Normal Adult Kidney Research Articles

Successful transplantation to two recipients after splitting a large horseshoe kidney with complicated anatomy

A 44-year-old male (blood group B) with no history of kidney or urinary tract disorders (creatinine 0.5 mg/dl) was diagnosed as being brain dead after spontaneous subarachnoidal bleeding. A horseshoe-shaped kidney was diagnosed intraoperatively, and after harvesting the liver and the pancreas nephrectomy of both kidneys together with the aorta and vena cava was performed by an external transplant team following a standard technique. Before the organs were offered to our centre, they had not been accepted for transplantation by two major transplant centres because of suspected complex anatomy, large mass (estimated to be twice as large per side when compared with a normal adult kidney) and an enlarged isthmus. After a long allocation process and time-consuming external cross match, inspection revealed a normal pancreas and the left kidney to have normal vascular anatomy, whereas the right kidney had one main with two accessory arteries and two separate veins. The isthmus contained normal kidney parenchyma. Injection of contrast medium into both ureters showed that each kidney had separate urinary-collecting systems. The kidney was divided sharply followed by suture ligation of all vascular and tubular structures. The cut surfaces were then oversewn using interrupted sutures. The left side of the kidney together with the pancreasduodenal graft [cold ischemia time (CIT) 19 h] from the same donor was transplanted after 21 h CIT using a transperitoneal standard technique, namely to a 54-yearold woman with type I diabetes and renal failure requiring hemodialysis. Initial function of both organs was normal with good diuresis and normal blood glucose levels. After one shot of antithymocyte globulin (7 mg/kg), immunosuppression was based on tacrolimus, mycophenolat mofetil and steroids.

Protective role of l-2-oxothiazolidine-4-carboxylic acid in cisplatin-induced renal injury

Oxidative stress and inflammation are implicated in the pathogenesis of cisplatin-induced nephrotoxicity. l-2-oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug, and increases cellular glutathione (GSH). OTC is converted to cysteine by the intracellular enzyme, oxoprolinase. To date, the protective role of OTC on cisplatin-induced renal injury has not been investigated. The purpose of the present study was to examine the protective effect of OTC on cisplatin-induced renal injury and to examine the mechanism of its protection. Mice were treated with cisplatin with or without administration of OTC. The generation of reactive oxygen species (ROS), expression of intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1 were determined in the kidney using 2',7'-dichlorofluorescein diacetate, immunostaining or western blot analysis. Nuclear factor (NF)-kappaB activity, infiltration of F4/80-positive cells and apoptosis were also investigated in addition to renal function and histology using electrophoretic mobility shift assay, immunostaining, western blot analysis, uridine triphosphate (dUTP) nick-end labelling or periodic acid-Schiff staining. The effect of OTC on superoxide dismutase activity and GSH level in cisplatin-treated normal adult human kidney (HK-2) cells were measured using assay kits. The administration of OTC resulted in a significant reduction of cisplatin-induced ROS production, the p65 subunit of NF-kappaB translocation into nucleus, expression of ICAM-1, caspase 3 activity, expression of MCP-1 and the infiltration of macrophages into renal tissue. OTC markedly ameliorated renal damage induced by cisplatin through antioxidant and anti-inflammatory effect. These results suggest that OTC can be a potential therapeutic agent in cisplatin-induced renal injury through decreasing the ROS levels and activation of NF-kappaB.

Decreased expression of the human stem cell marker, Rex-1 (zfp-42) , in renal cell carcinoma

The Rex-1 (Zfp-42) gene encodes a zinc finger family transcription factor which is highly expressed in mouse and human embryonic stem cells. It is one of several gene markers used to identify human stem cells. While several organs are known to harbor adult human stem cells, the presence and distribution of stem cells in both the normal and neoplastic adult kidney remains largely unknown. In this study we evaluated Rex-1 mRNA and protein expression in normal and malignant kidney tissue specimens from human patients. Rex-1 mRNA expression was determined using both reverse transcription and real-time PCR. REX1 protein expression was assessed by western analysis and immunohistochemistry, using an affinity-purified, polyclonal antibody to the REX1 protein. We found that 14 of 15 (93%) non-tumor renal parenchymal specimens demonstrated Rex-1 mRNA, compared with 5 of 14 (36%) renal tumors (P < 0.005). REX1 protein expression was detected in 21 of 23 (91%) non-tumor and in 7 of 19 (37%) tumor specimens (P < 0.001). Furthermore, in six of these seven renal tumor specimens where REX1 protein expression was detected, the levels were at least 3-fold lower than those in adjacent, normal kidney tissue. There were no differences in Rex-1 mRNA or protein expression among the various histologic subtypes of renal tumors (clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma). Immunohistochemical staining confirmed the absence of REX1 in three renal tumor specimens (two clear cell and one papillary carcinoma), while the REX1 protein was detected in a small percentage of proximal tubular cells in normal renal tissue. Immunohistochemical staining of another stem cell marker, OCT4, demonstrated a similar pattern of protein expression in a small percentage of normal renal proximal tubular cells. In summary, we were able to detect Rex-1 mRNA and protein expression in over 90% of normal renal parenchymal specimens and we observed a significant reduction in REX1 expression in renal tumor specimens of all histologic subtypes.

Adult Kidney Tubular Cell Population Showing Phenotypic Plasticity, Tubulogenic Capacity, and Integration Capability into Developing Kidney

Using in vivo bromodeoxyuridine (BrdU) labeling, a tubular cell population (label-retaining tubular cells [LRTC]) was identified recently in normal adult kidneys, which contributes actively to the regeneration process of the kidney after injury. Here, these LRTC are characterized in vitro. The LRTC population was isolated from BrdU-treated rat kidney by FACS. Both LRTC and non-LRTC underwent proliferation and maintained an epithelial phenotype in the presence of tubulogenic growth factors such as EGF, TGF-alpha, IGF-I, and hepatocyte growth factor. It is interesting that LRTC also proliferated without epithelial markers expression in the presence of soluble factors derived from an embryonic kidney metanephric mesenchyme cell line. The type of extracellular matrix strongly influenced the phenotype of LRTC. Furthermore, in three-dimensional collagen gel culture, LRTC formed tubule-like or tubulocystic structures in response to growth factors (hepatocyte growth factor and fibroblast growth factor) that are known to induce kidney cell tubulogenesis in vitro and/or participate in renal regeneration in vivo. In contrast, non-LRTC did not form these structures. When transplanted into the metanephric kidney, LRTC but not non-LRTC were integrated into epithelial components of nephron, including the proximal tubular cells and the ureteric bud. They also differentiated into fibroblast-like cells. Collectively, these findings suggest that LRTC are an adult kidney tubular cell population that shows phenotypic plasticity, tubulogenic capacity, and integration capability into the developing kidney.

Expression of PKD1 and PKD2 transcripts and proteins and its significance in different types of kidney tissues and kidney lines

To investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines. Immunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines. Coordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01). Similar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

The sialoadhesin (CD169) expressing a macrophage subset in human proliferative glomerulonephritis

Sialoadhesin (Sn; CD169) is a lectin-like receptor whose expression is restricted to subsets of tissue and inflammatory macrophages. We have previously identified accumulation of Sn+ macrophages as an important marker of disease progression versus remission in rat mesangial proliferative nephritis. The current study examined the significance of Sn+ macrophages in human proliferative glomerulonephritis. Frozen kidney sections from normal adult human kidney (n = 4) and pediatric nephropathy (n = 40) were stained for total macrophages (CD68+ cells), Sn+ macrophages, CD3+ T-cells and collagen type I by immunofluorescence. Leukocyte infiltration and the severity of glomerular lesions and interstitial damage were scored. A second protocol biopsy was performed in 27 cases and clinical and biopsy-based data obtained. Sn+ macrophages were absent from glomeruli in normal adult human kidney and in thin basement membrane disease (n = 4), but were detected in 4 of 9 cases of purpura nephritis; 7 of 17 IgA nephropathy; 5 of 5 membranoproliferative glomerulonephritis, and 5 of 5 lupus nephritis. Sn+ macrophages were localized in areas of focal glomerular and interstitial damage. Two-colour immunostaining confirmed that Sn+ cells are a subset of total CD68+ macrophages. The number of glomerular Sn+ macrophages correlated with the degree of proteinuria and glomerular lesions (r = 0.44, P = 0.0045 and r = 0.82, P<0.0001; respectively), while interstitial Sn+ macrophages correlated with the degree of proteinuria and interstitial damage (r = 0.59, P<0.0001 and r = 0.75, P<0.0001; respectively). Combined immunostaining revealed that interstitial Sn+ macrophages and CD3+ T-cells co-localized in areas of tubulointerstitial damage with increased type I collagen deposition. There was significant correlation between the number of interstitial Sn+ macrophages and CD3+ T-cells (r = 0.74, P<0.0001). Most patients responded to a 2 year period of glucocorticoid therapy with a reduction in proteinuria and glomerular lesions and this correlated with the reduction in the number of glomerular Sn+ macrophages. This study has identified Sn+ cells as a macrophage subset whose accumulation in the kidney correlates with proteinuria and histologic damage. These results, together with recent findings from animal studies, suggest that Sn+ macrophages may play an important role in progressive renal disease.

Expression of Gremlin, a Bone Morphogenetic Protein Antagonist, in Human Diabetic Nephropathy

We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo. Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease. Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score. In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.

Isolation of Renal Progenitor Cells from Adult Human Kidney

We describe here isolation and characterization of CD133+ cells derived from normal adult human kidney. These cells lacked the expression of hematopoietic markers and expressed PAX-2, an embryonic renal marker, suggesting their renal origin. Renal tissue-derived CD133+ cells and clones of individual cells were capable of expansion and limited self-renewal and differentiated in vitro into epithelial or endothelial cells. On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. At variance, when differentiated in endothelial cells, these cells formed functional vessels. On intravenous injection in SCID mice with glycerol-induced tubulonecrosis, the in vitro expanded renal-derived CD133+ cells homed into the injured kidney and integrated in tubules. We propose that CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.

Pax2 expression occurs in renal medullary epithelial cells in vivo and in cell culture, is osmoregulated, and promotes osmotic tolerance.

Pax2 is a transcription factor that is crucial for kidney development, and it is also expressed in the normal adult kidney, where its physiological function is unknown. In the present study, we find by cDNA microarray analysis that Pax2 expression in second-passage mouse inner-medullary epithelial cells is increased by a high NaCl concentration, which is significant because NaCl levels are normally high in the inner medulla in vivo, and varies with urinary concentration. Furthermore, a high NaCl concentration increases Pax2 mRNA and protein expression in mouse inner medullary collecting duct (mIMCD3) cells, and its transcriptional activity. Pax2 mRNA and protein expression is high in normal adult mouse renal inner medulla but much lower in renal cortex. Pax2 protein is present in collecting duct cells in both renal medulla and cortex and in thin descending limbs of Henle's loop in inner medulla. Treating Brattleboro rats with desamino-Cys-1,d-Arg-8 vasopressin, which increases inner-medullary NaCl concentration, causes a 4-fold increase in inner-medullary Pax2 protein. Treatment with furosemide, which decreases inner-medullary NaCl, reduces inner-medullary Pax2 mRNA and protein. Pax2-specific short interfering RNA increases high NaCl concentration-induced activation of caspase-3 and apoptotic bodies in mIMCD3 cells. We thus conclude that (i) Pax2 is expressed in normal renal medulla, (ii) its expression is regulated there by the normally high and variable NaCl concentration, and (iii) it protects renal medullary cells from high NaCl concentration-induced apoptosis.

Binding of the chemokine SLC/CCL21 to its receptor CCR7 increases adhesive properties of human mesangial cells

Adherence of human mesangial cells to the surrounding matrix contributes to glomerular homeostasis and is important for the maintenance of glomerular architecture and function in normal adult human kidney. The expression of chemokines and corresponding chemokine receptors on adjacent intrinsic renal cells indicates a novel chemokine/chemokine receptor function on nonimmune cells important for glomerular homeostasis. A constitutive expression of the chemokine SLC/CCL21 on human podocytes and of its corresponding receptor CCR7 on mesangial cells was shown before. SLC/CCL21 has a positive effect on proliferation and migration of mesangial cells and leads to increased cell survival in Fas-induced apoptosis. In leukocytes chemokines mediate integrin-dependent firm adhesion. Therefore, we examined the influence of chemokine receptor CCR7 activation by SLC/CCL21 on adhesive properties of human mesangial cells to matrix molecules. Adhesion assays, mechanical detachment assays, and evaluation of integrin activation by integrin-linked kinase activity were performed. Changes in the cytoskeletal F-actin were illustrated by phalloidin immunofluorescence staining. SLC/CCL21 stimulation enhanced adhesiveness to fibronectin in a time- and concentration-dependent manner. SLC/CCL21 also increased the firmness of mesangial cells adhesion as judged by detachment assays. Furthermore activation of integrin-linked kinase occurred with SLC/CCL21 addition to mesangial cells, resulting in increased phosphorylation of glycogen synthase kinase-3 (GSK-3) and protein kinase B (PKB/Akt). Exposure of mesangial cells to SLC/CCL21 also resulted in F-actin rearrangements with membrane ruffling and extensions leading to bridging between mesangial cells. Activation of CCR7 on mesangial cells by SLC/CCL21 enhances the degree and firmness of cell adhesion and increases cell spreading and the formation of cell-cell contacts. This includes integrin-linked kinase activation and F-actin rearrangements. Thus, local chemokine generation and chemokine receptor expression on mesangial cells may play an important role in the maintenance of glomerular homeostasis and in local remodeling processes.

Preparation of monoclonal antibody against LRR-WSC domain of polycystin-1 and distribution of polycystin-1 in kidney tissues and kidney cell lines

目的 用杂交瘤技术制备抗多囊蛋白-1 LRR-WSC区单克隆抗体,检测多囊蛋白-1在肾组织和肾细胞株中的分布和定位.方法用多囊蛋白-1 LRR-WSC区融合蛋白PC1-e免疫BALB/c小鼠,将其脾细胞与骨髓瘤细胞株SP2/0进行细胞融合,间接酶联免疫吸附试验(ELISA)筛选出阳性克隆,有限稀释法将杂交瘤细胞株单克隆化,间接ELISA法和免疫印迹法(WB)鉴定抗体的特异性.用制备的抗多囊蛋白-1 LRR-WSC区单克隆抗体,免疫组织化学和免疫细胞化学法检测多囊蛋白-1在不同肾组织和肾细胞株中的分布.结果细胞融合后经筛选和克隆得到的杂交瘤细胞株经WB分析表明,该细胞株分泌的单克隆抗体能特异地与多囊蛋白-1 LRR-WSC区结合.免疫组织化学显示,多囊蛋白-1主要分布于正常肾组织的远端肾小管和集合管,在胎肾囊肿组织中表达于近端肾小管,在人常染色体显性多囊肾病(ADPKD)肾囊肿组织中,表达于囊肿衬里上皮细胞,同时在ADPKD肾囊肿衬里上皮细胞系和猪近端肾小管细胞株(LLC-PK1)中也发现了多囊蛋白-1的表达.结论本实验成功制备了抗多囊蛋白-1 LRR-WSC区的单克隆抗体,该抗体对深入研究ADPKD的发病机制具有重要意义.多囊蛋白-1在肾组织中的表达模式对肾小管的形态发生、维持肾小管结构的完整性非常重要。

Cycloxygenase-2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors.

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henle's loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.

KIT expression in fetal, normal adult, and neoplastic renal tissues

Background: KIT is a transmembrane tyrosine kinase receptor, expressed in high amounts in various normal cells. In addition, c-kit mutation or activation is a major pathogenetic event in certain tumours...

Cloning of a new familial t(3;8) translocation associated with conventional renal cell carcinoma reveals a 5 kb microdeletion and no gene involved in the rearrangement.

This study describes the molecular cloning of a familial translocation, t(3;8)(p14.2;q24.2), that segregates with the conventional renal cell carcinoma (conventional RCC). We had previously reported the family history and, through loss of heterozygosity and comparative genomic hybridization, detected the loss of the 3p chromosome arm and somatic mutation in the retained von Hippel-Lindau gene in some members of the family. With the help of array painting and sequence tagged site-PCR on flow-sorted derivative chromosomes, we have cloned the breakpoints of the translocation. We have studied the junctions on both derivative chromosomes at the genomic and expression levels. The analysis of the sequence revealed a 5 kb microdeletion at the chromosome 3 breakpoint together with a high density of repetitive motifs (Alu, short interspersed nuclear element) and an AT-rich region. Both chromosome 3 and 8 rearranged regions were very poor in gene content. We tested an expressed sequence tag, two predicted genes, one novel gene and LRIG1, a gene located more than 200 kb apart from the breakpoint on chromosome 3. None of these genes, except LRIG1, showed expression in any of the tested tissues (including normal adult and fetal kidney, sporadic kidney tumours and tumour samples from the proband's family). Taken together, all these data suggest that, rather than deregulation of specific genes that may be rearranged by the translocation, the proposed three-step model of tumour development (translocation, loss of the 3p chromosome, and mutation in a tumour suppressor gene located within that region) could be the biological mechanism that takes place in this familial form of conventional RCC.

Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies.

To examine the role of nucleostemin in the growth regulation of gastric cancer, liver cancer and other cancers. RT-PCR was used to clone the fragment of nucleostemin cDNA from HEK 293 cells. Eighteen kinds of malignant tumor tissues including gastric adenocarcinoma and liver cancer tissues, 3 kinds of benign tumor tissues, 3 kinds of benign hyperplastic tissues and normal tissues were employed to examine nucleostemin gene expression by RT-PCR, Slot blot, Northern blot and in situ hybridization. We successfully cloned a 570 bp fragment of nucleostemin-cDNA from HEK-293 cells. All detected malignant tumor tissues, benign tumor tissues, and benign hyperplastic tissues had high levels of nucleostemin expression. Nucleostemin was also expressed in human placenta tissue at a high level. In terminally differentiated normal human adult kidney and mammary gland tissues, no nucleostemin expression could be detected. Nucleostemin can help regulate the proliferation of both cancer cells and stem cells. It might play an important role in the growth regulation of gastric cancer, liver cancer and other cancers.

Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells.

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na(+)-bicarbonate cotransporter, Na(+)-H(+) exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.

Gene expression in the normal adult human kidney assessed by complementary DNA microarray.

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.

Podocan, a Novel Small Leucine-rich Repeat Protein Expressed in the Sclerotic Glomerular Lesion of Experimental HIV-associated Nephropathy

Growing evidence suggests that human immunodeficiency virus (HIV)-1 infection of podocytes plays a central role in the glomerular disease of HIV-associated nephropathy (HIVAN). As an approach to identify host genes involved in the pathogenesis of the sclerotic glomerular lesion in HIVAN, representational difference analysis of cDNA was used to identify differentially expressed genes in HIV-1 transgenic and nontransgenic podocytes. We isolated a novel member of the small leucine-rich repeat (SLR) protein family, podocan, that is expressed at high levels in the HIV-1 transgenic podocytes. In normal embryonic kidney, a 3.2-kb podocan transcript was detected at low levels, and expression increased dramatically within 24 h following birth. Expression of a 2.3-kb transcript became evident after birth and gradually increased to 50% of the total podocan RNA in the mature kidney. Phylogenetically, podocan represents a new class in the SLR protein gene family, an expanding protein family sharing homology with the small leucine-rich repeat proteoglycans. The 3.2-kb transcript encodes a predicted 611-amino acid secretory protein with 20 leucine-rich repeats, a unique N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain. In situ hybridization of normal kidney revealed podocan mRNA expression in podocytes and likely vascular endothelial cells within the glomerulus. The immunohistochemical staining pattern of podocan protein in normal kidney glomeruli was consistent with that of the glomerular basement membrane, and staining was markedly increased in sclerotic glomerular lesions in the transgenic HIVAN model. Thus, podocan defines a new class within the SLR protein family and is a previously unrecognized component of the sclerotic glomerular lesion that develops in the course of experimental HIVAN.

PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases.

PDGF-C is a new member of the PDGF-family and has recently been identified as a rat mesangial cell mitogen. Its expression and function in human kidneys is unknown. Localization of PDGF-C protein was analyzed by immunohistochemistry using a rabbit polyclonal antibody directed against the core-domain of PDGF-C in human fetal kidneys (n = 8), normal adult human kidneys (n = 9), and in renal biopsies of patients with IgA nephropathy (IgAN, n = 31), membranous nephropathy (MGN, n = 8), minimal change disease (MC, n = 7), and transplant glomerulopathy (TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissected glomeruli by real-time RT-PCR in cases of normal adult kidneys (n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In the fetal kidney, PDGF-C localized to the developing mesangium, ureteric bud epithelium, and the undifferentiated mesenchyme. In the adult kidney, PDGF-C was constitutively expressed in parietal epithelial cells of Bowman's capsule, tubular epithelial cells (loops of Henle, distal tubules, collecting ducts), and in arterial endothelial cells. A marked upregulation of glomerular PDGF-C protein was seen in MGN and TxG with a prominent positivity of virtually all podocytes. In MC, PDGF-C localized to podocytes in a more focal distribution. In MGN, increased glomerular PDGF-C protein expression was due to increased mRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detected in microdissected glomeruli from MGN compared with normal. PDGF-C protein was additionally expressed in individual mesangial cells in TxG. Finally, upregulated PDGF-C protein expression was detected within sclerosing glomerular and fibrosing tubulointerstitial lesions in individual cases from all analyzed groups. We conclude that PDGF-C is constitutively expressed in the human kidney and is upregulated in podocytes and interstitial cells after injury/activation of these cells.

Localization of SPARC in developing, mature, and chronically injured human allograft kidneys

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is expressed during development, tissue remodeling and repair. It functions as an endogenous inhibitor of cell proliferation, regulates angiogenesis, regulates cell adhesion to extracellular matrix, binds cytokines such as platelet derived growth factor and stimulates transforming growth factor-beta (TGF-beta) production. This study describes the expression of SPARC during human renal development, in normal kidneys and during renal allograft rejection. A total of 60 renal specimens, including normal areas from tumor nephrectomies (N = 24), fetal kidneys (N = 27) and explanted renal allografts (N = 9), were included in the study. SPARC protein was localized by immunohistochemistry using two different antibodies. On consecutive sections SPARC mRNA was detected by in situ hybridization. In the normal adult kidney SPARC protein was expressed by visceral and parietal epithelial cells, collecting duct epithelium (CD), urothelium, smooth muscle cells of muscular arteries and focally in interstitial cells. During renal development immature glomeruli demonstrated a polarized SPARC expression in visceral epithelial cells at their surface abutting the capillary basement membranes. In the fully differentiated glomeruli the expression pattern mirrored that of the adult kidney. Furthermore, SPARC was abundantly expressed by derivatives of the ureteric bud, and smooth muscle cells of arterial walls. During chronic allograft rejection SPARC is expressed in neointimal arterial smooth muscle cells, infiltrating inflammatory cells as well as by interstitial myofibroblasts in areas of interstitial fibrosis. SPARC mRNA synthesis detected by in situ hybridization mirrored these protein expression patterns. These studies co-localize SPARC to several sites of renal injury previously shown to be sites of PDGF B-chain expression and/or activity. We speculate that SPARC could function as an accessory molecule in chronic PDGF-mediated sclerosing interstitial and vascular injury. SPARC localization to glomerular epithelial cells corresponds to similar findings in rodents, and may reflect its role in cell adhesion and /or regulation of cell shape.