Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.     

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the “turn-on” signal and understand the mechanism of early differentiation. Here, we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at ∼17,800 epitopes and ∼700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.     

Human embryonal carcinoma tumor antigen,Gp200/GCTM-2, is podocalyxin

We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as podocalyxin. Protein sequence results of gp200 peptides match with podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of podocalyxin expression on malignant cells.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency. Morphological, enzyme histochemical and immunohistochemical studies

An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were accompanied by hematuria and tubular necroses including and collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency

Summary An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were aecompanied by hematuria and tubular necroses including the collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.Dedicated to Professor Zdenek Lojda on the occasion of his 60th birthdaySupported by the German Research Foundation (Sfb 174)     

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 ? 3GlcNAc (type 1) or Galβ1 ? 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)₃-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 ? 4GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125–1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 ? 3GlcNAc(6S)β1 ? 3Galβ1 ? 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 ? 3GlcNAcβ1 ? 3Galβ1 ? 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only “type 2-type 2 keratan sulfate” but also “type 1-type 2 keratan sulfate”, significantly.     

Characterization and cell distribution of polycystin, the product of autosomal dominant polycystic kidney disease gene 1.

BACKGROUND: In a majority of cases, autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations within a putative open reading frame of the PKD1 gene. The encoded protein, polycystin, is predicted to span the plasma membrane several times and contains extracellular domains, suggestive of a role in cell adhesion. The cellular distribution and function of polycystin is not known. MATERIALS AND METHODS: We selected as immunogens two conserved 15 amino acid peptides: P1, located in a predicted extracellular region of polycystin, and P2, located in the C-terminal putative cytoplasmic tail. The anti-peptide antibodies from immunized rabbits were affinity purified on peptide-coupled resins and their specificity confirmed by their selective binding to recombinant polycystin fusion proteins. Western blotting and immunohistochemistry were used to characterize the size, tissue, and cell distribution of polycystin. RESULTS: A high-molecular mass protein (about 642 kD) was detected by Western blotting in rat brain tissue. A few additional bands, in the 100- to 400-kD range, probably representing tissue-specific variants and/or proteolytic fragments, were recognized in human and rat tissues. Polycystin was abundantly expressed in fetal kidney epithelia, where it displayed basolateral and apical membrane distribution in epithelial cells of the ureteric buds, collecting ducts, and glomeruli. In normal human adult kidney, polycystin was detected at moderate levels and in a cell surface-associated distribution in cortical collecting ducts and glomerular visceral epithelium. Expression of polycystin was significantly increased in cyst-lining epithelium in ADPKD kidneys, but was primarily intracellular. CONCLUSIONS: Polycystin appears to be a developmentally regulated and membrane-associated glycoprotein. Its intracellular localization in the cyst-lining epithelium of ADPKD kidneys suggests an abnormality in protein sorting in this disease.     

MUC1 marks collecting tubules,renal vesicles,comma- and S-shaped bodies in human developing kidney

MUC1 is a transmembrane glycoprotein, apically expressed in most epithelial cells, used in the differential diagnosis of carcinomas and for discrimination of tumors of non-epithelial origin showing epithelioid features. Little attention has been paid so far though, on its possible significance in embryonic tissues. A preliminary study from our group revealed MUC1 expression in the cap mesenchymal cells during human nephrogenesis, suggesting a role for MUC1 in the process of mesenchymal-to-epithelial transition. This study aimed at investigating the expression pattern of MUC1 in various developing structures of human fetal kidney. Expression of MUC1 was examined in kidneys of 5 human fetuses. MUC1 immunoreactivity was detected in ureteric bud tips, in collecting tubules, in cap mesenchymal cells undergoing the initial phases of mesenchymal-to-epithelial transition, in renal vesicles, comma-bodies, and S-shaped bodies. Our previous preliminary report suggested a role for MUC1 in the initial phases of the process of mesenchymal-to-epithelial transition. The present data suggest that MUC1 expression characterizes multiple structures during human nephrogenesis, from the ureteric bud, to the initial phases of mesenchymal-to-epithelial transition and that MUC1 should be added to the genes activated during the process of mesenchymal-to-epithelial transition in the cap mesenchyme of human kidney.Key words: MUC1, immunohistochemistry, fetal kdney, nephrogenesis, renal vesicles, comma and S-shaped bodies, collecting tubules.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.     

FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons

During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. Here, we have used an allelic series of mutations to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4 or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis.     

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

Aquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1?4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1?4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.     

Nephrogenesis and the renal renin-angiotensin system in fetal sheep: effects of intrauterine growth restriction during late gestation

Previous studies have shown that intrauterine growth restriction (IUGR) can impair nephrogenesis, but uncertainties remain about the importance of the gestational timing of the insult and the effects on the renal renin-angiotensin system (RAS). We therefore hypothesized that induction of IUGR during late gestation alters the RAS, and this is associated with a decrease in nephron endowment. Our aims were to determine the effects of IUGR induced during the later stages of nephrogenesis on 1) nephron number; 2) mRNA expression of angiotensin AT(1) and AT(2) receptors, angiotensinogen, and renin genes in the kidney; and 3) the size of maculae densae. IUGR was induced in fetal sheep (n = 7) by umbilical-placental embolization from 110 to 130 days of the approximately 147-day gestation; saline-infused fetuses served as controls (n = 7). Samples of cortex from the left kidney were frozen, and the right kidney was perfusion fixed. Total kidney volume, nephron number, renal corpuscle volume, total maculae densae volume, and the volume of macula densa per glomerulus were stereologically estimated. mRNA expression of AT(1) and AT(2) receptors, angiotensinogen, and renin in the renal cortex was determined. In IUGR fetuses at 130 days, body and kidney weights were significantly reduced and nephron number was reduced by 24%. There was no difference in renin, angiotensinogen, or AT(1) and AT(2) receptor mRNA expression levels in the IUGR kidneys compared with controls. We conclude that fetal growth restriction late in nephrogenesis can lead to a marked reduction in nephron endowment but does not affect renal corpuscle or macula densa size, or renal RAS gene expression.     

Embryonic stem cells markers are present within rabbit atherosclerotic plaques

Recent evidence suggests that smooth muscle cells within atherosclerotic plaques originate from vascular progenitor cells. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for factors of the renin angiotensin and nitric oxide systems as well as the hematopoietic stem-cell marker CD34 and the pan-leukocyte marker CD45. To explore the idea that these cells are of primitive types, immunohistochemistry was used to identify pluripotent embryonic stem cells (ESC) markers (Oct-4, SSEA1,3,4, TRA1-60, 81) in these plaques and to compare these to intimal thickening. Objective: To immunolocalise ESC markers in rabbit aortic intimal thickening and atherosclerotic plaques. Design: New Zealand White rabbits were fed either a control (Con) diet, 0.5% cholesterol (Chol) or 1% methionine (Meth) for 12 weeks. Animals were perfusion fixed, aortae excised and processed for paraffin. Immunohistochemistry was performed by standard techniques. Results: Oct-4, SSEA 1, 3 and 4, TRA-1-60 and TRA-1-81 were all present within in atherosclerotic plaques. However, some cells were not positive for TRA-1-60 and TRA-1-81. In fact, positive TRA-1-81 macrophages were uncommon, and positive TRA-1-81 smooth muscle cells were rare. Intimal thickening in Meth did not show any TRA-1-81 positive cells Conclusions: Macrophages and smooth muscle cells within atherosclerotic plaques express markers of ESC. These results suggest that cells within these plaques are primitive and might differentiate into other types of cells.     

Tubular changes in obstructed kidney of adult mice evaluated using immunohistochemistry for segment-specific marker

The main focus of the present investigation is to examine obstructed kidneys due to unilateral ureteral obstruction (UUO) model in adult mice using segment-specific tubular marker and to confirm the detailed morphological evaluation of UUO that is a typical model for the tubulointerstitial fibrosis which is an endpoint outcome of chronic renal diseases. Adult mice were subjected to UUO, and kidneys were harvested 1, 3, 7 days after surgical operation. Expansion of interstitial space both in the cortex and the medulla was confirmed 3 days after UUO by HE- and azan-staining. Interstitial fibrosis developed especially around dilated tubules. Immunohistochemistry for segment-specific antibodies revealed that the proximal tubules and the descending limb of Henle's loop did not dilate until 7 days after UUO, whereas initial dilation of the ascending limb of Henle's loop appeared to occur one day after surgery. The segment from the distal tubules to the collecting ducts began dilating one day after surgery and afterward significantly dilated. The downstream segment of nephron was involved in dilating earlier than the upstream of nephron in obstructed kidney examined in the present study. Moreover, the tubules accompanying apoptosis of tubular epithelia significantly dilated compared with those without apoptotic tubular epithelia. From the above-mentioned findings, we conclude that tubular dilatation of distal segment (from the ascending limb of Henle's loop to the collecting ducts) of nephron develops tubular epithelial apoptosis caused by accumulated urine, which would link to tubular disappearance and its replacement with fibrous tissue in UUO kidney of adult mice.     

Immunocytochemical localization of gamma-glutamyltranspeptidase during fetal development of mouse kidney

In the fully developed kidney, gamma-glutamyltranspeptidase is localized predominantly to the apical plasma membrane of the proximal tubules. The appearance of this activity during murine fetal nephrogenesis was quantitated using a sensitive fluorometric assay, and development of membrane polarity was assessed by immunocytochemistry. Specific activity of the transpeptidase in 13-day fetal kidney was approximately 1 mU/mg protein. Between 13-21 days of gestation, total transpeptidase activity increased 7500-fold, whereas specific activity increased 50-fold. At 13 days of gestation, gamma-glutamyltranspeptidase immunoreactivity is localized to the apical surfaces of developing renal vesicles and the proximal segment of the S-shaped tubules. The organized cell structures have tight tubular junctions but lack a well-defined brush-border membrane. By 15 days of gestation, immunostaining of the apical surface of developing proximal segments is more prominent, and slight reactivity of the basolateral membrane is evident. By 17 days of gestation, the kidney is organized into discrete zones. The large increase in gamma-glutamyltranspeptidase activity correlates with the appearance of increased immunostaining of the developing brush-border membranes of the proximal tubules contained in the inner cortex. A very similar although somewhat delayed pattern of appearance of transpeptidase activity and immunostaining was observed in metanephric organ culture. Induction of proximal tubular cyst formation had no effect on the increase in transpeptidase activity that occurred during organotypic nephrogenesis.     

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.