Tubular Fraction Research Articles

A Novel Approach for Assessing Mitochondrial Respiration in Freshly Isolated Glomerular and Tubular Fractions from Whole Mouse Kidney

Intro: Understanding mitochondrial function within specific renal compartments is crucial for unraveling the intricate metabolic processes underlying kidney physiology and pathophysiology. In this study, we present a novel method for accurately measuring mitochondrial respiration in freshly isolated glomerular and tubular fractions derived from dissected kidneys. Methods: Employing a differential adhesive sieving technique, we isolated glomerular and tubular fractions from freshly harvested whole kidneys (n=2) and subsequently utilized a standard substrate-inhibitor titration protocol to assess mitochondrial respiration in the Oroboros Oxygraph O2K (Oroboros Instruments, Innsbruck AT). Results: Notably, our findings revealed distinct state 3 respiration rates in various fractions, with the 70 μm tubule fraction exhibiting a rate of 2133.029 pmol/sec/mgprotein, the 40 μm tubule fraction displaying a rate of 522.4116 pmol/sec/mgprotein, the first glomerular fraction demonstrating a rate of 407.7911 pmol/sec/mgprotein, and the second tubular fraction manifesting a rate of 362.3286 pmol/sec/mgprotein. Moreover, these fragments exhibited differential substrate preferences, as the addition of succinate increased state 3 respiration by ~90% in the 70 μm tubule fraction. In contrast, succinate increased respiration by ~30% in the 40 μm tubule and glomerular fractions. Discussion: These ongoing experiments provide compelling evidence supporting the feasibility and reliability of our approach in quantifying mitochondrial respiration within isolated renal fractions. Our methodology not only enables precise measurements of mitochondrial function in distinct nephron segments but also lays the groundwork for further investigations into the physiological and pathological implications of mitochondrial dynamics in kidney health and disease. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensin II type 2 receptor and angiotensin-converting enzyme 2 mediate ischemic renal injury in diabetic and non-diabetic rats

AimDepressor arm of the renin-angiotensin system (RAS) exerts reno-protective effects in chronic kidney diseases like diabetic nephropathy. However, same is still elusive under AKI and hyperglycaemia comorbidity. Hence, the present study delineates the role of angiotensin-II type 2 receptor (AT2R) and angiotensin-converting enzyme 2 (ACE2) in AKI under normal and hyperglycaemia condition. MethodsNon-diabetic (ND) and Streptozotocin-induced diabetes mellitus (DM) rats were subjected to ischemic renal injury (IRI). Rats underwent IRI were treated with an AT2R agonist, C21 (0.3 mg/kg/day, i.p.) or ACE2 activator, Dize, (5 mg/kg/day, p.o.) either alone or as combination therapy. Renal histopathology and immunohistochemistry, proximal tubular fraction isolation, ELISA, immunoblotting and qRT-PCR were performed for subsequent analysis. Key findingsRats subjected to IRI displayed an increase in plasma ACE, AT1R, AT2R, Ang II, and reduction in ACE2, Ang-(1–7) expressions, with augmented renal inflammation and apoptosis. These changes were more prominent in diabetic rats with IRI. Co-administration of C21 and Dize augmented ACE2, Ang-(1–7), AT2R and MasR expressions, and attenuated tubular injury in both DM and ND rats. ConclusionWe demonstrated that pharmacological activation of AT2R and ACE2 protects DM and ND rats from IRI by preventing oxidative stress, inflammation and apoptosis-mediated tubular damage.

P1087New method for determination of tubular membrane capacity in intact cardiomyocytes

Introduction: Transverse tubular system (TTS) plays a key role in the fast transmission of action potential from the cell surface to the depth of cardiomyocytes, thus, near to calcium stores and contractile machinery. The function of TTS is highly dependent on the presence of transport proteins whose distribution considerably differs in TTS and surface cell membrane. Knowledge of TTS capacity is required for the proper analysis of TTS function. Determination of TTS capacity and its contribution to the total cell capacity may be done by measurement of the membrane capacity before and after the so called detubulation, i.e. detachment of TTS caused by osmotic shock. However, this method is irreversible and, moreover, may result in wrong data due to incomplete TTS detachment. Purpose: The aim of this work was to suggest an alternative method for determination of TTS capacity that preserves the measured cardiomyocyte intact and, thus, enables repetitive measurements in the same cell. Method: It is impossible to distinguish TTS capacity and surface membrane capacity during measurement of the cell membrane capacity in normal conditions because both membrane systems are connected closely one to another. The newly developed method is based on the alternative electrical cell circuit with two membrane systems separated by electrical resistance of TTS. Transient application of isotonic sucrose solution (ISS) on the measured cell causes a significant increase of TTS resistance and, therefore, electrical separation of the two systems. Analysis of current responses to the imposed rectangular voltage pulses (by a new quantitative model) then enables to reveal capacity of the surface membrane and TTS separately. In this work, the membrane capacities were determined on enzymatically isolated rat ventricular myocytes. Results: After ISS application, a transitory phenomenon was observed. It was registered by changes of the membrane current at the imposed constant voltage. The analysed parameters gradually changed, first only the surface membrane parameters and, with a delay, also TTS parameters. It enabled to visualize the process of solution diffusion into TTS by a newly suggested quantitative model. Capacities of TTS and surface membranes (CTTS = 68.0±4.8 pF a CS = 99.8±3.9 pF, respectively; n = 16) were determined in the steady-state from approximation of the responses to rectangular pulses by the sum of two exponential waveforms. Ratio of both surfaces was determined as SS/STTS = 0.40±0.02, assuming the same specific capacity. Conclusion: The newly suggested method leaves the measured cell intact and allows repetitive measurements. Determined value of the tubular capacity fraction (Ct/Cm = 0.43) falls into the range of published data obtained by various methods (0.21-0.64). Recent estimates are based on the model of cell membrane with concentrated parameters. Further improvement of this method may be achieved after implementation of TTS description as a system with distributed parameters.

Infertility in Fabry’s disease: role of hypoxia and inflammation in determining testicular damage

Fabry’s disease (FD) is a genetic X-linked systemic and progressive rare disease, which is characterized by the accumulation of glycolipid bodies (GB) into the lys- osomes of almost all cell types and consequently by a multiform clinical picture. Here we studied testicular biopsies of a 42 ys old FD patient, presenting infertility with reduced number of spermatozoa and preserved sexual activity. Testicular biopsies have been analyzed by optical microscopy (OM) and transmis- sion electron microscopy (TEM). OM, showed a severe involvement of testis inter- stitium blood vessels with reduced or closed lumen, an increased of connective tis- sue and a substantial thicketing of peritubular region. TEM, showed that GB were abundant in vessel wall cells and in myofibroblast of peritubular region. In contrast with literature reports, Leydig cells were constantly unaffected by GB accumulation showing well preserved ultrastructural organization. On the contrary, tubular cells, although not affected by GB accumulation, appeared severely damaged. These data led us to hypothesize that diffusion of oxygen and nutrients from blood to tubules could be impaired. To test this hypothesis we explored, by immunofluorescence (IF) and molecular biology (MB) coupled to laser capture micro-dissection (LCMD), the activation of HIF/NFkB pathway. IF showed increased signal for HIF1a in all stromal components, while it appeared almost absent in seminipherous tubules. On the contrary, NFkB fluores- cence was evident in tubules. mRNA of tubular and interstitial tissue fractions, separately extracted by LCMD, confirms that HIF1a and hypoxic-related genes such as alarmin recepters (RAGE, TLR4) were overexpressed in the interstitial cells. At the same time, NFkB and a number of proinflammatory genes such as HMOX1, PTGES, SAA1-SAA2 were up- regulated in the tubule microenviroment. Taken together, these results suggest that the GB accumulation in interstitium, reducing vessel lumen and increasing the distance between vessel and tubular cells, leads to chronic progressive hypoxia. Hypoxia has two effects: 1)Necrosis of cells more distant from vessels, especially germinative epithelium and Sertoli cells, releas- ing alarmins; 2)Adaptation to low levels of O2, with activation of HIF1a. In both cas- es a strong activation of NFkB occurs that trigger a inflammatory response (IR). We suggest a role for the IR activation in determining intratubular cells damage and con- sequently, infertility in FD.

Introduction to the HESI‐Sponsored Inhibin Consortium

The existence of a compound from the tubular fraction of the testis which acts on the pituitary was first proposed in writing by McCullagh in 1932. This was inferred by observing the differential effects on the prostate and the pituitary when animals were administered water-soluble or ether-soluble extracts of testis. Because the water-soluble extract inhibited pituitary hypertrophy, he dubbed this moiety inhibin (his androitin for the ether-soluble actor later became testosterone). The early literature has been nicely reviewed (Ying, 1988). The early assays for inhibin were bioassays (e.g., Steinberger and Steinberger, 1976), later superseded by the more-sensitive radioimmunoassays, and now ELISAs. Inhibin has been used for several years as a clinical tool to help diagnose and categorize male infertility patients (e.g., Pierik et al., 1998; reviewed in Lockwood 2004, Adamopoulos and Koukkou, 2010, and Anderson and Sharpe, 2000). There has been a robust literature on various aspects on the physiology of the hormone, for example, trying to discern what controls circulating levels of Inhibin B in males (e.g., Ramaswamy et al., 1999), or describing the relationship between Inhibin and FSH (e.g., von Eckerdstein et al., 1999; Kamischke et al., 2001), or determining the impact of different germ cell populations on circulating Inhibin B levels (e.g., Pineau et al., 1989; Allenby et al., 1991; Clifton et al., 2002), or outlining Inhibin B’s role in development (Byrd et al., 1998) or evaluating its use as an epidemiologic marker (e.g., Ellingsen et al., 2007). There are numerous excellent reviews on this subject, a few of which are Meachem et al. (2001), de Kretser et al. (2004), and Stewart and Turner (2005). Because the large interand intraindividual variability makes sperm count and semen studies quite insensitive (e.g., Schrader et al., 1988), toxicologists and epidemiologists have long been looking for a more sensitive (i.e., less noisy and more responsive) biomarker of damage to the seminiferous tubules. Inhibin B seemed to fill this need, and indeed was found to respond to several different types of toxicities, especially over sufficient periods of time (Wallace et al., 1997; Tsatsoulis et al., 1988; Foppiani et al., 1999; Hild et al., 2001; Suescun et al., 2001; rev. in Stewart and Turner, 2005; Tunc et al., 2006).

Potential effects of tooth‐brushing on human dentin wear following exposure to acidic soft drinks

This study used scanning electron microscopy and atomic force microscopy to examine the short-term potential effects of brushing time and the start-time of tooth-brushing after demineralization on primary dentin wear in vitro. Thirty-six noncarious primary central incisors were assigned to 12 experimental groups. Exposure to cola drinks was used to initiate the demineralization process. Three brushing times (5, 15 and 30 s) and four start-times of brushing (0, 30, 60 and 120 min) after an erosive attack were used for the abrasion process. Tooth-brushing the softened dentin surface led to increases in the open tubular fraction and microstructural changes on the dentin surface. Brushing immediately after exposure to cola resulted in the greatest irreversible dentin loss, whereas brushing 60 or 120 min after pretreatment resulted in the least irreversible dentin loss. However, brushing time had no effect on the irreversible loss of dentin wear. Based on these experimental results, tooth-brushing should be performed at least 60 min after consuming a cola drink to achieve the desired tooth cleaning and avoid the introduction of surface lesions on dentin.

Prediction of human pharmacokinetics — renal metabolic and excretion clearance

The kidneys have the capability to both excrete and metabolise drugs. An understanding of mechanisms that determine these processes is required for the prediction of pharmacokinetics, exposures, doses and interactions of candidate drugs. This is particularly important for compounds predicted to have low or negligible non-renal clearance (CL). Clinically significant interactions in drug transport occur mostly in the kidneys. The main objective was to evaluate methods for prediction of excretion and metabolic renal CL (CL(R)) in humans. CL(R) is difficult to predict because of the involvement of bi-directional passive and active tubular transport, differences in uptake capacity, pH and residence time on luminal and blood sides of tubular cells, and limited knowledge about regional tubular residence time, permeability (P(e)) and metabolic capacity. Allometry provides poor predictions of excretion CL(R) because of species differences in unbound fraction, urine pH and active transport. The correlation between fraction excreted unchanged in urine (f(e)) in humans and animals is also poor, except for compounds with high passive P(e) (extensive/complete tubular reabsorption; zero/negligible f(e)) and/or high non-renal CL. Physiologically based in-vitro/in-vivo methods could potentially be useful for predicting CL(R). Filtration could easily be predicted. Prediction of tubular secretion CL requires an in-vitro transport model and establishment of an in-vitro/in-vivo relationship, and does not appear to have been attempted. The relationship between passive P(e) and tubular fraction reabsorbed (f(reabs)) for compounds with and without apparent secretion has recently been established and useful equations and limits for prediction were developed. The suggestion that reabsorption has a lipophilicity cut-off does not seem to hold. Instead, compounds with passive P(e) that is less than or equal to that of atenolol are expected to have negligible passive f(reabs). Compounds with passive P(e) that is equal to or higher than that of carbamazepine are expected to have complete f(reabs). For compounds with intermediate P(e) the relationship is irregular and f(reabs) is difficult to predict. Tubular cells are comparably impermeable (for passive diffusion), and show regional differences in enzymatic and transporter activities. This limits the usefulness of microsome data and makes microsome-based predictions of metabolic CL(R) questionable. Renal concentrations and activities of CYP450s are comparably low, suggesting that CYP450 substrates have negligible metabolic CL(R). The metabolic CL(R) of high-P(e) UDP-glucuronyltransferase substrates could contribute to the total CL.

Defects in the regulatory clearance mechanisms favor the breakdown of self-tolerance during spontaneous autoimmune orchitis

We identified aberrations leading to spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and natural model for autoimmunity. This study provides evidence favoring the view that a malfunction of the clearance mechanisms for apoptotic cell debris arising from imbalances in phagocyte receptors or cytokines acting on Sertoli cells constitutes a major factor leading to breakdown of self-tolerance during spontaneous AIO. Serum anti-sperm antibody titers measured by ELISA reflected spermatogenic activity without causing immune inflammatory responses. Orchitic mink showed excess antibody production accompanied by spermatogenic arrest, testicular leukocyte infiltration, and infertility. AIO serum labeled the postacrosomal region, the mid and end piece of mink sperm, whereas normal mink serum did not. Normal serum labeled plasma membranes, whereas AIO serum reacted with germ cell nuclei. Western blot analyses revealed that AIO serum reacted specifically to a 23- and 50-kDa protein. The number of apostain-labeled apoptotic cells was significantly higher in orchitic compared with normal tubules. However, apoptosis levels measured by ELISA in seminiferous tubular fractions (STf) were not significantly different in normal and orchitic tubules. The levels of CD36, TNF-alpha, TNF-alpha RI, IL-6, and Fas but not Fas-ligand (L), and ATP-binding cassette transporter ABCA1 were changed in AIO STf. TNF-alpha and IL-6 serum levels were increased during AIO. Fas localized to germ cells, Sertoli cells, and the lamina propria of the tubules and Fas-L, to germ cells. Fas colocalized with Fas-L in residual bodies in normal testis and in giant cells and infiltrating leukocytes in orchitic tubules.

Phenotypic expression of factor H mutations in patients with atypical hemolytic uremic syndrome

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.

HCaRG, a Novel Calcium-regulated Gene Coding for a Nuclear Protein, Is Potentially Involved in the Regulation of Cell Proliferation

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Dopamine D3 receptor activation modulates renal function in anesthetized rats.

The renal effects of the D3 receptor agonist R(+)-7-hydroxy-dipropyl-aminotetraline (7-OH-DPAT) were studied in anesthetized Sprague-Dawley rats using standard clearance experiments. 7-OH-DPAT infusion (0.01, 0.1, and 1.0 microg kg(-1) min(-1)) dose-dependently increased glomerular filtration rate (GFR) compared to baseline by a maximum of 20+/-2% while arterial blood pressure was not affected. Heart rate was not altered during the two lower doses of 7-OH-DPAT whereas a slight reduction occurred due to infusion of 1.0 microg kg(-1) min(-1). In contrast, higher doses of 7-OH-DPAT, starting from 3 microg kg(-1) min(-1), markedly influenced systemic hemodynamics. In addition to the hyperfiltration, 7-OH-DPAT (1.0 microg kg(-1) min(-1)) also induced a significant diuresis (27.7+/-4.3 microl min(-1) 100 g(-1) vs 16.2+/-5.4 microl min(-1) 100 g(-1)) and increased both absolute (3.30+/-0.58 micromol min(-1) 100 g(-1) vs 0.95+/-0.26 micromol min(-1) 100 g(-1)) and fractional sodium excretion (2.48+/-0.32% vs 0.79+/-0.19%). These changes in renal function were not modulated by pretreatment with the D2 receptor antagonist S(-)-sulpiride but abolished by the D3 antagonist 5,6-dimethoxy-2-(di-n-propylamino)indane (U-99194A). In coincidence with the action of 7-OH-DPAT on both glomerular and tubular function, reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of D3 receptors in both glomerular and tubular fractions of kidneys taken from Sprague-Dawley rats. These data indicate that D3 receptors in the kidney are involved in the regulation of renal hemodynamics and tubular function.

Creatine metabolism in the seminiferous epithelium of rats. I. Creatine synthesis by isolated and cultured cells.

The testis synthesizes creatine from both arginine and glycine precursors, but when rat testicular tissue is separated into seminiferous tubules and interstitial cells, creatine synthesis occurs only in the tubular fraction. The purpose of the work presented here was to define the locus of creatine synthesis within the seminiferous tubules, by using cell separation and culture techniques to examine synthesis in the Sertoli cells and germ cells. The total creatine content, in the cellular compartment and incubation medium, of Sertoli-germ cell co-cultures and of Sertoli cell-enriched cultures, largely free of germ cells, increased by similar amounts over a 24 h incubation period. Sertoli cell-enriched cultures incorporated radioactivity from L-[guanidino-14C]arginine and [1-14C]glycine into both creatine and its biosynthetic precursor, guanidinoacetic acid. Isolated germ cells did not incorporate radioactivity from L-[guanidino-14C]arginine into either creatine or guanidinoacetic acid when incubated at a similar density and protein concentration under similar conditions. It is concluded that the synthesis of creatine observed in isolated rat seminiferous tubules occurs within the Sertoli cells and not the germ cells.

Effect of lowering serum cholesterol on the composition of surfactant in adult rat lung.

Treatment of rats with 10 mg.kg body wt-1 day-1 4-aminopyrazolo[3,4-d]pyrimidine (APP) for 2 days markedly reduced serum cholesterol and phospholipids. This was associated with large decreases in the principal component of alveolar surfactant, the disaturated phospholipids (DSP), in the lamellar body and in the tubular myelin-rich and -poor alveolar fractions, but with no concomitant change in cholesterol or surfactant protein A. These decreases in DSP were associated with a decrease in the synthesis of surfactant phospholipids. Despite these large changes in composition of alveolar surfactant, we could detect no change in either static or dynamic lung compliance. However, the treatment markedly increased both the minimum and maximum surface tension of the lipid extract of the tubular myelin-rich fraction, as measured by bubble surfactometry. Whereas these changes appeared unimportant in the isolated perfused lung at resting tidal volume, they were associated with edema after an increase in tidal volume. The ability of APP to inhibit phospholipid synthesis selectively makes it a useful tool in investigating the surfactant system.

Ontogenesis of the Nuclear 3,5,3’-Triiodothyronine Receptor in the Rat Testis

The aim of this study was to investigate the ontogenesis of the nuclear T3 receptor among the different cell types in the rat testis from fetuses at the 19th day of gestation and animals 1, 5, 15, 20, and 60 days after birth. Whole testis, tubular fraction, nontubular fractions, and Sertoli cells cultured in vitro or enriched in vivo by irradiation were used. The results demonstrate that high affinity, low capacity T3-binding sites are localized only in Sertoli cells; the binding specificity and affinity (Kd ranges from 0.8 +/- 0.2 to 2.6 +/- 0.4 nM) do not change significantly with the age of the animals and are comparable to those observed for T3 receptors in other mammalian tissues. In the whole testis, the concentration of receptors changes during gonadal development, being maximally expressed in the fetus (154.3 +/- 8.1 fg T3 bound/10(6) nuclei) and from 1 (203.4 +/- 10.9 fg T3 bound/10(6) nuclei) to 5 (185.3 +/- 15.1 fg T3 bound/10(6) nuclei) days of postnatal life, decreasing significantly at 15 and 20 days (65.4 +/- 2.0 and 57.9 +/- 1.9 fg T3 bound/10(6) nuclei, respectively) and being virtually absent in the adult. The same change in receptor concentration was found in Sertoli cells obtained by different techniques. This ontogenetic profile coincides with the pattern of Sertoli cell proliferation and differentiation, thus suggesting a role of thyroid hormones in the regulation of growth and maturation of the somatic cells of the seminiferous epithelium.

Glomerular leukotriene synthesis in Heymann nephritis

The glomerular synthesis of LTB4 was assessed in glomeruli isolated from rats with passive Heymann nephritis (PHN). PHN was induced by a single intravenous administration of proteinuric doses of immune sera raised in sheep against rat brush border tubular fraction Fx1A. At various time points following induction of the disease glomeruli were isolated and LTB4 synthesis was assessed under basal and phospholipase A2 activation conditions. LTB4 was measured by high pressure liquid chromatography and radioimmunoassay and was identified by UV spectroscopy. The role of complement system in mediating glomerular LTB4 synthesis was also assessed in a group of decomplemented rats using cobra venom factor and at various time points following administration of immune serum. Following induction of PHN, enhanced glomerular LTB4 synthesis was observed as early as one hour, peaked at five hours and returned toward control levels over the subsequent four days. The peak in glomerular LTB4 synthesis did not correlate with changes in glomerular neutrophiles or macrophages. A second increment of LTB4 synthesis occurred at the onset of heavy proteinuria (day 5). Complement depletion reduced proteinuria and the enhancement in LTB4 synthesis at day 5 but had no effect at earlier time points. The observations indicate that in non-inflammatory forms of glomerular immune injury the glomerular arachidonate 5-lipoxygenation is enhanced. This phenomenon has no apparent relationship with increased glomerular permeability to protein and may reflect the presence or activation of a leukotriene producing cell following intraglomerular interactions of Fx1A antigen, anti-Fx1A antibody and complement.

Renal Allograft Rejection: Protection of Renal Tubular Epithelial Cells from Lymphokine Activated Killer Cell Mediated Lysis by Pretreatment with Cytokines

Human renal tissue was disaggregated by passage through a stainless-steel mesh and the resultant material was separated on the basis of particle size. The tubular fraction recovered from a 45 microns mesh was cultured and after a period of between 5 and 7 days outgrowing adherent epithelial cells were identified. All of these cells contained cytokeratin and after culture for at least 14 days fewer than 5% showed surface HLA-DR antigens. Cultured renal epithelial cells were rapidly lysed by lymphokine activated killer (LAK) cells which were produced from peripheral blood lymphocytes of normal subjects by incubation for 5 days in the presence of either recombinant interleukin-2 (IL-2) or mixed lymphocyte reaction (MLR) supernatant. Stimulation of the cultured epithelial cells for 5 days with either gamma-interferon or MLR supernatant resulted in 67% +/- 9% (mean +/- SD; n = 8) of the cells showing an upregulation of HLA-DR expression. These treated cells were markedly less sensitive to lysis by LAK cells than the untreated cells. Thus, if renal cell damaging LAK cells are generated from lymphocytes in vivo by the action of IL-2 produced during rejection, then gamma-interferon, which is also produced during rejection, may partially protect the renal cells from such LAK cell mediated damage.

Alpha- and beta-adrenergic receptors in proximal tubules of rat kidney.

Proximal tubules were isolated from the rat kidney by collagenase digestion of the cortical tissue followed by Percoll gradient centrifugation. Microscopic and hormone-stimulated adenylate cyclase activity studies proved the purity of the preparation. [3H]Prazosin, [3H]rauwolscine, and [125I]iodocyanopindolol were used to identify and quantitate respectively the alpha 1-, alpha 2- and beta-adrenergic receptors. Proximal tubular (F4) particulate fraction was compared against other cortical nephron segment (F1, F2) fractions and the total collagenase-digested cortex particulate suspension (Ft). Proximal tubules were enriched in alpha 1- and alpha 2-adrenergic receptors compared with Ft (alpha 1-receptor, 100.4 +/- 4.5 vs. 87.4 +/- 4.9; alpha 2-receptor, 250 +/- 16.2 vs. 185.1 +/- 12 fmol/mg protein). The fractions enriched in glomeruli and distal tubular segments (F1, F2) had relatively low concentrations of alpha 1- and alpha 2-adrenergic receptors. In contrast, beta-adrenergic receptor concentration in the proximal tubules was approximately 25% of that in the Ft fraction and approximately 10% of that in the F1 fraction. Isoproterenol-stimulated adenylate cyclase activities in the different fractions corroborated well with the pattern suggested by the [125I]iodocyanopindolol binding studies. Our results suggest that whole-cortex preparation radioligand binding studies may reflect proximal tubular alpha 1- and alpha 2-adrenergic receptor changes quite well. They may, however, miss or give erroneous impressions about beta-adrenergic receptor changes occurring in different cortical nephron segments.

Physiologic Regulation and Tissue Localization of Renal Erythropoietin Messenger RNA

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

Physiologic regulation and tissue localization of renal erythropoietin messenger RNA

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

Stimulation of adenylate cyclase activity via A 2-adenosine receptors in isolated tubules of the rabbit renal cortex

Adenylate cyclase activity in a tubular fraction obtained from rabbit renal cortex was stimulated by typical adenosine receptor agonists with a rank order of potency NECA (5′-(N-ethyl-carboxamido)-adenosine) (EC 50 = 0.48 μmol/1) R-PIA[(−)N 6(R-phenylisoprophyl)-adenosine] (3.22 μmol.1). The stimulatory effect of NECA was competitively antagonized by 8-phenyltheophylline. Contamination of the tubular fraction with glomeruli and microvessels was less than 2%, as verified by tissue renin determination and could, therefore, be ruled out as being responsible for the observed effect. Tubular A 2-adenosine receptors are probably involved in the control of renal electrolyte secretion and may represent the site of action of methylxanthines.