessing the application of tissue microarray technology to kidney research.

Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and inner medulla fixed with different fixatives; (2) rat kidneys at different stages of development fixed with different fixatives; (3) mouse and rat kidneys with different physiological or pathophysiological treatments; and (4) built-in controls. As examples of the utility, immunostaining for cyclooxygenase-2, renin, Tamm Horsfall protein, aquaporin-2, connective tissue growth factor, and synaptopodin was carried out with kidney TMA slides. Quantitative analysis of cyclooxygense-2 expression in kidneys confirms that individual cores provide meaningful representations comparable to whole-kidney sections. These studies show that kidney TMA technique is a promising and useful tool for investigating the expression profiles of proteins of interest in rodent kidneys under different physiological and pathophysiological conditions.

Differential expression of the intermediate filament protein nestin during renal development and its localization in adult podocytes.

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.