Proximal Tubule Brush Border Research Articles

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

We examined the circulating levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 alpha, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), and the anti-inflammatory cytokine IL-10, and their expression in kidneys acutely infected with murine malaria parasite P. berghei ANKA in C57BL/6J mice. Groups of six mice sacrificed on days 5, 10, 15, and 20, and normal controls were used for cytokine analysis. High concentrations of TNF-alpha and IL-10 were detected in plasma as shown by ELISA, and elevated levels of mRNA specific for TNF-alpha and IL-10 in infected kidneys were demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Kidney sections stained with antibodies against TNF-alpha, IL-1 alpha, IL-6, GM-CSF and IL-10 for immunohistochemistry showed markedly enhanced staining for TNF-alpha, and progressively increased staining for IL-1 alpha and IL-6 both in the tubules and the walls of arteries during the course of infection. The endothelia of blood vessels and inflammatory cells located around small arteries showed positive staining for GM-CSF from day 10 onwards. Unlike the staining for proinflammatory cytokines, the anti-inflammatory cytokine IL-10 showed strongly positive staining in normal tubules and walls of arteries, especially in the brush border of proximal tubules, but the staining intensity decreased dramatically after day 15 post-infection. A strongly positive correlation was found between the antibody staining for TNF-alpha/IL-1 alpha in tubules, and the severity of proteinuria. In contrast, there was an inverse correlation between the staining for IL-10 with TNF-alpha/IL-1 alpha, and the degree of proteinuria. Plenty of pigmented macrophages showed positive staining both for proinflammatory and anti-inflammatory cytokines in the tubulointerstitium. Our findings imply that the up-regulation of proinflammatory cytokines and the dysregulation of anti-inflammatory cytokines are involved in the pathogenesis of tubulointerstitial nephritis associated with malaria.

Localization of the extracellular Ca2+/polyvalent cation-sensing protein in rat kidney.

We previously identified transcripts encoding a G protein-coupled, extracellular calcium/polyvalent cation-sensing receptor, RaKCaR, in rat kidney (D. Riccardi, J. Park, W.-S. Lee, G. Gamba, E. M. Brown, and S. C. Hebert. Proc. Natl. Acad. Sci. USA 92:131-135, 1994), which was proposed to provide the mechanism for modulating a variety of renal functions in response to changes in extracellular Ca2+ (E. M. Brown. In: Handbook of Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, vol. 2, chapt. 39, p. 1841-1916; and S. C. Hebert. Kidney Int. 50: 2129-2139, 1996). Here, we examine the cellular and regional distribution of receptor protein by immunofluorescence microscopy using a polyclonal antibody raised against a 22 amino acid region of the NH2 terminus of the receptor. The most intense fluorescence was seen at the basolateral border of cortical thick ascending limb cells. Basolateral staining for the receptor was also detected in medullary thick ascending limbs, in macula densa cells identified by costaining with antibody to brain nitric oxide synthase, NOS-B1, and in distal convoluted tubule cells distinguished by costaining for the apical thiazide-sensitive Na(+)-Cl- cotransporter. Apical anti-RaKCaR staining was detected at the base of the brush border of proximal tubules with decreasing intensity from S1 to S3 segments. In cortical collecting ducts, anti-RaKCaR staining was detected in some, but not all, type A intercalated cells identified by costaining with anti-H(+)-ATPase and anti-AE1 Cl-/HCO3- exchanger antibodies. The present study demonstrates that RaKCaR protein is expressed in many different nephron segments and that the polarity of receptor expression varies with cell type along the nephron. These results suggest potential roles for the extracellular Ca2+/ polyvalent cation-sensing receptor in responding to both circulating and urinary concentrations of divalent minerals and potentially other polyvalent cations (e.g., aminoglycoside antibiotics) to modulate nephron function.

Renal brush border sodium-sulfate cotransport in guinea pig: effect of age and diet.

Renal adaptation to changes in inorganic sulfate intake and age was studied by comparing sulfate uptake by proximal tubule brush border membrane vesicles (BBMV) from guinea pigs of different ages on relatively high- or low-sulfate diets. Adult (> 60 days) or young guinea pigs (< 25 days) were fed either a control diet (0.28% sulfur content), a sulfur-free diet, or a high-sulfate diet. After 5 days on the diet, BBMV were obtained and kinetic analysis of 35sulfate uptake was determined. In adult guinea pigs, the low-sulfate diet produced a significant increase in apparent maximal velocity (Vmax). In young guinea pigs, a lower sulfate intake did not appreciably increase Vmax, but a high-sulfate intake produced a reduction in Vmax. The affinity for sulfate (Km) was not changed in either age group. The dietary sulfate intake did not alter sodium gradient dependent-D-glucose or 32phosphate Vmax. In conclusion, our data indicate that renal inorganic sulfate BBMV uptake is regulated and responds to conditions of increased need (i.e., during the growth phase in young animals and during periods of decreased sulfate availability in adult animals) by increasing BBMV Vmax.

North American opossum Didelphis virginiana as a fetal nephrotoxicity model: histologic and ultrastructural assessment of uranyl nitrate (UN)-induced damage.

We have used our opossum model of fetal nephrotoxicity to investigate uranyl nitrate-induced morphologic changes in the developing kidney. The present study establishes a renal dose response curve for the uranyl nitrate (UN). We find that pups treated with nonlethal doses of UN do not demonstrate growth retardation compared to saline-treated controls. The kidneys of UN-treated pups are heavier than the control animals, an effect less apparent the longer the pups are followed. A low dose of 60 mg/kg of UN administered to small pups causes slight histologic derangement but nevertheless more change than the same dose administered to larger more mature pups. Using a dose of 100 mg/kg of UN that effectively causes nonfatal renal disruption, we examined the kidneys from 4 to 42 days following injection. We find that tubular dilation and epithelial necrosis starts soon after treatment (day 4) and reaches its maximum during the second and third week (11 and 22 days). Architectural restoration appears complete by the end of the third week. By electron microscopy, UN induces sequential structural damage with loss of proximal tubule brush border, epithelial necrosis with intact basement membranes and regeneration at 4, 11, and 22 days. Residual tubular mitochondrial damage is present at 42 days in spite of histologically normal tubules. No apparent lesions are seen in glomeruli. Fibroblastic interstitial proliferation in UN-treated kidneys at 11 days is not followed by appreciable fibrosis when assessed at 22 and 42 days. As the structural changes caused by 100 mg/ml UN administration in fetal opossum kidneys are reversible, this is a useful model to study the molecular mediators responsible for this form of renal damage and repair.

Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody.

Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT1 immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.

Immunolocalization of tissue non-specific alkaline phosphatase in mice.

Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-micron tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.

Proximal tubular injury in Chinese herbs nephropathy: Monitoring by neutral endopeptidase enzymuria

Neutral endopeptidase (NEP) is a 94 kDa ectoenzyme of the proximal tubule brush border, physiologically released into the urine with apical membrane fragments. As proximal tubular atrophy was a histological hallmark of Chinese herbs nephropathy (CHN), this study firstly determined renal excretion of NEP in healthy control subjects (N = 31), in patients with CHN (N = 26) and in women having consumed Chinese herbs and whose renal function was normal but running the risk of developing CHN (N = 27). Another patient group consisted of female patients with glomerular diseases (N = 12). At the same time, measurements of urinary microproteins (Clara cell protein, retinol binding protein, beta 2-microglobulin and alpha 1-microglobulin) were performed, as indicators of tubular dysfunction. Cell damage was estimated by the excretion of N-acetyl-beta-D-glucosaminidase (NAG). In the control group, the physiological NEP enzymuria was 43.1 micrograms/24 hr (geometric mean). In CHN patients, levels of urinary NEP were significantly decreased in those with moderate renal failure (26.7 micrograms/24 hr; N = 21; P < 0.05) and almost abolished in end-stage renal failure patients (4.35 micrograms/24 hr; N = 5; P < 0.05). In patients at risk as well as in patients with glomerular diseases, urinary NEP levels were not statistically different from those observed in control subjects (40.68 micrograms/24 hr and 48.5 micrograms/24 hr, respectively). Several degrees of tubular dysfunction and injury were noted in patients groups, as attested by increased urinary microproteins and NAG excretions. Considering the data from control and CHN patients, NEP enzymuria positively correlated with individual creatinine clearance values (r = 0.76; P = 0.0001) and negatively correlated with urinary microproteins levels (r = -0.55; P = 0.00001). Finally, NEP was regularly quantitated in the urine of 6 CHN patients for a period ranging from six months to two years and in 19 patients at risk during two years, respectively. In the first group, renal function progressively deteriorated in 3 patients, leading them to renal replacement therapy after 38 to 115 weeks. Stable parameters were observed in the remaining 3 patients. A direct correlation between creatinine clearance and NEP excretion was found longitudinally in each case. In the second group, no significant change of urinary NEP levels was observed (45.9 micrograms/24 hr), in parallel with stable renal function. Taken together, these results indicate that, in CHN patients, NEP enzymuria provides a rapid and noninvasive determination of the degree of structural impairment affecting the proximal tubular population and further reflecting the severity of the renal disease. The interest of this urinary marker in monitoring the progression of other tubulointerstitial diseases remains to be assessed.

Receptor-associated Protein and Members of the Low Density Lipoprotein Receptor Family Share a Common Epitope: AN EXTENDED MODEL FOR THE DEVELOPMENT OF PASSIVE HEYMANN NEPHRITIS

Heymann nephritis is an experimental rat model for human membranous glomerulonephritis. Two target antigens have been identified in the proximal tubule brush border of rat kidneys. One of them is megalin, a 600-kDa membrane protein that belongs to the family of low density lipoprotein receptor (LDLR)-related proteins. The other one is receptor-associated protein (RAP), a polypeptide of 40 kDa that associates with members of the LDLR family. Here we show that antibodies produced against recombinant human RAP strongly cross-react with the chicken oocyte receptor for very low density lipoprotein and vitellogenin (LR8), and with two other members of the LDLR family, LDLR-related protein and megalin. The interaction of this antibody with LR8 showed binding characteristics exactly as those demonstrated for the physiological ligands of this receptor, in that binding of the antibody: (i) is Ca2+-dependent; (ii) is abolished by unfolding of the cysteine-rich binding domain by reduction; and (iii) interferes with the binding of very low density lipoprotein and vitellogenin. Immunopurification of the LR8-specific subpopulation of the polyclonal antiserum yielded an IgG fraction strongly reacting with LR8 as well as with RAP. Using recombinant fragments of RAP and peptide mapping, the cross-reacting epitope(s) could be narrowed down to three short sequences (5-7 residues) in the COOH-terminal part of the protein. After immunization with RAP, anti-LR8 antibodies and anti-RAP antibodies arise simultaneously, indicating that the receptor-specific activity is not due to anti-idiotypic antibodies. These findings suggest the existence of a common epitope(s) on RAP and members of the LDL receptor family. Based on these results, we present an extended molecular model for the development of passive Heymann nephritis.

Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression.

cGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific CI- channel shown by others to be regulated by dehydration, are localized in the ATL. Whereas additional data suggest that cGK I may primarily mediate cGMP-related changes in renal hemodynamics, cGK II may regulate renin release and ATL ion transport.

Localization of authentic thromboxane A2/prostaglandin H2 receptor in the rat kidney

Using a polyclonal antibody against authentic thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptor protein, we assessed the distribution of this receptor in the normal rat kidney by routine methods of immunofluorescence microscopy. The receptor localized both in glomeruli and in tubules. In the former, the distribution of the receptor was most prominent along the lumen of glomerular capillary loops. Parietal epithelial cells of the Bowman's capsule, podocytes and mesangial cells also demonstrated immunostainable receptor. In the tubules, the receptor localized most prominently at the base of the brush border of proximal tubules and at the luminal surface of thick ascending limbs and distal convoluted tubules. These observations point to sites that are likely to be targeted by thromboxane A2 in forms of renal injury characterized by enhanced synthesis of this eicosanoid.

Evidence from incorporation and patch-clamp experiments for a nonselective channel of large conductance at the luminal membrane of rabbit proximal tubule

The presence of nonselective ionic channels at the apical membrane of rabbit proximal tubule cells was investigated by fusion of proximal tubule brush border membrane vesicles onto a planar bilayer and by means of patch clamp measurements on primary cultured proximal tubule cells. The fusion of brush border membrane vesicles onto a phosphatidylethanolamine-phosphatidylcholine (1:1) planar bilayer led to the identification of a nonselective channel of 115 pS in 200 mM NaCl conditions with an ionic permeability sequence for Na+, K+, and Cl- corresponding to PNa/PK = 0.3 and PNa/PCl = 3, respectively. This channel showed an increased open probability at depolarizing potentials, but its activity was independent of the Ca2+ concentration in both the cis and trans compartments. These findings were subsequently confirmed in inside-out patch clamp experiments carried out on primary cultured proximal tubule cells. This channel was seldom seen in cell-attached recordings on resting cells, but its presence was observed with a higher probability in patch excised experiments performed following superfusion of the cells with a hypotonic solution. It is suggested on the basis of these observations that the apical membrane of rabbit proximal tubule cells contains a voltage-dependent nonselective channel, the activity of which may contribute to the regulation of the membrane potential.

Acid/base transport in a model of the proximal tubule brush border: impact of carbonic anhydrase.

A mathematical model of the brush border of the proximal tubule (T. A. Krahn, P. S. Aronson, and A. M. Weinstein. Bull. Math. Biol, 56: 459-490, 1994) has been extended by the inclusion of CO2 and H2CO3 as diffusible species and by the inclusion of finite rate constants for the hydration of CO2. This permits the simulation of carbonic anhydrase (CA) activity and its inhibition. We confirm the result of our previous study, which is that, in the presence of CA, the unstirred layer has only a modest effect on the observed formic acid permeability. CA inhibition results in disequilibrium pH gradients, and the effect of these gradients on formic acid permeability depends on the presence of other membrane transport proteins. We also examined the impact of CA activity on the flux of total CO2 through the brush border. Under physiological conditions, CA inhibition depressed NaHCO3 reabsorption through the brush border by interfering with the HCO3(-)-facilitated diffusion of CO2. However, the determination of brush-border CO2 permeability, using an imposed CO2 gradient, was relatively uninfluenced by CA activity. Finally, we inserted a kinetic representation of the Na+/H+ exchanger into the brush-border model. Even when luminal and cytosolic diffusion coefficients were increased 1,000-fold, there was no effect on brush-border Na+ flux. This suggests that variations in the unstirred layer cannot be responsible for the flow dependence of Na+ reabsorption.

Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling.

Receptor-mediated endocytosis and recycling have been described for extrarenal angiotensin II (ANG II) receptors. In proximal tubule (PT) epithelia expressing polarized ANG II receptors, these processes have not been examined as thoroughly. We utilized a PT cell model, LLC-PKCl4 cells stably transfected with rabbit type 1 ANG II receptor (AT1R) cDNA, to investigate these properties. LLC-PK-AT1R cells expressed the rabbit AT1R transcript and displayed losartan-inhibitable specific 125I-labeled ANG II binding at apical (AP) and basolateral (BL) membranes when grown on permeable supports. AP AT1R internalized 125I-ANG II more rapidly than BL AT1R, and phenylarsine oxide treatment inhibited AP AT1R internalization without significantly affecting BL AT1R endocytosis. Pertussis toxin had no effect on AP or BL AT1R endocytosis. In addition, AP AT1R recovered specific 125I-ANG II binding after ANG II treatment (a measure of recycling). BL AT1R displayed minimal recovery of 125I-ANG II binding after ANG II pretreatment. These data suggested that AP AT1R enter endocytic/endosomal pathways. Phospholipase A2 (PLA2) activity has been linked to endosomal fusion in other systems, and PT brush-border membrane AT1R also have been associated with PLA2 activity. LLC-PK-AT1R cells were therefore treated with quinacrine, a nonspecific PLA2 inhibitor, or Compound I (CI), a selective Ca(2+)-independent PLA2 inhibitor, to determine if PLA2 activity was linked to AT1R recycling. Both quinacrine and CI decreased AP AT1R recycling without affecting BL AT1R recycling. Polarized AT1R in LLC-PKCl4 cells thus display differential rates of endocytosis and recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.

Cadmium-induced nephrotoxicity in rhesus monkeys ( Macaca, mulatta) in relation to protein calorie malnutrition

In this study, we compared results obtained in protein calorie malnourished (PCM) monkeys and controls given Cd 2+ (5 mg Cd 2+/kg body wt./day) orally for 24 weeks. After 16 weeks of Cd 2+ exposure, an indolent renal failure develops in PCM monkeys which resulted in significant increase in urinary excretion of total protein, Cd 2+, Zn 2+ and Ca 2+ as compared to corresponding Cd 2+-treated control group. In isolated proximal tubule brush border membrane vesicles (BBMV), Cd 2+, Zn 2+ and Ca 2+ transport were significantly impaired in Cd 2+-exposed PCM monkeys as compared to Cd 2+-treated controls. The mechanism of higher urinary excretion of Cd 2+, Zn 2+ and Ca 2+ was examined by analyzing the kinetic parameters of transport systems. The kinetic studies of Cd 2+, Zn 2+ and Ca 2+ transport systems in the BBMV preparations of Cd 2+-exposed PCM monkeys exhibited a significant decrease in V max and an appreciable increase in K m as compared to Cd 2+-treated controls. These findings suggested that Cd 2+ treatment of PCM monkeys caused either a decrease in the number of transporters in the brush border membrane or an increase in the number of less active transporters for Cd 2+, Zn 2+ and Ca 2+. Furthermore, brush border membrane-bound enzymes, viz. alkaline phosphatase and leucine aminopeptidase, activities were significantly impaired in Cd 2+-exposed PCM monkeys. Cadmium content in kidney cortex of Cd 2+-exposed PCM monkeys was 3.34-fold higher than Cd 2+-exposed controls. These findings also established that Cd 2+ not bound to metallothionein (MT) was significantly higher in Cd-exposed PCM monkeys, which may be an important determinant in renal toxicity by interacting with sensitive sites in the renal cells and causing renal damage in Cd-exposed PCM monkeys.

ICAM-1 and VCAM-1 in human renal allograft rejection

Light microscopy studies have demonstrated heightened ICAM-1 and VCAM-1 expression in renal allograft rejection in experimental animals and in humans, and administration of ICAM-1 blocking antibodies has been shown to prolong graft survival in nonhuman primates. We used a precise ultrastructural immunogold localization technique to identify the exact sites of expression of ICAM-1 and VCAM-1 in both normal human kidney and in renal allograft rejection. In the normal kidney ICAM-1 is moderately strongly expressed in glomeruli, on the endothelium and parietal epithelium and in the interstitium, on the endothelium of peritubular capillaries, arterioles and small arteries, on fibroblast-like interstitial cells and on the brush border of proximal tubules. In contrast, in normal kidney, VCAM-1 expression is restricted to the parietal epithelium and the basolateral surfaces of a few proximal tubule cells. In allograft rejection, although ICAM-1 expression appears to be increased, its pattern of distribution is similar to that seen in the normal kidney. However, VCAM-I in allograft rejection is widely expressed on the endothelium of peritubular capillaries and arterioles in association with adhesion of mononuclear leukocytes within these vessels. The tubular expression of VCAM-1, although still focal in nature, is increased on the basolateral surfaces in association with lymphocytic infiltration of tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

The Kinetics of the Pathogenic Pronase-Digested Renal Proximal Tubular Antigen and Antibody in Rat Active Heymann Nephritis

We investigated the pathogenesis of active Heymann nephritis in the rat by conducting immunofluorescent and immunoblotting studies of the pathogenic antigen and the autoantibody, and by detecting this antigen-bound IgGs. Rat IgG was detected along the glomerular basement membrane (GBM) and significant proteinuria was observed 6 weeks after the injection of rat pronase-digested tubular brush border antigen. Circulating antibody which bound only to the brush border of proximal tubules of normal rat, appeared 2 weeks after antigen injection. Eluted antibody from nephritic kidney 6 weeks after immunization bound exclusively to the brush border of the proximal tubules of normal rat kidney. Monoclonal antibody against the nephritogenic 0.3 M antigen, which bound exclusively to the brush border in the normal rat, bound to the GBM in a fine granular fashion, as well as to the brush border from nephritic rats, indicating the deposition of nephritogenic 0.3 M antigen in the GBM of nephritic rats. On immunoblotting, both the circulating antibody and eluted antibody obtained from the nephritic kidney 6 weeks after immunization recognized the 0.3 M antigen. This antigen-bound IgG appeared in circulation at 2 weeks, becoming smaller in size at 4 weeks and disappearing 12 weeks after immunization. Thus, it is suggested that active Heymann nephritis in rats was induced by deposition of the circulating 0.3 M antigen-bound IgG complexes in the subepithelial space of GBM.

Reduced proximal tubule angiotensin II receptor expression in streptozotocin-induced diabetes mellitus

Diabetes mellitus is characterized by alterations in the intrarenal renin-angiotensin system, including decreases in glomerular angiotensin II (Ang II) receptor density. Since Ang II regulates proximal tubule transport function, the present studies examined whether diabetes altered expression of proximal tubule receptors. In basolateral membranes from 14 day streptozotocin-induced diabetic rats, specific binding of 125I Ang II was decreased to 53 +/- 8% of control (3.2 +/- 0.5 vs. 1.5 +/- 0.2 fmol/mg protein; N = 7; P < 0.02). Similarly, in proximal tubule brush border membranes from diabetic animals, specific binding was decreased to 63 +/- 11% of control (1.1 +/- 0.2 vs 0.6 +/- 0.1 fmol/mg protein; N = 9; P < 0.05). Concomitant insulin treatment reversed the decrease in specific binding of 125I Ang II to basolateral membranes (109 +/- 26% of control; N = 3) and to brush border membranes (85 +/- 17% of control; N = 6). In order to determine if changes in expression of type-1 Ang II receptors (AT1R) accompanied the changes in binding, quantitative polymerase chain reaction of AT1R mRNA was performed and expressed as the ratio of the amplified AT1R to that of an Msc1/Msc1 internal deletion mutant and normalized to that of beta-actin. In total RNA from proximal tubule suspensions of diabetic animals, AT1R mRNA expression decreased by 38% (21 +/- 3 vs. 13 +/- 2 cpm AT1R/cpm deletion mutant/cpm beta actin/10(6); N = 4; P < 0.0025). Insulin treatment reverted AT1R mRNA expression to control levels (22 +/- 3 cpm AT1R/cpm deletion mutant/cpm beta actin/10(6); P < 0.001 compared to the untreated group).(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the complement regulatory proteins in the normal human kidney

The kidney is an organ where complement-mediated tissue injuries take place by various stimuli. To assess how the kidney is protected from the autologous complement attack, comparative localization of decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20) was studied in the normal human kidney. Specific monoclonal antibodies to DAF, MCP and HRF20 were used for the study. Studies by immunofluorescence and immunoelectron microscopy showed that the distribution of each protein in the kidney was complementary to each other in most parts. MCP and HRF20 were clearly seen in the glomerular capillaries, while DAF was only faintly observed. Juxtaglomerular apparatus was abundant in DAF and MCP but not in HRF20. HRF20 was most strongly expressed in the peritubular capillaries where MCP was not detectable. Basolateral membranes of the proximal tubules and collecting ducts expressed MCP strongly, while there was no expression of DAF in the proximal tubules. Interestingly, both DAF and MCP, which inhibit complement activation at C3/C4 level, were not expressed in the apical portion of the tubular cells including proximal tubule brush border. In contrast, HRF20 was expressed on the apical part of the tubules. Medullary interstitium strongly expressed MCP but not DAF. Based on these observations, we conclude that each segment of the kidney is protected from the complement attack by the different combination of complement regulatory proteins. We speculate that the tubular cells might be fragile when complements are activated inside the tubular lumen, because there is no expression of complement regulatory proteins which inhibit C3 convertase.