Intraluminal ATP concentrations in rat renal tubules.
It is becoming increasingly recognized that stimulation of apical P2 receptors can influence solute transport in the nephron, but, to date, no information is available on endogenous intraluminal nucleotide concentrations in vivo. This study measured intraluminal ATP concentrations in the renal tubules of anesthetized rats. Proximal tubular concentrations were found to be in the range of 100 to 300 nmol/L, with no significant variation along the S2 segment, whereas concentrations in the early distal tubule were markedly lower. Using collections of varying duration, the half-life of ATP in collected proximal tubular fluid was found to be 3.4 min, indicating significant breakdown by soluble nucleotidases. For assessment of whether proximal tubular ATP was filtered or secreted, experiments were performed in Munich-Wistar rats. The ATP concentration in midproximal tubules (142 +/- 23 nmol/L) was more than four-fold higher than in Bowman's space (32 +/- 7 nmol/L; P < 0.001), whereas fractional water reabsorption between the two sites was modest. In experiments that were designed to determine the effects of (patho)physiologic disturbances on intraluminal ATP, rats were either volume expanded or subjected to hypotensive hemorrhage. Neither maneuver affected proximal tubular luminal ATP concentrations significantly; rapid degradation of secreted ATP by ecto- and soluble nucleotidases is a possible explanation. It is concluded that the proximal tubule secretes ATP into the lumen, where it may have an autocrine/paracrine regulatory role. (+info)
Fluid flow in the juxtaglomerular interstitium visualized in vivo.
Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 +/- 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 +/- 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function). (+info)
Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys.
Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowman's capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases. (+info)
Gut-like structures from mouse embryonic stem cells as an in vitro model for gut organogenesis preserving developmental potential after transplantation.
Recently, we reported the formation of gut-like structures from mouse ESCs in vitro. To determine whether ESCs provide an in vitro model of gastrointestinal (GI) tracts and their organogenesis, we investigated the morphological features, formation process, cellular development, and regional location within the GI tract by immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction. We also examined the developmental potential by transplantation into kidney capsules. The results demonstrated that Id2-expressing epithelium developed first, alpha-smooth muscle actin appeared around the periphery, and finally, the gut-like structures were formed into a three-layer organ with well-differentiated epithelium. A connective tissue layer and musculature with interstitial cells of Cajal developed, similar to organogenesis of the embryonic gut. Enteric neurons appeared underdeveloped, and blood vessels were absent. Many structures expressed intestinal markers Cdx2 and 5-hydroxytryptamine but not the stomach marker H(+)/K(+) ATPase. Transplants obtained blood vessels and extrinsic nerve growth from the host to prolong life, and even grafts of premature structures did not form teratoma. In conclusion, gut-like structures were provided with prototypical tissue components of the GI tract and are inherent in the intestine rather than the stomach. The formation process was basically same as in gut organogenesis. They maintain their developmental potential after transplantation. Therefore, gut-like structures provide a unique and useful in vitro system for development and stem cell studies of the GI tract, including transplantation experiments. (+info)
Parietal podocytes in normal human glomeruli.
Although parietal podocytes along the Bowman's capsule have been described by electron microscopy in the normal human kidney, their molecular composition remains unknown. Ten human normal kidneys that were removed for cancer were assessed for the presence and the extent of parietal podocytes along the Bowman's capsule. The expression of podocyte-specific proteins (podocalyxin, glomerular epithelial protein-1, podocin, nephrin, synaptopodin, and alpha-actinin-4), podocyte synthesized proteins (vascular endothelial growth factor and novH), transcription factors (WT1 and PAX2), cyclin-dependent kinase inhibitor p57, and intermediate filaments (cytokeratins and vimentin) was tested. In addition, six normal fetal kidneys were studied to track the ontogeny of parietal podocytes. The podocyte protein labeling detected parietal podocytes in all of the kidneys, was found in 76.6% on average of Bowman's capsule sections, and was prominent at the vascular pole. WT1 and p57 were expressed in some parietal cells, whereas PAX2 was present in all or most of them, so some parietal cells coexpressed WT1 and PAX2. Furthermore, parietal podocytes coexpressed WT1 and podocyte proteins. Cytokeratin-positive cells covered a variable part of the capsule and did not express podocyte proteins. Tuft-capsular podocyte bridges were present in 15.5 +/- 3.7% of the glomerular sections. Parietal podocytes often covered the juxtaglomerular arterioles and were present within the extraglomerular mesangium. Parietal podocytes were present in fetal kidneys. Parietal podocytes that express the same epitopes as visceral podocytes do exist along Bowman's capsule in the normal adult kidney. They are a constitutive cell type of the Bowman's capsule. Therefore, their role in physiology and pathology should be investigated. (+info)
Expression of the chemokine receptor CCR1 in human renal allografts.
BACKGROUND: Chemokines are involved in the recruitment of leukocytes to vascularized allografts. CCR1 is a receptor for various proinflammatory chemokines and CCR1 blockade reduces renal allograft injury in rabbits. The purpose of the study was to characterize CCR1-positive cells in human renal allografts. METHODS: Formalin-fixed, paraffin-embedded allograft nephrectomies (n = 9) and non-involved parts of tumour nephrectomies (n = 10) were studied. Immunohistochemistry for CCR1, CD3 and CD68 was performed on consecutive sections. Double immunofluorescence for CCR1 and CD3, CD20, CD68, DC-SIGN and S100 was used on selected cases. Expression of CCR1 mRNA and the ligands CCL3 and CCL5 was studied in renal allograft biopsies with acute rejection (n = 10), with chronic allograft nephropathy (n = 8) and controls (n = 8). RESULTS: CCR1 protein was expressed by circulating cells in glomerular and peritubular capillaries, colocalizing with CD68. In renal allografts CCR1-positive cells were present within glomerular tufts, but only scattered CCR1-positive cells were found in tubulointerstitial infiltrates. CCR1 did not colocalize with the majority of CD68-positive cells in the interstitium. The small number of CCR1-positive interstitial cells were identified as CD20- or DC-SIGN-positive by double immunofluorescence. CCR1 mRNA was significantly increased in renal biopsies with acute allograft rejection (P < 0.001), and with chronic allograft nephropathy (P < 0.05), it correlated with the expression of CCL3 and CCL5, and with serum-creatinine. CONCLUSIONS: CCR1 mRNA expression was associated with renal function in allografts. CCR1 protein expression was restricted to monocytes, CD20-positive B cells and DC-SIGN-positive dendritic cells. Thus most interstitial macrophages were CCR1 negative, which may relate to down-regulation after migration into the interstitium in human renal allografts. (+info)
A pitfall of glomerular sieving: profibrotic and matrix proteins derive from the Bowman's capsule and not the glomerular tuft in rats with renovascular hypertension.
BACKGROUND: The glomeruli in the non-clipped kidney of rats with 2-kidney, 1-clip hypertension are a classical model for studying the mechanisms of glomerular injury. METHODS: In the present study, we compared the glomerular expression of PAI-1 and collagen I alpha1 mRNA from glomeruli isolated by the classic technique of sieving with the recently developed technique of tissue laser microdissection. For quantification of mRNA from both methods, real-time PCR was used. RESULTS: Real-time PCR revealed a 9.0 +/- 1.3- and a 7.1 +/- 0.2-fold induction of PAI-1 and collagen I alpha 1, respectively, in the glomeruli from hypertensive rats isolated by sieving. However, in situ hybridization and microdissection revealed that expression of both mRNAs was mainly from the Bowman's capsule and not from the glomerular tuft (10.7 +/- 1.3- and 7.2 +/- 0.6-fold higher induction in whole glomeruli compared with tuft alone). CONCLUSION: This emphasizes that studies focusing on processes in the mesangium, endothelial cells or podocytes should not rely on glomeruli obtained by sieving. Rather, a technique like the laser microdissection or in situ hybridization should be applied which allows the clear separation of different glomerular and periglomerular compartments. (+info)
Alterations in renal cilium length during transient complete ureteral obstruction in the mouse.