Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.     

Immunohistochemical localization of ghrelin in rodent kidneys

Ghrelin is a novel peptide hormone, originally identified in the rat and human stomach that plays various important roles. In the present study, we report the intra-renal localization of ghrelin in laboratory rodents. Kidneys from 3 month-old mice, rats and hamsters of both sexes were analyzed by immunohistochemistry. Positive signals were clearly observed in the epithelium of the distal tubules, whereas other segments of the nephron or interstitial cells, including juxtaglomerular cells, showed negative reactions. Pre-embedding immunoelectron microscopy revealed positive signals exclusively on the basolateral membrane in the distal tubular cells and in the collecting ducts. In addition, prepro-ghrelin gene expression was assessed by RT-PCR, and the expected 329-bp prepro-ghrelin mRNA was clearly detected in the kidney. On Western blot analysis, although a specific band for ghrelin (3 kDa) was not detected in the kidney, the expected band for prepro-ghrelin (13 kDa) was clearly detected in both the stomach and the kidney. This paper clarified the intra-renal localization of ghrelin.     

Sites of urokinase-type plasminogen activator expression and distribution of its receptor in the normal human kidney

The urokinase-type plasminogen activator (uPA) is secreted into the urine at high concentrations and both the uPA protein and mRNA are present in human renal tissue. Normal kidney tissue also expresses the receptor for uPA. Neither the precise sites of uPA mRNA expression, nor the distribution of the uPA-receptor antigen, have been elucidated in the human kidney. In the present study, the sites of uPA mRNA expression were identified by in situ hybridization, and the cellular localization of both uPA and uPA-receptor was determined by immunohistochemical analysis. High-level uPA mRNA expression was restricted to epithelial cells of the convoluted proximal tubules and the thick ascending limb of Henle's loop (the straight part of the distal tubule). However, uPA immunoreactivity was not confined to sites of uPA mRNA expression, but was present in all segments of the tubular epithelium. Tubular epithelial cells also exhibited a consistent immunoreactivity with uPA-receptor antibody, indicative of a co-localization of the uPA antigen and its receptor in the uriniferous epithelium. We propose that the uPA antigen expression in nephron segments lacking demonstrable endogenous uPA synthesis may be the result of a uPA-receptor-mediated uptake of uPA.     

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.     

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.     

CFTR structure and function: is there a role in the kidney?

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective protein processing that leads to changes in function and regulation of this chloride channel. Despite of the expression of CFTR in the kidney, patients with CF do not present major renal dysfunction, but it is known that both the urinary excretion of proteins and renal capacity to concentrate and dilute urine are altered in these patients. CFTR mRNA is expressed in all nephron segments of rat and human, and this abundance is more prominent in renal cortex and outer medulla renal areas. CFTR protein was detected in apical surface of both proximal and distal tubules of rat kidney but not in the outer medullary collecting ducts. Studies have demonstrated that CFTR does not only transport Cl⁻ but also ATP. ATP transport by CFTR could be involved in the control of other ion transporters such as Na⁺ (ENaC) and K⁺ (renal outer medullary potassium) channels, especially in TAL and CCD. In the kidney, CFTR also might be involved in the endocytosis of low-molecular-weight proteins by proximal tubules. This review is focused on the CFTR function and structure, its role in the renal physiology, and its modulation by hormones involved in the control of extracellular fluid volume.