Munich-Wistar Rats Research Articles

Quantification of Renal Albumin Filtration and FcRn‐Mediated Transcytosis via 2‐Photon Microscopy

The kidney filters and reclaims both ions and macromolecules of various size. Endocytosis is known to play a key role in protein reabsorption by proximal tubule cells (PTC) yet the fate of endocytosed molecules remains unknown. We have utilized and maximized intravital 2‐photon microscopy to optimize the signal from filtered Texas Red – rat serum albumin (TR‐RSA) and follow its endocytosis and intracellular trafficking in PTC compared with fluorescent dextrans and beta‐2‐microglobulin (B2M). Studies in Munich‐Wistar rats (MW), that have surface glomeruli, under optimal conditions using 12‐bit non‐descanned detectors gave background corrected glomerular sieving coefficient (GSC) values of 0.030 +/−0.005. Studies conducted in fasted vs fed MW rats revealed a drop in the GSC to 0.011 ± 0.005 in fasted rats. Endocytosis of RSA by proximal tubules (PT) consistently occurred within minutes after i.v. infusion followed by rapid and prolonged transcytosis of TR‐RSA via both intracellular vesicles and tubular projections at the basolateral‐interstitial junction of PT's. This was not true for filtered fluorescent dextrans or B2M. FcRn was visualized in the apical membrane of PTC with indirect IF and PT: apical membrane preparations were Western blot positive of FcRn. These studies indicate there is physiologic regulation of glomerular permeability and that FcRn mediates the transcytosis of albumin within PTC.

Automated quantification of microstructural dimensions of the human kidney using optical coherence tomography (OCT)

Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can non-invasively provide cross-sectional, high-resolution images of tissue morphology in situ and in real-time. We previously demonstrated that OCT is capable of visualizing characteristic kidney anatomic structures, including blood vessels, uriniferous tubules, glomeruli, and renal capsules on a Munich-Wistar rat model. Because the viability of a donor kidney is closely correlated with its tubular morphology, and a large amount of image datasets are expected when using OCT to scan the entire kidney to provide a global assessment of its viability, it is necessary to develop automatic image analysis methods to quantify the spatially-resolved morphometric parameters such as tubular diameter to provide potential diagnostic information. In this study, we imaged the human kidney in vitro and quantified the diameters of hollow structures such as blood vessels and uriniferous tubules automatically. The microstructures were first segmented from cross-sectional OCT images. Then the spatially-isolated region-of-interest (ROI) was automatically selected to quantify its dimension. This method enables the automatic selection and quantification of spatially-resolved morphometric parameters. The quantification accuracy was validated, and measured features are in agreement with known kidney morphology. This work can enable studies to determine the clinical utility of OCT for kidney imaging, as well as studies to evaluate kidney morphology as a biomarker for assessing kidney's viability prior to transplantation.

Increased tissue acid mediates a progressive decline in the glomerular filtration rate of animals with reduced nephron mass

The combination of an acid-inducing diet and reduced nephron mass is associated with a progressive decline in glomerular filtration rate (GFR) that can be corrected by dietary alkali. Here we determined whether the higher tissue acid content mediates the decline in GFR. Using Munich-Wistar rats we induced sub-total nephrectomy and measured by microdialysis the tissue acid content in the kidney cortex and in the paraspinous muscle. The GFR was lower in the rats with reduced nephron mass at 1 and 13 weeks following subtotal nephrectomy compared to the sham-operated rats. Both groups of rats ate the same acid-inducing casein-based diet and had similar plasma acid-base parameters and net urine acid excretion. However, rats with reduced nephron mass had higher tissue acid content compared to control animals and had a lower GFR at week 13 compared to that measured at week 1. Adding dietary acid to the casein diet led to an even higher tissue acid and lower GFR by week 13. By contrast, adding alkali to the casein diet or placing animals with reduced nephron mass on a soy-based diet led to a lower tissue acid content and no decline in GFR. Animals with reduced nephron mass on a soy-based diet given dietary acid had a higher tissue acid content and a decline in GFR. These studies show that dietary maneuvers that increase the tissue acid content reduce GFR, whereas diets that lower the tissue acid level preserve GFR during chronic kidney failure.

Independent two-photon measurements of albumin GSC give low values

Independent two-photon measurements of albumin GSC give low values

Glomerular sieving coefficient of serum albumin in the rat: a two-photon microscopy study

Recent studies of the sieving of serum albumin in the rat kidney using a two-photon microscope suggested that the glomerular sieving coefficient (GSC) of albumin is 0.034, much higher than earlier micropuncture determinations. In the present study, we critically evaluated the use of the two-photon microscope to measure the GSC of albumin in the Munich-Wistar rat in vivo. The albumin GSC averaged 0.004 (SD 0.004), n = 34 glomeruli, when determined with a Zeiss two-photon microscope system and 0.002 (SD 0.002), n = 5, when determined with an Olympus two-photon microscope system. These values are close to the lower limit of detection of GSC, which we estimate to be approximately 0.001-0.003. We identified several factors that were likely responsible for the higher albumin GSCs reported earlier using two-photon microscopy. These include animal conditions (i.e., low glomerular filtration rate) and failure to recognize the role of out-of-focus fluorescence in contaminating the fluorescence signal from the urinary space of Bowman's capsule. We observed that hypothermia plus dehydration or a low blood pressure led to an increased albumin GSC. High levels of illumination (high laser outputs) resulted in a falsely elevated albumin GSC. Use of external, instead of internal, photodetectors resulted in an exaggerated albumin GSC because of greater collection of out-of-focus fluorescence. In conclusion, the albumin concentration in the glomerular filtrate of the normal rat, determined by two-photon microscopy, is exceedingly low (5-10 mg/dl).

Regression of glomerular injury by losartan in experimental diabetic nephropathy

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowman's capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.

Effect of Total Glucosides of Paeony on the Expression of Nephrin in the Kidneys from Diabetic Rats

Total glucosides of paeony (TGP), extracted from the traditional Chinese herb root of Paeonia lactiflora pall, have been shown to have a therapeutic role in experimental diabetic nephropathy including albuminuria. Recent investigation has identified nephrin, a podocyte-specific transmembrane protein, as a key regulator in the pathogenesis of diabetic albuminuria. The aim of this study was to investigate whether TGP can attenuate albuminuria through prevention of nephrin loss in the experimental diabetic nephropathy. Fifty male Munich-Wistar rats were obtained from the Experimental Animal Center of Anhui Medical University. These rats were divided into 5 groups (n = 10); normal group, control diabetic group, and 3 TGP treated diabetic groups at different concentrations. Diabetes was induced by streptozotocin, and TGP (50, 100, 200 mg/kg) was orally administered to the 3 TGP treated diabetic groups once a day for 8 weeks, respectively. Blood glucose and 24 hour urinary albumin excretion rate (AER) were measured. The expressions of nephrin, tumor necrosis factor-alpha (TNF-alpha), NF-kappaB p65 and 3-nitrotyrosine (3-NT) protein were determined by immunoinfluorescence or Western blot analysis in the kidneys. Elevated AER was markedly attenuated by TGP treatment in diabetic rats. There was a finely dotted linear epithelial staining of nephrin in normal group glomeruli. In contrast, the staining of glomeruli from untreated diabetic rats was attenuated, more diapersed, and clustered. This diabetic-induced loss of glomerular nephrin expression was prevented in a large degree in TGP-treated diabetic rats. Western blot analysis showed that the expression of nephrin protein was reduced in the kidneys of diabetic rats, but significantly increased in the TGP treatment groups. The expressions of TNF-alpha, NF-kappaB p65 and 3-NT protein were significantly increased in the kidneys of diabetic rats, which were all significantly inhibited by TGP treatment. Our results showed that TGP could decrease AER in diabetic rat, and that its mechanism may be at least partly correlated with upregulation of the expression of nephrin in the kidney.

Impaired Tubular Uptake Explains Albuminuria in Early Diabetic Nephropathy

Understanding the pathogenesis of albuminuria in diabetic nephropathy is important to improve methods for early diagnosis and treatment. In this study, we addressed whether albuminuria in diabetes results from altered glomerular filtration and/or altered processing of filtered albumin by the proximal tubule. Type 1 diabetic Munich Wistar rats developed albuminuria after 12 wk of diabetes. Intravital two-photon microscopy revealed similar glomerular permeability in the diabetic and control animals, assessed using both albumin-Alexa568 and 69-kD FITC-dextran; however, diabetic animals demonstrated significantly less filtered fluorescent albumin in renal proximal tubule (PT) cells compared with control animals. We also observed increased albumin-derived urinary peptide excretion in diabetic animals, and hyperglycemia modulated this peptideuria. In conclusion, in the early stages of diabetic nephropathy, the PT plays a major role in the development of albuminuria, which may be preceded by peptideuria.

Loop of Henle interaction with interstitial nodal spaces in the renal inner medulla

Understanding dynamics of NaCl reabsorption from loops of Henle, and cellular and physiological consequences, requires a clear understanding of the structural relationships of loops with other functional elements of the inner medulla (IM). Pathways taken by ascending thin limbs (ATLs) and prebend segments along the corticopapillary axis were evaluated for the outer zone of the IM of the Munich-Wistar rat. Connectivity between these segments and microdomains of interstitium adjacent to collecting ducts (CDs) and abutting ascending vasa recta (interstitial nodal spaces) was assessed by evaluating their physical contacts. For each secondary CD cluster, the number of contacts made between the total population of ATLs and interstitial nodal spaces declines as a function of depth below the outer medulla (OM)-IM boundary at near the same exponential rate that loop number declines. The proportion of each loop that makes contact with nodal spaces is inversely related to loop length. Prebend and postbend equivalent length ATL segments lie in contact with an interstitial nodal space along nearly their entire lengths. The number of contacts made by the total population of prebend or postbend segments exhibits a marked, periodic increase and decrease as a function of depth below the OM-IM boundary; this number of contacts correlates with equivalent periodic changes in prebend number. Simulations of loop distribution indicate that small discontinuities in loop distribution contribute to periodic changes in prebend number. Convergence of IM loop bends within CD clusters likely plays an essential role in NaCl compartmentalization by promoting NaCl reabsorption near interstitial regions lying adjacent to CDs and ascending vasa recta.

The Collecting Duct Is the Major Source of Prorenin in Diabetes

In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.

High-resolution optical coherence tomography imaging of the living kidney

Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can provide non-invasive, cross-sectional, high-resolution images of tissue morphology in situ and in real-time. In the present series of studies, we used a high-speed OCT imaging system equipped with a frequency-swept laser light source (1.3 μm wavelength) to study living kidneys in situ. Adult, male Munich–Wistar rats were anesthetized, a laparotomy was performed and the living kidneys were exposed for in situ observation. We observed the kidneys prior to, during and following exposure to renal ischemia induced by clamping the renal artery. The effects of intravenous mannitol infusion (1.0 ml of 25%) prior to and during renal ischemia were also studied. Finally, living kidneys were flushed with a renal preservation solution, excised and observed while being stored at 0–4°C. Three-dimensional OCT data sets enabled visualization of the morphology of the uriniferous tubules and the renal corpuscles. When renal ischemia was induced, OCT revealed dramatic shrinkage of tubular lumens due to swelling of the lining epithelium. Three-dimensional visualization and volumetric rendering software provided an accurate evaluation of volumetric changes in tubular lumens in response to renal ischemia. Observations of kidneys flushed with a renal preservation solution and stored at 0–4°C also revealed progressive and significant loss of tubular integrity over time. Intravenous infusion of mannitol solution resulted in thinning of the tubular walls and an increase in the tubular lumen diameters. Mannitol infusion also prevented the cell swelling that otherwise resulted in shrinkage of proximal tubule lumens during ischemia. We conclude that OCT represents an exciting new approach to visualize, in real-time, pathological changes in the living kidney in a non-invasive fashion. Possible clinical applications are discussed.

PERSISTENT HYPERTENSION AND PROGRESSIVE RENAL INJURY INDUCED BY SALT OVERLOAD AFTER SHORT TERM NITRIC OXIDE INHIBITION

Administration of the NO inhibitor N(wdelta)-nitro-L-arginine methyl ester (NAME) and a high-salt diet (HS) promotes severe albuminuria and renal injury, which regresses upon discontinuation of treatments. We investigated whether these changes reappear after reinstitution of HS, and whether they are prevented by treatment with the antilymphocyte agent mycophenolate mofetil (MMF) or the AT-1 receptor blocker losartan (L). Adult male Munich-Wistar rats received NAME and HS. A control Group (C) received only HS. After 20 days, rats receiving HS and NAME exhibited severe hypertension and albuminuria. After a 30-day recovery period, hypertension was attenuated and albuminuria had virtually disappeared. Rats were then distributed among the following groups: HS, receiving HS; NS, receiving a normal salt (NS) diet; HS-MMF, receiving HS and MMF; HS-LOS, receiving HS and L; HS-HDZ, receiving HS and hydralazine (HDZ). Sixty days later, NS rats showed only slight albuminuria and renal damage or inflammation. In contrast, HS rats developed severe hypertension, marked glomerulosclerosis with interstitial expansion and renal infiltration by macrophages and angiotensin II-positive cells. The group treated with losartan had lowered blood pressure and a lack of albuminuria or renal injury. MMF provided similar protection without altering blood pressure, suggesting a nonhemodynamic effect, a hypothesis reinforced by the finding that HDZ lowered blood pressure without preventing renal injury. These results indicate that treatment with HS and NAME predisposes to the development of hypertension and renal injury upon salt overload, characterizing a new model of chronic nephropathy. The response to MMF or L, but not HDZ, suggests a key role for inflammatory rather than hemodynamic factors.

Losartan-hydrochlorothiazide association promotes lasting blood pressure normalization and completely arrests long-term renal injury in the 5/6 ablation model

The possible long-term renoprotective effects of treatment with thiazides, either as monotherapy or associated with renin-angiotensin suppressors, have not been assessed. We investigated the effect of hydrochlorothiazide (H), alone or combined with losartan (L), in the 5/6 renal ablation model (Nx). Adult male Munich-Wistar rats underwent Nx, remaining untreated for 1 mo. At this time, functional and morphological studies were performed in 21 rats (group Nx(pre)). The remaining rats were distributed among groups: Nx, no treatment; Nx+L, receiving L, 50 mg kg(-1) day(-1) in the drinking water; Nx+H, receiving H, 6 mg kg(-1) day(-1) in drinking water; and Nx+L+H, receiving both L and H as described. At 30 days of treatment, systemic and glomerular pressures were markedly elevated in group Nx. Both H and L attenuated hypertension, whereas combined L+H treatment completely normalized both pressures. Eight months after Nx, mortality approached 70% in untreated rats, whereas severe albuminuria, hypertension, glomerulosclerosis, and interstitial expansion were observed. H and L attenuated, but did not prevent, mortality, hypertension, and renal injury. Combined L+H treatment completely prevented mortality, normalized albuminuria and blood pressure, and arrested renal injury at levels found 1 mo after ablation, despite the unusually long period of observation. Combined L+H treatment may represent an effective therapeutic alternative to prevent progression of chronic nephropathies.

Dietary protein induces endothelin-mediated kidney injury through enhanced intrinsic acid production

Dietary protein as casein (CAS) augments intrinsic acid production, induces endothelin-mediated kidney acidification, and promotes kidney injury. We tested the hypothesis that dietary CAS induces endothelin-mediated kidney injury through augmented intrinsic acid production. Munich-Wistar rats ate minimum electrolyte diets from age 8 to 96 weeks with 50 or 20% protein as either acid-inducing CAS or non-acid-inducing SOY. Urine net acid excretion and distal nephron net HCO3 reabsorption by in vivo microperfusion (Net J(HCO3)) were higher in 50 than 20% CAS but not 50 and 20% SOY. At 96 weeks, 50% compared the 20% CAS had higher urine endothelin-1 excretion (U(ET-1)V) and a higher index of tubulo-interstitial injury (TII) at pathology (2.25+/-0.21 vs 1.25+/-0.13 U, P<0.03), but each parameter was similar in 50 and 20% SOY. CAS (50%) eating NaHCO3 to reduce intrinsic acid production had lower Net J(HCO3), lower U(ET-1)V, and less TII. By contrast, 50% SOY eating dietary acid as (NH4)2SO4 had higher Net J(HCO3), higher U(ET-1)V, and more TII. Endothelin A/B but not A receptor antagonism reduced Net J(HCO3) in 50% CAS and 50% SOY+(NH4)2SO4 animals. By contrast, endothelin A but not A/B receptor antagonism reduced TII in each group. The data support that increased intake of acid-inducing dietary protein induces endothelin B-receptor-mediated increased Net J(HCO3) and endothelin A-receptor-mediated TII through augmented intrinsic acid production.

DIETARY PROTEIN INDUCES REMNANT KIDNEY INJURY BY INCREASED INTRINSIC ACID PRODUCTION MEDIATED THROUGH ENDOTHELIN RECEPTORS.

Purpose We tested the hypothesis that acid-producing dietary protein induces endothelin-mediated parenchymal injury in the 5/6 nephrectomized rat by increasing intrinsic acid production. Methods Munich-Wistar rats underwent surgical 5/6 nephrectomy (Nx) and were studied 12 weeks after eating diets with either acid-producing protein (casein) or non-acid-producing protein (soy). Intrinsic acid production in the casein group was reduced by added Ca ++ citrate with Ca ++ phosphate as a control. Intrinsic acid production in the soy group was increased by added (NH 4 ) 2 SO 4 with Na 2 SO 4 as a control. Both casein-eating Nx and soy + (NH 4 ) 2 SO 4 -eating Nx received either the oral endothelin A/B receptor antagonist bosentan or the oral endothelin A antagonist darunsentan. Distal nephron Net HCO 3 reabsorption (Net J HCO3 ) was measured at 4 weeks by in vivo microperfusion. Urine endothelin 1 excretion (U ET-1 V), urine albumin excretion (U alb V), kidney glomerulosclerosis (GS), and tubulointerstitial injury (TII) were measured at 12 weeks. Results (1) Casein-eating Nx versus soy-eating Nx had higher U ET-1 V (315 ± 52 vs 78 ± 13 fmol/d, p alb V (180 ± 22 vs 74 ± 10 mg/d, p p p ++ citrate versus Ca ++ phosphate had lower U ET-1 V (89 ± 22 vs 341 ± 56 fmol/d, p alb V (105 ± 11 vs 177 ± 20 mg/d, p p p 4 ) 2 SO 4 versus Na 2 SO 4 had higher U ET-1 V (350 ± 40 vs 211 ± 26 fmol/d, p alb V (145 ± 16 vs 86 ± 10 mg/d, p p p HCO3 in acid-ingesting groups. By contrast, the endothelin A but not endothelin A/B antagonist reduced U alb V and TII in acid-ingesting groups. Conclusions Acid-producing dietary protein induces endothelin B-mediated increased acidification and endothelin A-mediated TII in the 5/6 nephrectomized rat through increased intrinsic acid production.

Chronic inhibition of nuclear factor-κB attenuates renal injury in the 5/6 renal ablation model

Recent studies indicated that the nuclear transcription factor, NF-kappaB, activates a number of proinflammatory genes in subjects with progressive nephropathies. We investigated whether NF-kappaB inhibition limits progressive renal injury in the 5/6 renal ablation model (Nx). Adult male Munich-Wistar rats were subdivided in four groups: S (n = 16), subjected to sham operation; S+PDTC (n = 18), sham-operated rats receiving the NF-kappaB inhibitor pyrrolidine-dithiocarbamate (PDTC; 60 mg x kg(-1) x day(-1)) in drinking water; Nx (n = 16), Nx rats receiving vehicle only; and Nx+PDTC (n = 19), Nx rats given PDTC as above. Thirty days after renal ablation, Nx rats exhibited systemic and glomerular hypertension. Only the former was attenuated by PDTC treatment. Sixty days after renal ablation, Nx rats exhibited marked hypertension, albuminuria and creatinine retention, as well as glomerulosclerosis and cortical interstitial expansion/inflammation. Immunohistochemical analysis of Nx rats showed renal interstitial infiltration by macrophages and by cells staining positively for ANG II and its receptor, AT(1). Glomerular and interstitial cells expressing the p65 subunit of the NF-kappaB system were also found. PDTC treatment attenuated renal injury and inflammation, as well as the density of cells staining positively for the p65 subunit. Activation of the NF-kappaB system plays an important role in the pathogenesis of renal injury in the Nx model. Inhibition of this system may represent a new strategy to prevent the progression of chronic kidney disease.

Fluid flow in the juxtaglomerular interstitium visualized in vivo

Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 +/- 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 +/- 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function).

Intraluminal ATP Concentrations in Rat Renal Tubules

It is becoming increasingly recognized that stimulation of apical P2 receptors can influence solute transport in the nephron, but, to date, no information is available on endogenous intraluminal nucleotide concentrations in vivo. This study measured intraluminal ATP concentrations in the renal tubules of anesthetized rats. Proximal tubular concentrations were found to be in the range of 100 to 300 nmol/L, with no significant variation along the S2 segment, whereas concentrations in the early distal tubule were markedly lower. Using collections of varying duration, the half-life of ATP in collected proximal tubular fluid was found to be 3.4 min, indicating significant breakdown by soluble nucleotidases. For assessment of whether proximal tubular ATP was filtered or secreted, experiments were performed in Munich-Wistar rats. The ATP concentration in midproximal tubules (142 +/- 23 nmol/L) was more than four-fold higher than in Bowman's space (32 +/- 7 nmol/L; P < 0.001), whereas fractional water reabsorption between the two sites was modest. In experiments that were designed to determine the effects of (patho)physiologic disturbances on intraluminal ATP, rats were either volume expanded or subjected to hypotensive hemorrhage. Neither maneuver affected proximal tubular luminal ATP concentrations significantly; rapid degradation of secreted ATP by ecto- and soluble nucleotidases is a possible explanation. It is concluded that the proximal tubule secretes ATP into the lumen, where it may have an autocrine/paracrine regulatory role.

Effect of Cyclosporine A on Glucose Interstitial Concentration in Renal Cortex and Medulla from Rats

Aim: To standardize microdialysis in rat kidneys and address cyclosporine A (CsA) effects on renal cortex and medulla interstitial glucose. Methods: Munich-Wistar rats were treated with vehicle or CsA (15 mg/kg/day) for 3 weeks. Glucose was assessed by spectrophotometry in dialysate samples from cortex, medulla and arterial plasma. Plasma insulin was measured by radioimmunoassay. Renal blood flow (RBF) was measured by Doppler ultrasound. Creatinine and urea were measured by spectrophotometry. Results: CsA significantly increased the plasma levels of urea and creatinine (1.5 ± 0.20 vs. 0.73 ± 0.03 mg/dl in controls, p < 0.05). Medullary glucose in control was 44% lower than arterial glucose (56 ± 6 vs. 101 ± 8 mg/dl, p < 0.05). At the same time, CsA increased arterial (163 ± 35 vs. 101 ± 8 mg/dl in controls, p < 0.05) and medullary interstitial glucose (100 ± 18 vs. 56 ± 6 mg/dl in controls, p < 0.05), but did not affect cortical glucose (114 ± 21 vs. 90 ± 11 mg/dl in controls). These changes occurred in the presence of a decreased plasma insulin level (2.7 ± 0.2 vs. 9.3 ± 0.4 µU/ml in controls, p < 0.05). The increment in medullary glucose in CsA group occurred despite a reduction in RBF (4.6 ± 0.8 vs. 6.5 ± 1.0 ml/min/kidney in controls, p < 0.05). Conclusions: Microdialysis was an adequate tool to investigate in vivo regulation of renal glucose metabolism. Renal glucose uptake was dependent on medullary cells and CsA treatment induced diabetogenic effects on renal medulla in situ.

Relaxin Improves Renal Function and Histology in Aging Munich Wistar Rats

Administration of recombinant human relaxin (rhRLX) to conscious, chronically instrumented rats increases GFR and effective renal plasma flow (ERPF) and decreases effective renal vascular resistance (ERVR) with no significant change in mean arterial pressure. The Munich Wistar albino rat shows progressive chronic nephrosis with age and therefore was used to determine the functional and histologic consequences of rhRLX on matrix remodeling in the kidney of older rats. RLX-infused rats showed increased GFR and ERPF with decreased ERVR. Furthermore, in a double-blinded examination, the renal histology showed a significant decrease in glomerular and tubular collagen deposition in the rhRLX-infused aged rats. During short-term rhRLX administration (24 h), gelatinase activity was found to be essential for renal vasodilation and hyperfiltration. Surprisingly, after 20 d, improved renal function was insensitive to the inhibition of gelatinase activity, suggesting that collagen degradation in these rats had permanently altered the matrix of the renal vasculature. In conclusion, long-term administration of rhRLX improves renal function and ameliorates renal pathology in an aging rat model. The biphasic action of rhRLX on the kidney indicates that, acutely, the vessels dilate, causing increased filtration and renal blood flow with decreased vascular resistance as a result of upregulation of gelatinase activity. Subsequently, the renal vessels undergo alteration in supporting matrix, showing increased blood supply even in the face of acute matrix metalloproteinase inhibition, most likely as a result of the inhibitory properties of RLX on collagen production or increased collagen breakdown.