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作者：KeithDiPetrillo, BonitaCoutermarsh, FrankA.Gesek作者单位：Department of Pharmacology and Toxicology, DartmouthMedical School, Hanover, New Hampshire 03755 / K+ f" _1 Z) `" d
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【摘要】$ N4 R! R& i/ W/ I
Nephropathy is a major contributor tooverall morbidity and mortality in diabetic patients. Early renalchanges during diabetes include Na retention and renal hypertrophy.Tumor necrosis factor (TNF) is elevated during diabetes and isimplicated in the pathogenesis of diabetic nephropathy. We tested thehypothesis that TNF contributes to Na retention and renal hypertrophyduring diabetes. Rats with streptozotocin-induced diabetes exhibitincreased urinary TNF excretion, Na retention, and renal hypertrophythrough the first 20 days of diabetes. Administration of a soluble TNFantagonist (TNFR:Fc) to diabetic rats reduces urinary TNF excretion andprevents Na retention and renal hypertrophy. TNF stimulates Na uptakein distal tubule cells isolated from diabetic rats, providing apossible mechanism for TNF-induced Na retention. We conclude thaturinary TNF contributes to early diabetic nephropathy and may serve as a valuable diagnostic marker. Furthermore, inhibition of TNF during diabetes may attenuate early pathological changes in diabetic nephropathy. % ^. C# o) ]# f5 O
【关键词】 distal tubule albuminuria sodium retention hypertrophy TNFRFc
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DIABETES IS THE LEADING CAUSE of end-stage renal disease in the United States andEurope ( 24 ). Diabetic nephropathy contributes substantially to overall morbidity and mortality in diabetic patients, either directly or indirectly as a risk factor for cardiovascular disease. Microalbuminuria (defined as urinary albumin-to-creatinine ratio of 300 mg/g) is the earliest clinical sign of diabeticnephropathy ( 24 ) and is associated with an accelerateddecline in glomerular filtration rate in diabetic patients comparedwith normoalbuminuric patients ( 24, 33 ). Microalbuminuriais an independent risk factor for the development of ischemicheart disease ( 4 ) and cardiovascular events (myocardialinfarction, stroke, hospitalization for congestive heart failure, ordeath) ( 11 ). Nephropathy is associated with leftventricular hypertrophy and impaired diastolic relaxation in type Idiabetic patients ( 39 ). Nephropathy also underlies thedevelopment of hypertension in type I diabetic patients ( 24 ). Improved understanding of the early pathologicalchanges in diabetic nephropathy may identify novel clinical markers for detecting early renal disease, as well as elucidate new therapeutic strategies to diminish these changes and reduce the progression toend-stage renal disease.
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) b# s" j @ v$ GThe principal renal alterations that occur during the initial stage ofdiabetic nephropathy are hypertrophy and hyperfunction (see Ref. 23 for review). Renal enlargement is common in diabetic patients ( 3, 21 ) and may predict progression to overtdiabetic nephropathy ( 3 ). Na retention is a manifestationof hyperfunction and is observed in diabetic patients before the onsetof albuminuria ( 29, 41, 45 ). Na retention likelycontributes to the onset of hypertension ( 41, 44, 46 ),which develops from underlying renal dysfunction in type I diabeticpatients ( 24 ). Experimental evidence suggests that Naretention may also contribute to organ hypertrophy. Elevated dietary Nacan lead to renal hypertrophy and induce transforming growth factor- (TGF- ) expression in nondiabetic rats ( 48 ). TGF- hasbeen identified as a hypertrophic factor during diabetic nephropathy(see Ref. 36 for review). Moreover, Na restriction reducesrenal hypertrophy in diabetic rats ( 1 ). Becausehypertension ( 24 ) and renal hypertrophy ( 3, 21 ) are frequently observed in diabetic patients, Na retentionmay represent a significant manifestation of altered renal functionthat occurs during diabetes.' d/ b" M4 u' U- w& i. B' r
9 B3 ?, \1 v+ m( [& DRenal enlargement during early diabetic nephropathy results primarilyfrom tubular hypertrophy ( 34 ). Proximal and distal tubulesare enlarged as a result of hypertrophy and hyperplasia ( 34 ). Early degenerative changes in diabetes occur in thedistal tubule (DT) and precede changes in proximal tubules (PT) andglomeruli ( 47 ). Tubular function is also altered early inthe course of diabetes. Urinary excretion of 1 -microglobulin and Tamm-Horsfall protein (THP), markersof PT and DT dysfunction, respectively, are increased in diabeticpatients before the development of albuminuria ( 35 ).Altered tubular function during diabetes likely contributes to Na retention.
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The pathological events underlying the initial changes ofdiabetic nephropathy are not well defined. Inhibitory monoclonal antibodies against TGF- attenuate kidney enlargement in diabetic mice, illustrating that TGF- participates in renal hypertrophy during diabetes ( 50 ). Thomson et al. ( 43 )proposed that hypertrophy of the PT early in diabetes increasesreabsorption and, subsequently, elevates glomerular filtration rate.They demonstrated that enhanced ornithine decarboxylase activitycontributes to renal hypertrophy and hyperfunction. However, inhibitionof either pathway does not fully reduce renal hypertrophy, suggestingthe existence of additional pathogenic mechanisms.. h, u2 H' I' S7 ~! b
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Tumor necrosis factor (TNF) is elevated during diabetes ( 2, 9, 22, 25 ), and TNF is implicated in the development of diabeticnephropathy ( 7, 18, 27, 42 ). In the present study, wetested the hypothesis that TNF underlies Na retention and renalhypertrophy during diabetes. To test this hypothesis, we administered aspecific inhibitor of TNF to diabetic rats and measured Na balance andwet kidney weight. We also examined the effects of TNF inhibition onthe urinary excretion of TNF and investigated the ability of TNF todirectly stimulate Na uptake in isolated DT cells.
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Animals. Twelve-week-old male Sprague-Dawley rats were purchased from CharlesRiver Laboratories (Wilmington, MA), and diabetes was induced by tailvein injection of streptozotocin (STZ; 50 mg/kg; Sigma, St. Louis, MO)dissolved in 0.9% sodium chloride. Blood glucose levels were measuredbefore and 3-5 days after STZ injection to ensure the onset ofhyperglycemia. Hyperglycemia was also confirmed by measuring urineglucose using Ketodiastix (Bayer, Elkhart, IN) in rats housed inmetabolic cages. Control and diabetic rats were treated with theanti-TNF agent TNFR:Fc, a soluble TNF receptor fusion protein.Subcutaneous TNFR:Fc (2 mg/kg; Immunex, Seattle, WA)injections started 1 day before STZ injection and continued twiceweekly for 20 days.$ b' T9 y7 y' T' w& n* e3 T
/ C1 g' l- j/ ~6 v, iMetabolic cage studies. Animals were housed in metabolic cages to determine whole animal Nabalance. Food intake was determined by carefully weighing the amount offood provided to each rat housed in a metabolic cage. After 24 h,the remaining food was collected. Small particles of chow that fellthrough the floor of the metabolic cage were collected with feces bydesign of the metabolic cage. These small pieces were separated fromfeces, weighed with the remaining food, and subtracted from thestarting weight to determine total food intake per 24 h. Na intakewas subsequently calculated as a percentage of food intake( 49 ) based on the Na content of the chow provided by themanufacturer (Harlan-Teklad, Madison, WI). All animals received thesame lot of rat chow, ensuring consistent Na content in the food.8 T5 _% m+ C0 k% L6 `2 G# I, T, Q
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Water intake and urine output were also measured in animals housed inmetabolic cages. Urine glucose and ketone concentrations weredetermined using Ketodiastix. Urine Na, creatinine, and albumin concentrations were quantified at the clinical laboratory atDartmouth-Hitchcock Medical Center (Lebanon, NH). Na balance wascalculated as the difference between Na intake (determined as apercentage of food intake) and Na output (urine Na concentration × urine volume). This method has been used successfully to determineNa balance in rats ( 49 ). For albumin measurements, astandard curve ( r 2 = 0.99) was generatedusing rat albumin (Sigma) with a lower limit of 5 µg/ml. At theconclusion of metabolic cage experiments ( day 20 ), kidneyswere excised and weighed to determine wet kidney weight, an establishedindex of renal hypertrophy during diabetes ( 43, 50 ).7 k1 G+ ]. z' j/ K8 m3 e1 y% _
- n" v/ o9 e- f; U7 i9 zDT cell isolation. An in situ enzyme digestion procedure previously described was used toisolate PT and DT cells from diabetic and control rat kidneys( 16 ). Briefly, kidneys were perfused with a mixture ofcollagenase (1 mg/ml) and hyaluronidase (3 mg/ml) and subsequently excised. The kidneys were sliced longitudinally, and the renal medullawas removed. The remaining renal cortex contained primarily PT and DTsegments. The cortex was sliced and incubated with collagenase (1 mg/ml) for 10 min at 37°C. The mixture was filtered through wire meshto remove tissue remnants, and the cells were incubated with a magneticparticle coupled to a primary antibody recognizing THP (Polysciences,Warrington, PA) ( 32 ). The flasks were affixed to a magnetto selectively remove THP-containing DT cells, comprising cells fromthe thick ascending limb and distal convoluted tubule ( 30, 40 ). The remaining cells represent primarily PT cells. DT cellsisolated by this procedure exhibited characteristic DT Na and Catransport pathways, including calcitonin- and parathyroid hormone-stimulated Ca transport ( 10, 13 ), amiloride- andchlorothiazide-inhibitable Na uptake ( 14 ), andphenylephrine-stimulated Na transport ( 12 ). Thespecificity of PT cells isolated by this method was confirmed by thepresence of ethylisopropyl amiloride-inhibitable andphloridzin-sensitive Na uptake ( 15 ).: W- V0 K- T# I# n# X* H
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Na uptake assay. To quantify Na entry, uptake of 22 Na into isolated DT cellswas measured using a rapid filtration technique described in previous reports ( 15 ). DT cells were incubated with recombinant ratTNF (Research Diagnostics, Flanders, NJ) at 37°C before addition of 22 Na. Entry of 22 Na into cells was terminatedafter 1 min by rapid addition of ice-cold isosmotic buffer, and cellswere filtered onto Whatman GF/C filters using a Millipore 12-portmanifold. Nonspecific binding of 22 Na to filters and cellswas determined and subtracted to calculate uptake. Uptake measurementswere normalized to total cellular protein quantified by the Piercebicinchoninic acid protein assay (Pierce, Rockford, IL), using BSA as a standard.
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Detection of TNF and THP. Urine TNF levels were measured by ELISA using the OptEIA rat TNF set(Pharmingen, San Diego, CA) according to the accompanying protocol,using recombinant rat TNF as a standard. Urinary THP was also measuredby ELISA. Briefly, Nunc Maxisorp 96-well ELISA plates were coated withgoat anti-human THP antibody (ICN, Irvine, CA; 1:1,000) diluted incoating buffer (0.1 M carbonate, pH 9.5). Urine samples were incubatedat room temperature for 3 h, each well was washed, and horseradishperoxidase-conjugated sheep anti-human THP antibody (Biogenesis,Kingston, NH) was added for 1 h. A3,3',5,5'-tetramethylbenzidine-based substrate set (Pharmingen)was used for color development. A standard curve using rat THP(Biogenesis) was generated for each experiment.' `! H) b, y2 B
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Immunofluorescence and confocal microscopy. DT cells were grown on Transwell filters to maintain membranepolarization (Costar, Cambridge, MA) for confocal microscopy or onglass coverslips for fluorescence microscopy. The cells were fixed withmethanol-free paraformaldehyde in phosphate-buffered saline solution.DT cells were incubated overnight at 4°C with primary antibodies toTNF type 1 (TNFR1) or type 2 receptor (TNFR2; Santa Cruz Biotechnology,Santa Cruz, CA). DT cells were incubated with secondary antibodycoupled to Alexa 568 (Molecular Probes, Eugene, OR) and treated withProLong Antifade reagent (Molecular Probes). Confocal images werecaptured using a Zeiss microscope attached to a scanning confocalsystem (model MRC-1024, Bio-Rad) followed by three-dimensionalreconstruction using Bio-Rad imaging software. Immunofluorescenceimages were visualized using a fluorescence microscope (OlympusAmerica, Melville, NY).1 M6 Y# [) e, l+ Q, b9 @! y
( |, _; q2 F7 ~* h' P n( uStatistical analysis. Metabolic cage data for each treatment group are presented asmeans ± SE for control ( n = 3), diabetic( n = 8), diabetic TNFR:Fc ( n = 9), and control TNFR:Fc ( n = 3). Statisticalsignificance was determined using ANOVA followed by aStudent-Newman-Keuls multiple comparisons test. P significant.
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RESULTS; U/ I1 e+ r9 G# m7 D
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Urinary TNF excretion is increased during diabetes. Renal TNF expression is increased in animal models of diabetes( 26, 42 ). To determine whether renal TNF protein isincreased and released during diabetes, urinary TNF excretion wasmeasured. A significant increase in urinary TNF excretion was observedwithin the first 3 days after induction of diabetes (Fig. 1 ). Elevated urinary TNF excretioncontinued through the first 20 days of diabetes. Urinary TNF excretionwas also measured in diabetic TNFR:Fc rats. TNFR:Fc is a solubleTNF antagonist consisting of two TNFR2 extracellular domains fused tothe Fc portion of human IgG1 ( 5 ). TNFR:Fc binds to solubleTNF and prevents TNF from interacting with its cognate cell surfacereceptors. In diabetic TNFR:Fc rats, TNFR:Fc administrationsignificantly reduced urinary TNF excretion, nearly to control levels,throughout the 20-day study period (Fig. 1 ).
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Fig. 1. Soluble tumor necrosis factor (TNF) antagonist (TNFR:Fc)reduces urinary TNF excretion during diabetes. Urinary TNF was detectedby ELISA and expressed as total TNF/24 h to account for markeddifferences in urine volume between control and diabetic rats. Valuesare means ± SE of 3 control (Cont) rats, 8 diabetic (Diab) rats,and 9 diabetic rats treated with TNFR:Fc (Diab TNFR:Fc).* P
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, ]. l/ P8 @$ n2 t+ \$ ?TNFR:Fc does not alter the metabolic profile of diabetic rats. To accurately determine the role of TNF in the development of diabeticnephropathy, we first determined whether TNF inhibition alleviated themetabolic abnormalities of diabetes, because improved metabolic controlcould prevent the development of diabetic nephropathy ( 8, 24, 37 ). Urine glucose was significantly enhanced in diabetic ratsfrom day 3 through day 20 (Fig. 2 A ). TNFR:Fc therapy did notaffect urinary glucose at any day of the study period. The urinaryketone concentration in the diabetic group was not significantlyincreased above control until days 17 and 20 (Fig. 2 B ). TNFR:Fc administration did not alter ketonuria indiabetic rats throughout the first 17 days of diabetes but didsignificantly reduce ketonuria at day 20 (Fig. 2 B ). Water intake, food intake, and urine output wereincreased equally in the diabetic and diabetic TNFR:Fc groups(Fig. 2, C-E ). Although food intake increased, diabeticrats lost weight during the initial week of diabetes and subsequentlymaintained stable body weight throughout the rest of the study period(Fig. 2 F ). TNFR:Fc treatment did not alter body weightchanges during diabetes. Urine glucose and ketones were virtuallyundetectable in control rats. Water intake, urine output, and foodintake remained stable in control rats as they progressively gainedbody weight throughout the 20-day study period. TNFR:Fc administrationdid not affect these metabolic indexes in control TNFR:Fc rats.
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Fig. 2. TNFR:Fc does not affect urine glucose concentration ( A ),ketonuria ( B ), water intake ( C ), urine output( D ), food intake ( E ), or body weight[ F, expressed as percentage of body weight on day0 (baseline)]. Values are means ± SE of 3 Cont andCont TNFR:Fc, 8 Diab, and 9 Diab TNFR:Fc rats.* P- B; U6 m. K9 o& I5 j) U1 S9 h
! @: U ]- s9 k/ U( z+ k' z4 w4 p0 _TNFR:Fc reduces Na retention in diabetic rats. To investigate the role of TNF in the maintenance of Na homeostasisduring diabetes, we measured dietary Na intake and urinary Na output indiabetic and diabetic TNFR:Fc rats. Dietary Na intake remainedstable in control and control TNFR:Fc rats throughout the 20-daystudy period (Fig. 3 A ). Incontrast, dietary Na intake was increased above control equally indiabetic and diabetic TNFR:Fc rats starting at day 7 and continuing through day 20 (Fig. 3 A ).
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6 i6 U. D. F. U- v$ {1 s" |# pFig. 3. TNFR:Fc augments urinary Na output and reduces Naretention during diabetes. A and B : effect ofTNFR:Fc treatment on dietary Na intake and urinary Na output in controland diabetic rats. C : effect of TNFR:Fc therapy on Nabalance in control and diabetic rats. Na balance was calculated as thedifference between dietary Na intake and urinary Na output forindividual rats on each day. Values are means ± SE of 3 Cont andCont TNFR:Fc, 8 Diab, and 9 Diab TNFR:Fc rats.* P
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. l4 K7 s; R/ e% dUrinary Na output was constant throughout the study period in controlrats (Fig. 3 B ). TNFR:Fc therapy did not alter urinary Naoutput in control TNFR:Fc rats. Na output was elevated in diabetic rats compared with controls, probably because of markedly increased dietary Na intake in diabetic rats (Fig. 3 B ).TNFR:Fc administration significantly enhanced urinary Na outputin diabetic TNFR:Fc rats throughout the first 17 days of thestudy period (Fig. 3 B ). The ability of TNFR:Fc to augmenturinary Na excretion during diabetes was independent of therapeuticeffects on the metabolic state of the animals, because TNFR:Fc did notalter metabolic indexes of diabetes through day 17 (Fig. 2 ).& H, _6 [) T- x9 G) n
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After quantifying dietary Na intake and urinary Na output, wecalculated the difference between Na intake and output to determine Nabalance. Na balance was stable in control and control TNFR:Fc rats during the study period (Fig. 3 C ). Diabetic ratsexhibited significant Na retention compared with control as early as day 7 of diabetes and continued to retain Na through day 20 of the study. TNFR:Fc treatment significantly reducedNa retention in diabetic TNFR:Fc rats (Fig. 3 C ).
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TNFR:Fc reduces renal hypertrophy during diabetes. Experimental studies demonstrate that excess Na contributes to renalhypertrophy ( 1, 48 ). Increased kidney size is common indiabetic patients ( 3, 21 ). On the basis of our findings that TNF inhibition reduced Na retention during diabetes, we tested thehypothesis that TNF inhibition would reduce renal hypertrophy duringdiabetes. As an index of renal hypertrophy, we measured average wetkidney weight from TNFR:Fc-treated and untreated control and diabeticrats. Wet kidney weight is an accurate measure of renal hypertrophyduring diabetes ( 43, 50 ). Kidney weight was significantlyincreased in diabetic rats ( day 20 ) compared with controls(Fig. 4 ). TNFR:Fc did not affect wetkidney weight in control TNFR:Fc rats. In contrast, TNFR:Fctherapy significantly reduced renal hypertrophy in diabetic TNFR:Fc rats (Fig. 4 ).
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Fig. 4. TNFR:Fc prevents renal hypertrophy during diabetes.Effect of TNFR:Fc treatment on wet kidney weight is shown in controland diabetic ( day 20 ) rats. Values are means ± SE of 3 Cont and Cont TNFR:Fc, 8 Diab, and 9 Diab TNFR:Fc rats.* P+ O7 L) e6 A* t& s. y% ] Q ^; X% w
- G7 H$ P" C" mNa retention and renal hypertrophy precede albuminuria. The development of microalbuminuria, defined as urinaryalbumin-to-creatinine ratio of 30-300 mg/g ( 24 ), isused as a marker of glomerular dysfunction during diabetes( 24 ). Albuminuria is the earliest clinical marker ofdiabetic nephropathy and correlates with progression to end-stage renaldisease ( 24, 33 ) and cardiovascular morbidity andmortality ( 4, 11, 39 ). We measured urinary albumin-to-creatinine ratios on days 10 and 20 after the onset of diabetes to determine whether Na retention and renalhypertrophy precede albuminuria during diabetes. Control rats did notexhibit measurable urinary albumin excretion on day 10 or 20. None of the diabetic rats displayed microalbuminuria on day 10, at which point Na retention was established. On day 20, when diabetic rats exhibited significant renalhypertrophy, only two of eight diabetic rats exhibited microalbuminuria(albumin-to-creatinine ratios of 191.3 and 146.0 mg/g), whereas theremaining diabetic rats were normoalbuminuric.: V9 p, e6 c C" h
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TNF stimulates Na uptake in DT cells isolated from diabetic rats. Tubular structure and function are altered in the early stages ofdiabetes ( 31 ). To determine whether TNF induces Naretention by stimulating tubular Na uptake, we measured Na uptake in PT and DT cells freshly isolated from control and diabetic rats. Basal Nauptake in PT cells was unchanged after 10 days of diabetes (Fig. 5 A ). Acute exposure to 10 or100 ng/ml TNF did not stimulate Na uptake in PT cells isolated fromcontrol or diabetic rats (Fig. 5 A ).: ~7 P- {* ~1 V8 v7 f" |
7 j5 u6 h; F0 j5 `& n6 |Fig. 5. TNF stimulates Na uptake selectively in distal tubule(DT) cells during diabetes. A : active Na transport inproximal tubule cells isolated from control and diabetic ( day10 ) rats (Diab 10d). Cells were exposed to TNF for 15 min. Valuesare means ± SE of triplicate determinations of each experimentalcondition for 3 rats. B : basal and TNF-stimulated (10 ng/ml;15 min) Na uptake in DT cells isolated from control and diabetic rats. C : TNF-stimulated Na uptake is selective in DT cells fromdiabetic rats over a wide dose range. Values are means ± SE fortriplicate determinations of 5 diabetic rats on day 20 (Diab20d) or 6 Cont and Diab 10d rats. * P # P
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DT function is important for the regulation of Na homeostasis and isaltered during diabetes ( 31, 35 ). We confirmed DT tubuledysfunction in our animal model of diabetes by measuring urinary THPexcretion by ELISA. Diabetic rats exhibited a significant increase inTHP excretion from day 3 through day 20 of thestudy period (data not shown). Because DT function was altered early indiabetes, we hypothesized that TNF induced Na retention by stimulatingNa transport in DT cells. To test this hypothesis, we isolated DT cellsfrom control rats or after 10 or 20 days of diabetes and quantifiedTNF-stimulated Na uptake. Basal rates of Na transport were unaltered inDT cells after 10 or 20 days of diabetes (Fig. 5 B; controlbasal rate = 256.6 ± 5.7 nmol · min 1 · mgprotein 1 ). TNF had no effect on Na transport in DT cellsfrom control rats. In contrast, TNF stimulated Na uptake in DT cellsisolated from diabetic rats after 10 and 20 days. This differentialactivation of Na transport by TNF occurred over a wide concentrationrange. TNF at 10 ng/ml stimulated Na uptake in DT cells from diabetic rats, whereas DT cells from control rats responded minimally to TNFconcentrations up to 1 µg/ml (Fig. 5 C ).
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+ Z) w$ M$ y1 {3 N, b( T PExpression and cellular localization of TNF receptors. TNF modulates cellular function through distinct membrane receptors,TNFR1 and TNFR2 (see Ref. 38 for review). To examine theexpression and cellular localization of TNF receptors in DT cells,immunofluorescence and confocal studies were conducted with antibodiesspecific for TNFR1 and TNFR2 ( 6 ). In immunofluorescence studies using nonpolarized DT cells grown on glass coverslips, TNFR1and TNFR2 were observed in DT cells isolated from control rats (Fig. 6 ). TNFR1 and TNFR2 were also expressedin DT cells isolated from diabetic rats (data not shown). TNF receptorlocalization was examined by confocal microscopy using DT cells grownon filters to maintain polarity of apical and basal membranes. DT cellsare polarized in vivo, such that the apical membranecontacts the urine and the basal membrane faces the blood. In confocalimages, TNFR1 was clearly localized to the apical portion of DT cells, whereas TNFR2 was expressed diffusely throughout the cell (Fig. 6 ).Membrane localization of either TNF receptor subtype was not alteredduring diabetes (data not shown).4 h9 p0 e- ~3 g$ C
5 Z5 @" X, A+ b4 u# K2 \0 _Fig. 6. DTcells express TNF type 1 and 2 receptors (TNFR1 and TNFR2). TNFreceptors were visualized by immunofluorescence ( top ) andconfocal microscopy ( bottom ). Nonpolarized cells weregrown on glass coverslips for immunofluorescence images. Confocalimages represent Z -sections through polarized cells grown onfilters.1 B# O3 m8 h- l+ Q# h4 U' _
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The focus of this study was to elucidate the role of TNF in thedevelopment of early diabetic nephropathy. Our results implicate TNF asan important factor underlying diabetic nephropathy. Diabetic ratsexhibited enhanced urinary TNF excretion, Na retention, and renalhypertrophy. Administration of the TNF antagonist TNFR:Fc significantlydecreased urinary TNF and prevented Na retention and renal hypertrophy.These observations imply that enhanced TNF production and release arenecessary to induce Na retention and renal hypertrophy in diabetes.
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The mechanism of TNF-induced Na retention during diabetes is unclear,although several findings suggest that augmented tubular Na transportunderlies Na retention in response to elevated TNF. First, TNF directlystimulated Na uptake in DT cells isolated from diabetic rats. Second,TNFR:Fc treatment augmented urinary Na output during diabetes comparedwith untreated diabetic rats, despite equivalent Na and waterintake and urine output, implying that TNFR:Fc inhibited tubular Natransport. Finally, TNF was undetectable in serum samples from any ofthe groups, whereas urinary TNF was markedly increased in diabeticrats, suggesting that TNF actions were mediated from the lumen of thenephron. Taken together, these findings implicate enhanced tubular Natransport as the mechanism underlying TNF-induced Na retention.# ~8 C e$ G: F7 K$ G& m' z% H
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The role of DT cells underlying Na retention during diabetes isintriguing. Our data show that DT cells are sensitized to the acuteeffects of TNF on Na transport during diabetes. TNF does not activateNa uptake in DT cells isolated from control rats, but TNF significantlyincreases Na transport in DT cells from diabetic rats. The factor(s)leading to DT sensitization during diabetes is unclear but may includeincreased urinary TNF or glucose excretion. Second, DT cells normallyreabsorb only a small percentage of the filtered Na load but arecritical for the fine regulation of Na homeostasis. Our findings showthat perturbations in DT Na transport may induce profound changes in whole animal Na retention. However, TNFR:Fc treatment does not fullyreverse Na retention in diabetic rats, which suggests that additionalmechanisms may in part contribute to Na retention. The ornithinedecarboxylase-mediated increase in PT Na reabsorption during diabetesmay contribute to net Na retention ( 43 ). Alternatively, elevated urinary glucose concentrations may enhance proximal tubule Nareabsorption via constitutively expressed Na-glucose transporters.
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TNF contributes to Na retention and renal hypertrophy in the earlystages of diabetes; however, the mechanisms for TNF actions have notbeen determined. Our experiments do not fully differentiate betweenrenal hypertrophy induced by direct TNF actions and effects secondaryto Na retention. High-Na feeding can induce renal TGF- expressionand hypertrophy in rats ( 48 ). In theory, the hypertrophic effects of TNF during diabetes could be mediated by Na retention, withsubsequent TGF- effects. Experiments to inhibit Na retention withoutaffecting urinary TNF excretion in diabetes are needed to distinguishbetween these possibilities. At the cellular level, TNF-stimulated Nauptake could be mediated by bumetanide-sensitive Na-K-2Clcotransporters in thick ascending limb cells or by amiloride-sensitive epithelial Na channels or thiazide-inhibitable Na-Cl cotransporters indistal cortical tubule cells. In other studies, we show that TNF-stimulated Na uptake was inhibited by amiloride, implicating theepithelial Na channel as the Na transport mechanism underlying TNF-stimulated Na uptake in DT cells (unpublished observations).; G6 h+ t" _5 @
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Elevated urinary TNF correlated with Na retention and renalhypertrophy, and reduction of urinary TNF with TNFR:Fc therapy ameliorated Na retention and hypertrophy. Serum TNF was undetectable inall the treatment groups, implicating urinary TNF in these early renalchanges during diabetes. The finding that TNF receptors localize to theapical portion of DT cells further supports the theory that urinary TNFregulates renal function during diabetes. Although urinary TNF appearsto be a critical mediator of diabetic nephropathy, the source ofurinary TNF remains to be elucidated. Nakamura et al. ( 26 )demonstrate increased TNF mRNA in glomeruli during diabetes. PT cellsare also capable of producing TNF ( 19, 20 ). TNF secretionfrom PT segments could subsequently affect DT function in a paracrinemanner. TNF could also be produced in DT cells and act on nearby DTcells in an autocrine fashion. PT cells exhibited enhanced TNF mRNA andprotein expression after 10 days of diabetes in preliminary experimentsin our laboratory, whereas TNF mRNA and protein expression in DT cellswas unchanged (unpublished observations). These findings implicate thePT as an important source of urinary TNF and a paracrine regulator of DT function during diabetes. We propose that therapies targeted atreducing PT TNF production should decrease urinary TNF excretion and replicate the therapeutic effects of TNFR:Fc.Pentoxifylline, an agent that inhibits TNF synthesis, may be effectivein this regard.& \/ s8 f; X5 L1 l
8 z2 _2 }, V3 LOur results obtained using an animal model of type I diabetes correlatewell with clinical observations of Na retention ( 29, 45 )and renal hypertrophy ( 3, 21 ) in diabetic patients beforethe onset of microalbuminuria. Navarro et al. ( 28 )implicate TNF in the development of nephropathy in diabetic patients by demonstrating that the TNF inhibitor pentoxifylline reducesalbuminuria. Pentoxifylline also decreases microalbuminuria and overtproteinuria in type II diabetic patients ( 17 ). Thepresence of urinary albumin is the earliest clinical marker of renaldysfunction in diabetic patients ( 24 ). Our finding thaturinary TNF excretion was significantly increased early in diabetesbefore the development of microalbuminuria suggests that TNF may be auseful marker of renal dysfunction during diabetes. Elevated urinaryTNF may be a clinically relevant marker for early detection ofnephropathy on the basis of evidence we provide that TNF participatesin renal hypertrophy, which is a predictor of the severity of renaldysfunction in diabetic patients ( 3 ).
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In summary, these findings provide novel information regarding the roleof TNF in early diabetic nephropathy by examining the effects of TNF onisolated kidney cells and utilizing a specific TNFinhibitor (TNFR:Fc) in vivo. We demonstrate that TNF is a critical factor underlying the early pathological changes during diabetic nephropathy, including Na retention and renal hypertrophy. Urinary TNF excretion is increased during diabetes, and reduction ofurinary TNF by administration of TNFR:Fc prevents Na retention andhypertrophy. On the basis of these findings, urinary TNF excretion islikely a valuable diagnostic marker for renal dysfunction during theearly stages of diabetes. Our finding that increased urine TNFexcretion precedes proteinuria is particularly important, becauseproteinuria is the earliest clinical marker of renal dysfunction duringdiabetes. Furthermore, inhibition of TNF may represent a noveltherapeutic target for preventing the progression of diabetic nephropathy.
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ACKNOWLEDGEMENTS7 X; b$ u* m4 o3 z, K, [ r
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We thank Dr. B. Stanton [Dartmouth Cystic Fibrosis CellBiology/Cell Culture Core (STANTO97RO)] for scientific and technical support; we also thank K. Picha (Immunex) for helpful discussions andfor providing TNFR:Fc.
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