NAD+-15-hydroxyprostaglandin dehydrogenase distribution in rat kidney.

Rat kidney NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was measured in zones and substructure of the rat kidney nephron. This was accomplished utilizing an assay procedure based upon determining the amount of prostaglandin E1 present before and after the reaction with the 15-hydroxyprostaglandin dehydrogenase contained in the tissue sample. The enzyme activity was assayed in freeze dried, quick frozen rat kidney sections and its distribution within the rat kidney was determined. In kidney zones, it was localized to medullary rays and inner cortex. In kidney substructure, activity was highest in collecting tubule, pars recti tubule, distal convoluted tubule and the ascending limb of Henle (14.2, 11.5, 6.4 and 9.2 mM kg-1hr-1, respectively). Activity in glomeruli, proximal convoluted tubule and small arteries was lower (2.1, 2.8 and 2.1 mM kg-1hr-1, respectively). The assay procedure was verified by established assays (spectrophotometric, fluorometric and radiometric TLC) which are often used in homogenate and purified PGDH preparations.     

Mouse retinal dehydrogenase 4 (RALDH4), molecular cloning,cellular expression,and activity in 9-cis-retinoic acid biosynthesis in intact cells

This study describes cDNA cloning and characterization of mouse RALDH4. The 2.3-kb cDNA encodes an aldehyde dehydrogenase of 487 amino acid residues, about two-orders of magnitude more active in vitro with 9-cis-retinal than with all-trans-retinal. RALDH4 recognizes as substrate 9-cis-retinal generated in transfected cells by the short-chain dehydrogenases CRAD1, CRAD3, or RDH1, to reconstitute a path of 9-cis-retinoic acid biosynthesis in situ. Northern blot analysis showed expression of RALDH4 mRNA in adult mouse liver and kidney. In situ hybridization revealed expression of RALDH4 in liver on embryo day 14.5, in adult hepatocytes, and kidney cortex. Immunohistochemistry confirmed RALDH4 expression in hepatocytes and showed that hepatocytes also express RALDH1, RALDH2, and RALDH3. Kidney expresses the RALDH4 protein primarily in the proximal and distal convoluted tubules of the cortex but not in the glomeruli or the medulla. Kidney expresses RALDH2 in the proximal convoluted tubules of the cortex but not in the distal convoluted tubules or glomeruli. Kidney expresses RALDH1 and RALDH2 in the medulla. The enzymatic characteristics of RALDH4, its expression in fetal liver, and its unique expression pattern in adult kidney compared with RALDH1, -2, and -3 suggest that it could meet specific needs for 9-cis-retinoic acid biosynthesis.     

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.     

Histochemical localization of enzyme activity in the kidneys of male marmosets (Callithrix jacchus and Callithrix penicillata).

The distribution of several hydrolases and oxidoreductases was studied in the renal parenchyma of adult male marmosets (Callithrix jacchus and Callithrix penicillata). The oxidative enzymes showed a high reactivity in the proximal and distal tubules, whereas the hydrolases reacted strongly in the proximal tubules but only weakly or not at all in the thick limb of Henle's loop, distal tubules and collecting ducts. The NAD-dependent enzymes (except alpha-GPDH) showed a stronger reactivity in the proximal tubules, while the NADP-dependent ones were more reactive in the thick limb of Henle's loop and distal convoluted tubules. Two groups of interstitial cells were found in the medulla. A first group inside the outer medulla, showing cells rich in acid phosphatase and nonspecific esterases and a second group, close to the papilla, reactive to a certain number of oxidative enzymes. A different reactivity in cells of the distal convoluted tubules, thick limb of Henle's loops and collecting ducts (dark cells) was seen in the case of some enzymes like nonspecific esterase, alpha-GPDH and SDH.     

The polyol pathway in the bovine oviduct

The oviducts likely provide optimized micro‐environments for the final maturation of gametes, fertilization, and early embryo development. Hexoses, including glucose, fructose, and sorbitol, are involved in these critical reproductive events. Monosaccharide production is controlled, in part, by the polyol pathway and requires two enzymes: an aldose reductase (AR) that reduces glucose into sorbitol, followed by its oxidation into fructose by sorbitol dehydrogenase (SDH). We analyzed the expression of AR and SDH in the isthmus and ampulla of the bovine oviduct at the proliferative, mid‐luteal, and late‐luteal phases of the estrous cycle by quantitative PCR and immunoblots. Immunochemistry and an assay of SDH activity were also performed. The quantity of hexoses in whole sections of isthmus and ampulla were determined by liquid chromatography coupled to mass spectrometry. In sum, AR expression was restricted to the isthmus, while SDH was mostly expressed in the isthmic–ampullary junction and the ampulla, specifically concentrated in the luminal epithelium of the oviduct. The estrous cycle had no impact on protein expression of AR and SDH. Instead, the levels of AR and SDH expression were associated with higher ratios of sorbitol to fructose in the isthmus (1.6) than in the ampulla (4.1; P = 0.005). These results are discussed in light of physiological events occurring in the oviduct. Mol. Reprod. Dev. 79: 603–612, 2012. © 2012 Wiley Periodicals, Inc.     

Comparison of the distribution of tissue kallikrein and esterase A, a kallikrein-like enzyme, in rat kidney using specific monoclonal antibodies

Tissue kallikrein (E.C. 3.4.21.35) and arginine esterase A, another closely related, kinin-generating serine protease, have been localized by immunocytochemistry in rat kidney, using monoclonal antibodies that do not crossreact with other kallikrein-related enzymes or with tonin. Kallikrein was present primarily in the apical cytoplasm of the connecting tubule and the cortical collecting duct. Esterase A, on the other hand, was present primarily in the basolateral region of both proximal and distal straight tubules in the outer medulla and medullary rays. In addition, esterase A was demonstrable in distal convoluted tubules and, to a lesser extent, in proximal convoluted tubules. The presence of different kinin-generating enzymes at these sites would permit the formation of kinins from appropriate substrates on both the vascular and luminal poles of separate segments of the kidney tubule.     

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.     

Polyol determination along the rat nephron

The polyols sorbitol and inositol were determined in single freshly microdissected tubule segments of rat kidney. Twenty different structures were separated from six different kidney zones reaching from cortex to papillary tip. Picomol amounts of sorbitol and inositol were quantitated by use of an enzymatic bioluminescence procedure. Experimental conditions (700 mosmol/kg, 4 degrees C) were chosen to assure constant polyol concentrations over 3 h dissection period. Sorbitol exhibited a concentration gradient in the collecting duct system from the outer/inner medullary border (3.9 +/- 0.5 pmol/mm) to the papillary tip (78.8 +/- 6.9 pmol/mm). In the same region descending and ascending limbs of Henle's loop contained 1.5 +/- 0.5 to 5.3 +/- 1.6 pmol/mm and 2.5 +/- 0.8 to 8.35 +/- 1.5 pmol/mm, respectively. In contrast, all outer medullary and cortical structures had lower sorbitol concentrations. Inositol amounts increased continuously in the collecting duct from cortex (5.3 +/- 0.5 pmol/mm) to inner medulla (30.7 +/- 3.8 pmol/mm). This polyol was also found in thick ascending limb of Henle's loop (6.2 +/- 1.1 pmol/mm in cortex to 11.2 +/- 1.4 pmol/mm in outer medulla) and in proximal tubules (5.6 +/- 1.2 pmol/mm in S1 and 4.5 +/- 1.5 pmol/mm in S3). When related to cellular volume measured by planimetry, intracellular sorbitol concentration was calculated to be 51 mmol/l in papillary collecting duct and inositol 28 mmol/l in outer medullary thick ascending limb cells. These data confirm the role of sorbitol in the renal concentrating process in papilla. Inositol seems to have additional function in thick ascending limb of Henle's loop and the proximal tubule.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

The localisation of sorbitol pathway activity in the rat renal cortex and its relationship to the pathogenesis of the renal complications of diabetes mellitus.

A series of in vivo and in vitro investigations was performed to examine the localisation of sorbitol pathway activity in the rat renal cortex and to investigate the possible relation that the acculumation of sorbitol pathway intermediates in renal cortical tissue may have to the pathogenesis of renal complications in diabetes mellitus. Neither of the sorbitol pathway intermediates, sorbitol or fructose, were detected either in intact glomeruli which had been isolated from rats rendered chronically diabetic with streptozotocin, or in metabolically active glomeruli which had been incubated in vitro in high glucose media. Such data agreed with previously published observations that the enzyme aldose reductase is not present in renal glomeruli, and suggested that changes in sorbitol pathway activity cannot be directly related to the pathogenesis of diabetic glomerulosclerosis. Sorbitol was detected in low concentrations (3.1 mu-mol/g protein) in cortical tubules which had been isolated from the renal cortex of rats rendered chronically diabetic with streptozotocin. This concentration of sorbitol was higher than that in the intact renal cortex of the diabetic animal (0.3 mu-mol/g protein) or in the cortical tubules of non-diabetic animals (0.5 mu-mol/g protein). It is apparent that the renal cortical tubule is a major site of sorbitol pathway activity in the renal cortex. However, there is presently no obvious causal relationship between the accumulation of such relatively low concentrations of sorbitol in the renal cortical tubule and the pathogenesis of glomerulosclerosis or cortical tubular lesions in diabetes.     

Quantitative histochemistry of the sorbitol pathway in glomeruli and small arteries of human diabetic kidney.

Recent evidence has suggested a role for the polyol pathway in pathogenesis of cell damage in diabetes Glucose may be phosphorylated to glucose-6-phosphate via hexokinase and enter glycolysis or reduced to sorbitol via aldose reductase to enter the polyol pathway. The poorly diffusible sorbitol is converted via sorbitol dehydrogenase to fructose. Hexokinase, aldose reductase and sorbitol dehydrogenase activities were measured in glomeruli (G) and small arteries (SA) taken from normal and diabetic human kidneys, Hexokinase in diabetic G was 1688, which was significantly decreased from normal, 3147 mmoles/kg-1/h-1. Alodse reductase was significantly elevated in diabetic G,56-6, compared to normal G,10-8 mmoles/kg-1/h-1. In contrast, sorbitol dehydrogenase was significantly depressed in diabetic G, 3-7 VERSUs 10-9 mmoles/kg-1/h-1. The enzymatic changes observed in diabetic G would facilitate accumulation of sorbitol and therefore could contribute to the progression of glomerulosclerosis. The activity of hexokinase was also significantly reduced in SA, whereas aldose reductase and sorbitol dehydrogenase were unchanged.     

Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase-3β-positive epithelium

It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3β (GSK-3β). GSK-3β immunoreactivity was associated with glomeruli, the thick ascending limb of Henle's loop, and collecting ducts in the developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3β (pGSK-3β) protein decreased during postnatal development. After feeding of dams with litters lithium [50 mmol Li/kg chow, postnatal (P) days 7-28], the offspring showed plasma lithium concentration of 1.0 mmol/l. Kidneys from lithium-treated rat pups exhibited dilated distal nephron segments with microcysts. Stereological analysis showed reduced cortex and outer medullary volumes. Lithium increased pGSK-3β and the proliferation marker proliferating cell nuclear antigen (PCNA) protein abundances in the cortex and medulla. After lithium treatment, pGSK-3β-immunopositive cells exhibited restricted distribution and were associated primarily with subsets of cells in dilated and microcystic segments of cortical collecting ducts. After 6 wk of lithium discontinuation, adult rats exhibited attenuated urine concentration capacity and diminished outer medullary volume. Histological sections of two nephrectomy samples and a biopsy from three long-term lithium-treated patients showed multiple cortical microcysts that originated from normally appearing tubules. Microcysts were lined by a cuboidal PCNA-, GSK-3β-, and pGSK-3β-immunopositive epithelium. The postnatal rat kidney may serve as an experimental model for the study of lithium-induced human kidney injury. The data are compatible with a causal relationship between epithelial entry of lithium into cells of the aldosterone-sensitive distal nephron, inactivation of GSK-3β, proliferation, and microcysts.     

ELECTRON MICROSCOPE STUDIES ON THE SURFACE COAT OF THE NEPHRON

Attempts to make visible the carbohydrate coat at the free cell surface of glomeruli as well as the tubules of rabbit kidney were undertaken. The ruthenium red procedure was performed, according to Luft, at various pH values. Moreover, the colloidal iron and the colloidal thorium methods were used. Neuraminidase digestion was also performed. In the ruthenium red procedure the luminal face of the epithelial cells of the nephron was coated distinctly with reaction product. The results obtained revealed that some of the differences at various levels of the nephron depended on the pH values. In glomeruli and proximal convoluted tubules the optimum pH value was 7.4; in the ascending limb of Henle loops and distal convoluted tubules the optimum pH value was 6.8. The ruthenium red-positive surface coat was either closely connected with, or appeared as a part of, the outer leaflet of the unit membrane. The slit pores of glomeruli were also covered by a coat continuous with the surface coat of the adjacent foot processes. The coat lining the microvilli of proximal convoluted tubules completely filled the intervillous spaces. Also, both the colloidal iron method and the colloidal thorium method evidenced the presence of surface coat. Pre-treatment with neuraminidase abolished the effect of the Hale reaction. These results may indicate that the surface coat of the epithelia of the nephron shows the presence of glycoproteins containing siliac acid residues.     

Succinate dehydrogenase activity in rat kidney after repeated administrations of p-aminohippurate or sodium chloride

During PAH excretion and 18 h after repeated PAH administrations to rats, renal cortical SDH activity was unchanged in comparison with untreated controls. On the other hand, 18 h after repeated administrations of 0.9% or 1.8% NaCl solution, SDH activity was decreased by about 20% in kidney cortex. In outer medulla SDH activity was decreased 18 h after all pretreatments. The decline of SDH activity was observed whenever an increased urinary Na excretion occurred, except that PAH was repeatedly administered. The probability is discussed that substrate-induced stimulation of the carrier system for weak organic acids is accompanied by an increase of mitochondrial activity in kidney cortex.     

Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney

Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli.     

Hypoplasia of one kidney in a Sprague-Dawley rat

Pathological examination of a 4-week-old male Sprague-Dawley rat revealed hypoplasia of the left kidney. Grossly, the left kidney exhibited hypoplasia associated with absence of the ureter on the same side. Histologically, components of the cortex and medulla were mingled in the tissue, and the glomeruli and convoluted tubules were scattered in disorder, and connective tissue proliferation was also observed. The papilla and pelvis could not be identified.     

Angiotensin II receptors in the kidney

Angiotensin II (AngII) receptors have been localized in rat kidney by using the high-affinity agonist analog 125I-labeled [Sar1]AngII as a probe for in vitro autoradiography. Receptors were associated with four morphologically distinct patterns of distribution. First, a high density of receptors occurs in glomeruli. These are diffusely distributed, consistent with a mesangial localization. AngII receptor density shows a cortical gradient, which is highest in superficial and midcortical glomeruli and lowest in juxtamedullary glomeruli. Receptors associated with both superficial and deep glomeruli show down-regulation during low-sodium intake. Second, low levels of tubular AngII binding were seen in the outer cortex. Third, a very high density of AngII receptors occurs in longitudinal bands in the inner zone of the outer medulla in association with vasa recta bundles. Receptors in this site also show down-regulation during low dietary sodium intake. Fourth, a moderate density of receptors occurs diffusely throughout the inner zone of the outer medulla in the interbundle areas. These results suggest that AngII exerts a number of different intrarenal regulatory actions. In addition to the known vascular, glomerular, and proximal tubular effects of AngII, these findings focus attention on possible actions of AngII in the renal medulla where it could regulate medullary blood flow and thereby modify the function of the countercurrent concentrating system.     

Study of the polyol pathway in the porcine epididymis

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.     

Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.