S3 Segment Of Proximal Tubule Research Articles

Role of the kidney in iron homeostasis: renal expression of Prohepcidin, Ferroportin, and DMT1 in anemic mice

It is known that renal tissue plays a role in normal iron homeostasis. The current study examines kidney function in iron metabolism under hemolytic anemia studying renal expression of Prohepcidin, Ferroportin (MTP1), and divalent metal transporter 1 (DMT1). The relationship between these proteins and iron pigments was also investigated. Immunohistochemical procedures to study renal expression of Prohepcidin, MTP1, and DMT1 were performed in healthy and anemic mice. Renal tissue iron was determined by Prussian blue iron staining. To assess anemia evolution and erythropoietic recovery, we used conventional tests. In healthy mice, Prohepcidin expression was marked in proximal tubules and inner medulla and absent in outer medulla. Cortical tissue of healthy mice also showed MTP1 immunostaining, mainly in the S2 segment of proximal tubules. Medullar tissue showed MTP1 expression in the inner zone. In addition, S2 segments showed intense DMT1 immunoreactivity with homogeneous DMT1 distribution throughout renal medulla. The main cortical findings in hemolytic anemia were in S2 segments of proximal tubules where we found that decreased Prohepcidin expression coincided with an increment in Ferroportin and DMT1 expression. This expression pattern was concomitant with increased iron in the same tubular zone. However, in medullar tissue both Prohepcidin and MTP1 decreased and DMT1 was detected mainly in larger diameter tubules. Our findings clearly demonstrate that in hemolytic anemia, renal Prohepcidin acts in coordination with renal Ferroportin and DMT1, indicating the key involvement of kidney in iron homeostasis when iron demand is high. Further research is required to learn more about these regulatory mechanisms.

Novel NaPi-2c mutations that cause mistargeting of NaPi-2c protein and uncoupling of Na-Picotransport cause HHRH

hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive inherited disorder of mineral and bone metabolism. It is characterized by hypophosphatemia, rickets, and increased serum 1,25-dihydroxyvitamin D concentration, resulting in secondary absortive hypercalciuria and

Cisplatin-induced cytoxicity: is the nucleus relevant?

cisplatin is a potent chemotherapeutic agent widely used for the treatment of cancer. However, cisplatin is frequently associated with nephrotoxicity, manifesting as acute kidney injury accompanied by hypokalemia and hypomagnesemia ([6][1]). Overdose of cisplatin has been reported to cause a wide

Differential gene expression of 36-kDa microfibril-associated glycoprotein (MAGP-36/MFAP4) in rat organs

By using quantitative Western blot analysis and the real time polymerase chain reaction technique, we investigated the differential gene expression of microfibril-associated glycoprotein (MAGP-36) in rat organs. The gene was expressed highly in sites rich in elastic fibers, such as aorta, skin, and esophagus. However, MAGP-36 was also expressed highly in some other sites containing no elastic fibers. In lung and trachea, the expression levels of MAGP-36 mRNA were about seven times higher than those in other elastic tissues, although the protein abundances were almost at the same levels as other elastic tissues. MAGP-36 seemed to be secreted outside these organs. In brain, kidney, and spleen, although the expression levels of MAGP-36 mRNA were low, substantial amounts of MAGP-36 protein were detected. An immunohistochemical study revealed that MAGP-36 was present at the brush border of the S3 segment of proximal tubules in kidney. Since MAGP-36 is known to bind to mannan, MAGP-36 might be involved in mannose transport in the S3 segment. Thus, MAGP-36 might be multifunctional and present in a wide variety of sites in various organs.

Role of angiotensin II in the enhancement of ammonia production and secretion by the proximal tubule in metabolic acidosis

Acidosis and angiotensin II stimulate ammonia production and transport by the proximal tubule. We examined the modulatory effect of the type 1 angiotensin II receptor blocker losartan on the ability of metabolic acidosis to stimulate ammonia production and secretion by mouse S2 proximal tubule segments. Mice given NH(4)Cl for 7 days developed metabolic acidosis (low serum bicarbonate concentration) and increased urinary excretion of ammonia. S2 tubule segments from acidotic mice displayed higher rates of ammonia production and secretion compared with those from control mice. However, when losartan was coadministered in vivo with NH(4)Cl, both the acidosis-induced increase in urinary ammonia excretion and the adaptive increase in ammonia production and secretion of microperfused S2 segments were largely blocked. In renal cortical tissue, losartan blocked the acid-induced increase in brush-border membrane NHE3 expression but had no effect on the acid-induced upregulation of phosphate-dependent glutaminase or phosphoenolpyruvate carboxykinase 1 in cortical homogenates. Addition of angiotensin II to the microperfusion solution enhanced ammonia secretion and production rates in tubules from NH(4)Cl-treated and control mice in a losartan-inhibitable manner. These results demonstrate that a 7-day acid challenge induces an adaptive increase in ammonia production and secretion by the proximal tubule and suggest that during metabolic acidosis, angiotensin II signaling is necessary for adaptive enhancements of ammonia excretion by the kidney and ammonia production and secretion by S2 proximal tubule segments, as mediated, in part, by angiotensin receptor-dependent enhancement of NHE3 expression.

Targeted disruption of the meprin metalloproteinase β gene protects against renal ischemia-reperfusion injury in mice

Meprins are membrane-bound and secreted metalloproteinases consisting of alpha- and/or beta-subunits that are highly expressed in mouse kidney proximal tubules. Previous studies have implied that the meprin alpha/beta-isoform is deleterious when renal tissue is subjected to ischemia-reperfusion (I/R). To delineate the roles of the meprin isoforms in renal disease, we subjected mice deficient in meprin-beta (KO) and their wild-type (WT) counterparts to I/R. WT mice were markedly more susceptible to renal injury after I/R than the meprin-beta KO mice as determined by blood urea nitrogen levels. Urinary levels of inflammatory cytokines IL-6 and KC (CXCL1) were significantly higher in WT compared with meprin-beta KO mice by 6 h post-I/R. At 96 h postischemia, kidney mRNA expression levels for tumor necrosis factor-alpha, transforming growth factor-beta, inducible nitric oxide synthase, and heat shock protein-27 were significantly higher in the WT than meprin-beta KO mice. For WT mice subjected to I/R, there was a rapid (3 h) redistribution of meprin beta-subunits in cells in S3 segments of proximal tubules, followed by shedding of apical cell membrane and detachment of cells. These studies indicate that meprin-beta is important in the pathogenesis of renal injury following I/R and that the redistribution of active meprin-alpha/beta is a major contributor to renal injury and subsequent inflammation.

Amelioration of Cisplatin-Induced Acute Renal Injury by Renal Progenitor-Like Cells Derived from the Adult Rat Kidney

The replacement of a necrotic tubular epithelium with functional tubular epithelial cells is required for recovery from acute renal failure (ARF). A rat renal progenitor-like (rKS56) cell line was recently established derived from the S3 segment of renal proximal tubules. The therapeutic efficacy of rKS56 cells was examined in a rat model of cisplatin-induced ARF. rKS56-lacZ cells expressing 3-galactosidase were injected into SD rats either at the subcapsule of the left kidney (rKS-SC) or via the left renal artery (rKS-IA) 2 days after the injection of cisplatin. Bluo-gal(+) rKS56-lacZ cells were observed in the subcapsule in the rKS-SC group on day 5, and were further increased in number on day 9, accompanied by partial distribution in the corticomedullary junction, but not in the rKS-IA group. A portion of Bluo-gal(+) cells coexpressed Ki-67, aquaporin-1, hepatocyte growth factor (HGF), and c-Met. rKS-SC treatment significantly improved the tubular injury scores, ameliorated tubular cell apoptosis, and induced cell proliferation. The renal function also significantly improved in the rKS-SC group on day 5. These results demonstrate that locally implanted rKS56 cells could differentiate into tubular epithelial cells, thereby accelerating the recovery from tubular injury, most likely by producing tubular trophic factors. These results suggest the therapeutic potential of this novel approach for patients with end-stage renal failure.

Slc26a6 (PAT1) Deletion Downregulates the Apical Na+/H+ Exchanger in the Straight Segment of the Proximal Tubule

Background/Aim: Slc26a6 (PAT1, CFEX) is a major chloride/base exchanger located on the apical membrane of the kidney proximal tubule. The purpose of the present study was to examine the effect of Slc26a6 deletion on the apical Na⁺/H⁺ exchanger 3 (NHE3) in the straight segment (S3) of the proximal tubule, which is the major site for the reabsorption of filtered chloride in the kidney. Methods: The proximal tubule S3 segment was perfused and the intracellular pH and apical Na⁺/H⁺ exchanger activity and expression were measured. Results: In the proximal tubule straight segments that were microperfused in vitro, baseline intracellular pH, measured by BCPCF-AM, was 7.10 ± 0.02 in Slc26a6–/– and 7.33 ± 0.02 in Slc26a6+/+ animals, a significant reduction in Slc26a6 mutant mice (p < 0.00001). The activity of the apical Na⁺/H⁺ exchanger was 0.49 ± 0.02 pH units/min in Slc26a6+/+ and 0.26 ± 0.03 pH units/min in Slc26a6–/– animals, a significant reduction in Slc26a6–/– mice (p < 0.0001). Formate-induced intracellular alkalinization, which is mediated via NHE3, was significantly blunted in Slc26a6–/– animals, with an alkalinization magnitude of 0.16 pH unit in Slc26a6–/– versus 0.37 in Slc26a6+/+ animals (p < 0.00001, n = 5 separate animals). Angiotensin II stimulation of NHE3 activity was intact in Slc26a6–/– animals. Buffering capacity was comparable in Slc26a6+/+ and Slc26a6–/– mice. Immunoblotting and immunofluorescent labeling demonstrated comparable NHE3 abundance and distribution in kidney proximal tubules of Slc26a6+/+ and Slc26a6–/– mice. Conclusion: In conclusion, Slc26a6 deletion downregulates the apical Na⁺/H⁺ exchanger activity in the straight segment of the proximal tubule. The absence of a significant renal sodium loss in Slc26a6-null mice, despite NHE3 downregulation in the in vitro perfused tubules, points to possible activation of signaling pathways that can stimulate the apical Na⁺/H⁺ exchanger in vivo.

Mechanisms of basolateral base transport in the renal proximal tubule.

Renal proximal tubules absorb HCO3- by secretion of H+ into the tubular lumen. This paper focuses on the mechanisms of HCO3- exit across the basolateral cell membrane. The major exit pathway is rheogenic sodium bicarbonate co-transport. This system transports Na+ and HCO3-, but not Cl-, in obligatory coupling at a fixed overall stoichiometry of three HCO3- to one Na+. The fact that HCO3- flux is reduced after inhibition of cytoplasmic and/or membrane-bound peritubular carbonic anhydrase indicates that HCO3- is not transported as such but is split during permeation into its buffer subspecies from which it is regenerated on the other side of the membrane. Since flow of OH- or of H+ (in opposite directions) can be excluded, it appears most likely that one HCO3- and one CO3(2-) move together with one Na+. Besides carbonic anhydrase inhibitors, disulphonic stilbenes and harmaline are known to block the co-transporter. In addition to rheogenic Na+ (HCO3-)3 co-transport, Na+-dependent and Na+-independent electroneutral Cl-/HCO-3 exchange have been identified. The latter mechanisms are particularly important in S3 segments of proximal tubule where Na+ (HCO3-)3 co-transport is missing. Further mechanisms which operate in parallel, but at lower rates, are electroneutral SO4(2-)/HCO3- exchange and, in some species, lactate/HCO3- exchange. Moreover, there may be some uncoupled OH- flux and it is reasonable to assume that OH- (or H+) flux is involved in the transport of dicarboxylic acids across the basolateral cell membrane.

Role of monocarboxylate transport in the regulation of intracellular pH of renal proximal tubule cells.

Traditional models of acid-base transport and intracellular pH (pHi) regulation in the renal proximal tubule have been based on the existence of a Na+/H+ exchanger at the luminal membrane and a simple HCO3- conductance at the basolateral membrane. Our recent work, in which we used pH-sensitive microelectrodes or dyes to monitor pHi in isolated renal tubules perfused in the nominal absence of HCO3-, has demonstrated the existence of a novel mechanism of acid extrusion in amphibian and mammalian proximal tubule cells. The salamander proximal tubule, for example, possesses an electroneutral Na+ monocarboxylate (Na+-X-) co-transporter, but only at the luminal membrane. It also possesses an electroneutral H+-X- co-transporter, but only at the blood side or basolateral membrane. In the presence of lactate, the luminal Na+-lactate co-transporter mediates a net influx of lactate, driven by the Na+ gradient. The cell-to-blood lactate gradient, in turn, drives the coupled efflux of H+ and lactate across the basolateral membrane. The net effect is the reabsorption of lactate, the luminal uptake of Na+ and the basolateral extrusion of H+. Acid extrusion mediated by this monocarboxylate system in the salamander is comparable in magnitude to that mediated by the Na+/H+ exchanger. In the S3 segment of the rabbit proximal tubule, a similar monocarboxylate system (studied with acetate instead of lactate) extrudes acetate at twice the rate of the Na+/H+ exchanger. Thus, monocarboxylate transport, at least in the nominal absence of HCO3-, can have a major impact on pHi regulation.

Why isd-serine nephrotoxic and α-aminoisobutyric acid protective?

D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.

Role of the AT1Areceptor in the CO2-induced stimulation of HCO3−reabsorption by renal proximal tubules

The proximal tubule (PT) is major site for the reabsorption of filtered HCO(3)(-). Previous work on the rabbit PT showed that 1) increases in basolateral (BL) CO(2) concentration ([CO(2)](BL)) raise the HCO(3)(-) reabsorption rate (J(HCO(3))), and 2) the increase that luminal angiotensin II (ANG II) produces in J(HCO(3)) is greatest at 0% [CO(2)](BL) and falls to nearly zero at 20%. Here, we investigate the role of angiotensin receptors in the [CO(2)](BL) dependence of J(HCO(3)) in isolated perfused PTs. We found that, in rabbit S2 PT segments, luminal 10(-8) M saralasin (peptide antagonist of ANG II receptors), lowers baseline J(HCO(3)) (5% CO(2)) to the value normally seen at 0% in the absence of inhibitors and eliminates the J(HCO(3)) response to changes in [CO(2)](BL). However, basolateral 10(-8) M saralasin has no effect. As with saralasin, luminal 10(-8) M candesartan (AT(1) antagonist) reduces baseline J(HCO(3)) and eliminates the [CO(2)](BL) dependence of J(HCO(3)). Luminal 10(-7) M PD 123319 (AT(2) antagonist) has no effect. Finally, we compared PTs from wild-type and AT(1A)-null mice of the same genetic background. Knocking out AT(1A) modestly lowers baseline J(HCO(3)) and, like luminal saralasin or candesartan in rabbits, eliminates the J(HCO(3)) response to changes in [CO(2)](BL). Our accumulated evidence suggests that ANG II endogenous to the PT binds to the apical AT(1A) receptor and that this interaction is critical for both baseline J(HCO(3)) and its response to changes in [CO(2)](BL). Neither apical AT(2) receptors nor basolateral ANG II receptors are involved in these processes.

Renal Proximal Tubule Segment-Specific Nephrotoxicity: An Overview on Biomarkers and Histopathology

The correspondence between histopathological findings and segment-specific biomarkers was investigated in rats treated with segment-specific nephrotoxicants. Male Wistar rats were treated with a single injection of K2Cr2O7 (25 mg/kg s.c. in saline), cis-Pt (10 mg/kg i.p. in buffered MSO) or HCBD (100 mg/kg i.p. in corn oil). Twenty-four and 48 hours after treatment, the rats were sacrificed and the kidneys were drawn for histopathological and biochemical evaluation, i.e., GS activity in renal cortex and PAH uptake in renal cortical slices. Histopathological findings show that cis-Pt and HCBD cause diffuse necrosis of S3 segment of proximal tubules in the outer stripe of outer medulla, respectively. On the contrary, K2Cr2O7 damages exclusively S1-S2 segments, inducing vacuolization at 24 hr and diffuse necrosis at 48 hr after treatment. GS activity in renal tissue is significantly decreased after HCBD and cis-Pt, but not K2Cr2O7 treatment. In contrast, PAH uptake is significantly reduced by K2Cr2O7, but not by cis-Pt or HCBD treatment (even if HCBD causes a slight decrease 48 hr after treatment). The evidence of this study confirms the high specificity of GS activity as marker of S3 segment injury, that PAH uptake is prevalently active in the S1-S2 segments, and that there is complete correspondence among segment-specific nephrotoxicants, biomarkers of segment-specific damage, and histopathological findings.

Regulation of renal amino acid transporters during metabolic acidosis

The kidney plays a major role in acid-base homeostasis by adapting the excretion of acid equivalents to dietary intake and metabolism. Urinary acid excretion is mediated by the secretion of protons and titratable acids, particularly ammonia. NH(3) is synthesized in proximal tubule cells from glutamine taken up via specific amino acid transporters. We tested whether kidney amino acid transporters are regulated in mice in which metabolic acidosis was induced with NH(4)Cl. Blood gas and urine analysis confirmed metabolic acidosis. Real-time RT-PCR was performed to quantify the mRNAs of 16 amino acid transporters. The mRNA of phosphoenolpyruvate carboxykinase (PEPCK) was quantified as positive control for the regulation and that of GAPDH, as internal standard. In acidosis, the mRNA of kidney system N amino acid transporter SNAT3 (SLC38A3/SN1) showed a strong induction similar to that of PEPCK, whereas all other tested mRNAs encoding glutamine or glutamate transporters were unchanged or reduced in abundance. At the protein level, Western blotting and immunohistochemistry demonstrated an increased abundance of SNAT3 and reduced expression of the basolateral cationic amino acid/neutral amino acid exchanger subunit y(+)-LAT1 (SLC7A7). SNAT3 was localized to the basolateral membrane of the late proximal tubule S3 segment in control animals, whereas its expression was extended to the earlier S2 segment of the proximal tubule during acidosis. Our results suggest that the selective regulation of SNAT3 and y(+)LAT1 expression may serve a major role in the renal adaptation to acid secretion and thus for systemic acid-base balance.

Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones

The renal reabsorption and/or excretion of various organic anions is mediated by specific organic anion transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences [females (F) > males (M)]. The exact localization of OAT2 protein in the mammalian kidney has not been reported. Here we studied the expression of OAT2 mRNA by RT-PCR and its protein by Western blotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult M and F mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS) tissue, exhibiting 1) gender dependency (F > M), 2) upregulation by castration and downregulation by ovariectomy, and 3) strong downregulation by testosterone and weak upregulation by estradiol and progesterone treatment. A polyclonal antibody against rat OAT2 on WB of isolated renal membranes labeled a approximately 66-kDa protein band that was stronger in F. By IC, the antibody exclusively stained brush border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F > M). In variously treated rats, the pattern of 66-kDa band density in the OS membranes and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender independent. In mice, the expression pattern largely resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F > M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.

Targeted disruption of the meprin beta gene results in decreased renal ischemia/reperfusion injury in mice

Meprins are metalloproteinases consisting of alpha and/or beta subunits and are highly expressed in kidneys of mice. Inbred mouse strains that express very low levels of meprin alpha have been shown to be less susceptible to renal ischemia reperfusion (I/R) injury than mice expressing normal levels of meprin alpha. Bilateral renal I/R in normal meprin expressing mice resulted in a rapid [3 h] redistribution of meprin alpha and beta in cells in S3 segments of proximal tubules, followed by shedding of apical cell membrane and detachment of cells. Meprin redistribution and juxtamedullary tubular damage co-localized with activated leukocytes. To explore the role of meprins in disease, mice with targeted disruption of the meprin beta gene were generated. These mice expressed the same levels of meprin alpha mRNA as wild-type mice, however, the meprin alpha protein was not localized on the cell membrane and was secreted in an inactive form. By six hours of reperfusion, meprin beta deficient mice had better preservation of renal function than wild-type mice as determined by lower plasma blood urea nitrogen (BUN) and lower Levels of IL-6 and osteopontin mRNA. BUN levels were significantly lower in KO mice at all subsequent time points. At 96 hr post ischemia, mRNA expression levels for iNOS, TNF alpha, and HSP-27 (indicators of inflammation) were significantly lower in the meprin beta deficient mice. These results indicate that meprins are important in the pathogenesis of renal injury following ischemia/reperfusion. We propose that the redistribution of active meprin alpha is a major contributor during I/R to renal injury and subsequent inflammation. (Supported by NIH DK 19691)

Identifying site‐specific chemical modifications of urinary excreted proteins using a proteomics approach

Hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis and 2,3,5-tris-(glutathion-S-yl)hydroquinone is nephrocarcinogenic in the Eker rat. Previous studies have shown that HQGSH adducted proteins were localized in the S3 segment of the renal proximal tubules in the outer stripe of the outer medulla, which is the target region for toxicity. Recent technological advancements allow for the detection of chemical-induced post-translational modifications (PTMs) that may either indirectly alter cell signaling pathways or alter the structure and function of the modified proteins. We have now identified such modified proteins following administration of 2-(glutathion-S-yl)HQ (GSHQ) (400mmol/kg, iv, 2hr) to Long Evans (wild-type Eker) rats. Excreted urinary proteins were acetone precipitated, separated on a 1D gel, in-gel digested and analyzed by MudPIT analysis. pMOD and SALSA, in addition to DeNovo sequencing were utilized to identify site-specific chemical-induced PTMs of at least 40 proteins. 2-(Cystein-S-yl)HQ (CSHQ), a metabolite of GSHQ, was identified as the post-translational adduct on several selective solvent-exposed E, K, and R residues on various proteins, including g-actin and hemopexin. GSHQ is adducted through the carboxyl group of E but with the e-amino group of K and R. Oxidative modifications on M residues were also identified in >30 proteins. The site-specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response. (GM39338, GM070890, ES06694).

ROLE OF ANGIOTENSIN II IN MODULATING FLOW‐DEPENDENT BICARBONATE TRANSPORT IN MOUSE PROXIMAL TUBULES

We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ and HCO3− absorption and that flow-dependent modulation of proximal tubule bicarbonate reabsorption is due to changes in both NHE3 and H-ATPase activity within the luminal cell membrane. Since Angiotension II is one of the important autocrine hormones that regulate Na+ and HCO3− transport in the proximal tubule, we examined flow-dependent transport in AT1 receptor knockout mice as well as the effect of blockers of AT1, NHE3 and H-ATPase. Mouse proximal tubule S2 segments were microperfused in vitro at flow rates of 5 and 20nl/min in the absence and presence of losartan, EIPA or bafilomycin in the lumen. HCO3− (JHCO3) absorption was significantly lower under both flow rates in AT1 knockout compared to the wild-type mice (see table). Table 1. Flow-dependent Bicarbonate Absorption The fractional increases in JHCO3 in the AT1 KO, the losartan treated, and the wild type mice were 100%, 130% and 94%, the absolute changes were 42.74, 57.76 and 58.5pmole/min/mm and were all comparable. When the components of JHCO3 were assessed using inhibitors, it was found that the fractional increases in JHCO3 were reduced 49% by EIPA plus losartan or by bafilomycin plus losartan in the wild-type mice. We conclude that angiotensin II is not critical to the signal cascade that mediates flow-dependent proton secretion, and that flow impacts transport by both NHE3 and the H-ATPase comparably.

Establishment of renal stem/progenitor-like cell line from S3 segment of proximal tubules in adult rat kidney

Establishment of renal stem/progenitor-like cell line from S3 segment of proximal tubules in adult rat kidney

Establishment of renal stem/progenitor-like cell line from S3 segment of proximal tubules in adult rat kidney

Significant attention has been directed to the biologic and therapeutic capabilities of stem cells. However, it is still unknown whether stem/progenitor cells exist in adult kidney. We attempted to isolate and establish of an epithelial cell capable of growth from a single cell, which may indicate a potential for self renewal as stem/progenitor cell population. We dissected various segments of nephron from an adult Sprague-Dawley rat kidney. Each segment was cultured separately. Outgrowing cells were harvested and replated after limiting dilution so that each well contained a single cell. Cell colonies were observed only in the wells contained S3 segment-derived cells. One of cell lines most potent to grow was designated as rKS56. rKS56 cells showed cobblestone appearance and they were positive for pan-cytokeratin and vimentin by immunnocytechemistry. Reverse transcription-polymerse chain reaction (RT-PCR) study revealed rKS56 expressed both immature cell markers such as Pax-2, Wnt-4, and WT-1 and mature tubular cell markers like aquaporin-1 and chloride channel. rKS56 cells grew exponentially and could be maintained over 300 days without transformation. In different culture conditions, rKS56 cells differentiated into mature tubular cells like proximal or distal tubular cells defined by aquaporin-1 and -2 expression and responsiveness to parathyroid hormone or vasopressin. Engrafted to kidney in rat ischemic reperfusion model, rKS56 cells labeled with dioctadecyl tetramethylindocarbocyanine perchlorate (DiI) replaced in injured tubules in part at 7 days after implantation. These results suggest rKS56 possess characteristic of self-renewal, multiplasticity, and direct contribution to tissue repair and this step will potentially contribute to the development of novel therapies for end-stage renal disease (ESRD).