Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (mean +/- SE, fmol/mm): S-1 proximal, 10.8 +/- 2.1; S-2, 10.0 +/- 2.3; S-3, 21.3 +/- 3.1; type 1 thin descending limb (DTL), 12.9 +/- 4.6; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.     

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.     

Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury.

Previous studies have suggested that EGF or other members of the EGF family of mitogenic proteins are involved in proliferation of renal tubular epithelial cells occurring during recovery from injury to the kidney. The present studies examined whether expression of mRNA for the recently identified heparin-binding EGF-like growth factor (HB-EGF) is regulated in response to renal injury induced by either ischemia/reperfusion or mercuric chloride. Increased expression of HB-EGF mRNA was demonstrated in the post-ischemic kidney within 45 min of unilateral ischemia/reperfusion in the rat. Induction of HB-EGF mRNA occurred only when ischemia was followed by reperfusion, and was not eliminated by removal of blood cells from the post-ischemic kidney by saline perfusion. In situ hybridization with 35S-labeled antisense riboprobes of HB-EGF indicated that compared with control, there was increased HB-EGF mRNA expression in the 6 h post-ischemic kidney in the inner cortex and outer medulla in a patchy distribution, with the greatest expression in the inner stripe of the outer medulla. Expression occurred primarily in tubular epithelial cells. Recombinant human HB-EGF stimulated [3H]-thymidine incorporation in both primary cultures of rabbit proximal tubule cells and NRK 52E normal rat kidney epithelial cells, with potency similar to that of EGF. Induction of HB-EGF mRNA was observed in tubules freshly isolated from rat renal cortex or outer medulla when the tubules were subjected to reoxygenation after incubation in anoxic conditions. The nephrotoxin, mercuric chloride, also caused induction of HB-EGF mRNA both in vivo and in isolated rat cortical tubules. The anoxia/reoxygenation-induced expression of HB-EGF mRNA in isolated tubules was inhibited by the free radical scavengers, di- and tetra-methylthiourea, indicating involvement of reactive oxygen species. These findings indicate that HB-EGF mRNA is inducible in the kidney in vivo by acute tubular injury and suggest that HB-EGF may act as an autocrine/paracrine growth factor involved in proliferation of tubular epithelial cells and repair of the kidney.     

Tacrolimus and cyclosporinein vitro and in vivo induce osteopontin mRNA and protein expression in renal tissues

The mechanism of immunosuppression-linked nephrotoxicity in organ transplantation remains to be solved. Expression of osteopontin (OPN), a multifunctional secreted glycoprotein, has been associated with various forms of renal injuries. In this study, using in vitro and in vivo models, we examined the effects of cyclosporine (CsA) and tacrolimus (TAC) on OPN mRNA and protein expression. We also examined if CsA- and TAC-induced OPN expression is dependent on transforming growth factor (TGF)-beta expression. For in vivo experiments mice and rats were injected with CsA (25 mg/kg) and TAC (0.2 mg/kg). For in vitro experiments, human proximal tubular epithelial (PTE) cells were treated with CsA and TAC for 4 h. To study the in vivo effect of TGF-beta on OPN mRNA, mice were injected with recombinant TGF-beta protein (3 mg/kg). The expression of OPN was also studied in CsA-treated PTE cells with and without anti-TGF-beta antibody. At the end of in vitro and in vivo treatments, RNA was isolated from kidney tissue and renal cells reverse transcribed to cDNA and amplified for OPN mRNA. Using immunochemistry and Western blot analysis OPN protein expression was also studied in vivo and in vitro, respectively. Both in vitro and in vivo treatment with CsA and TAC resulted in significantly increased OPN mRNA and protein expression. TGF-beta treatment in vivo also resulted in a significantly increased OPN mRNA expression and anti-TGF-beta antibody but not the control antibody in vivo in CsA-treated mice, and in vitro in CsA-treated PTE cells inhibited OPN mRNA expression. OPN may contribute to the CsA- and TAC-induced nephrotoxicity in organ transplant recipients and the increased OPN expression might be mediated by TGF-beta.     

骨桥蛋白在糖尿病大鼠肾组织中的表达及意义

目的 观察糖尿病大鼠肾组织中骨桥蛋白(OPN)的表达及其与巨噬细胞(CD68)、细胞增殖核抗原(PCNA)之间的关系,初步探讨其在糖尿病肾损害中的意义.方法 雄性Wistar大鼠被随机均分为对照组和糖尿病组,每组24只.腹腔单次注射链脲佐菌素(STZ)诱发糖尿病大鼠模型,采用免疫组化和蛋白质免疫印迹法(Western blotting)分别检测OPN、CD68及PCNA在肾组织的表达,原位杂交检测肾组织中OPN mRNA的表达.结果 与对照组比较,糖尿病组大鼠在注射STZ后1、2、4和8周,血糖、尿素氮(BUN)和24 h尿蛋白定量显著增高,肌酐清除率(CCr)显著降低;OPN表达呈进行性增加并明显高于对照组(P均＜0.05);1周和2周时PCNA表达高于对照组,4周和8周时CD68表达较对照组略有增高.结论 OPN在糖尿病肾损害过程的早期可能与肾小管上皮细胞增殖有关,后期可能趋化巨噬细胞浸润.     

Evidence for beta adrenoceptors in proximal tubules. Isoproterenol-sensitive adenylate cyclase in pars recta of canine nephron.

Observations in vivo suggest that catecholamines modulate reabsorptive functions of proximal tubules by acting on beta-adrenoceptors. However, beta-catecholamine binding sites or beta-adrenoceptor-sensitive adenylate cyclase (AdC) has not been found in segments of proximal tubules of rat, rabbit, or mouse kidney. In the present study, we investigated the responsiveness of AdC to catecholamines, [8-Arg]vasopressin (AVP), and to parathyroid hormone (PTH) in proximal convoluted tubules (PCT), proximal straight tubules (PST), and in late distal convoluted tubules (LDCT) microdissected from canine kidney. Isoproterenol (ISO) caused a marked and dose-dependent stimulation of AdC in PST (maximum: delta + 850%; half maximum stimulation at 10(-7) M ISO), but ISO had no effect on AdC in PCT. The AdC in both PCT and PST was markedly stimulated by PTH; AVP stimulated the AdC in LDCT but not in PST or in PCT. The stimulatory effect of 10(-5) M ISO in PST (delta + 725%) was significantly greater than in LDCT (delta + 307%); norepinephrine and epinephrine had stimulatory effects in PST similar to ISO. The stimulation of AdC in PST by ISO was blocked by propranolol and by beta 2-blocker ICI-118551. On the other hand, alpha-blocker phentolamine and beta 1-blocker metoprolol did not abolish the stimulation of AdC in PST by ISO. The accumulation of cAMP in intact PCT and PST incubated in vitro was stimulated by PTH both in PST and in PCT, but ISO elevated cAMP (delta + 683%) only in PST. Our results show that proximal tubules of canine nephron, PST but not PCT, contain beta-adrenoceptors of beta 2 subtype coupled to AdC. These observations provide direct evidence that the effects of catecholamines, either released from renal nerve endings or arriving from blood supply, can act directly on beta 2-adrenoceptors located in proximal tubules, and also suggest that at least some of the catecholamine effects in proximal tubules are mediated via cAMP generation.     

The metabolism of labeled parathyroid hormone. IV. Autoradiographic studies.

Autoradiographs were prepared from tissues of rats sacrificed 10 minutes after injection of biologically active 125I-labeled parathyroid hormone. No radioactivity was seen in intestine and muscle. Deposition in liver was diffuse showing some sinusoidal concentrations. Depostion in kidney was high and, nearly all activity appeared in selected tubules (presumably proximal tubules) in the outer third of the cortex. Specific localization was also seen in bone particularly in the cellular layers of periosteum and endosteum adjacent to bony matrix and to some extent in osteocytes.     

Anatomical and developmental patterns of facilitative glucose transporter gene expression in the rat kidney.

In situ hybridization was used to map cellular patterns of gene expression for facilitative glucose transporters (GTs) 1-5 in the developing and adult rat kidney. GT3 was not detected. GT1 mRNA was present in the proximal straight tubule (PST), distal nephron and collecting duct. GT2 mRNA was localized in both proximal convoluted and PST, while GT5 mRNA was detected only in the PST. GT4 mRNA and immunoreactivity were focally localized in the thick ascending limb of Henle's loop and were coexpressed with IGF-I. Thus, each of the four different isoforms demonstrated a distinct renal distribution, with GTs 1, 2, and 5 coexpressed in the PST. Renal GT1 and GT5 gene expression were unchanged throughout development, while GT2 was most abundant before weaning and GT4 was first detected after weaning. Only GT4 appeared to be hormonally regulated: It was decreased after hypophysectomy and increased after vasopressin treatment, but was not affected by 1 or 4 d of insulinopenic diabetes mellitus. The coexpression of GT4 and IGF-I in the thick ascending limb segment of the nephron suggests a novel autocrine/paracrine mechanism by which cells may control local fuel economy independently from that of the larger structure to which they belong and from the systemic hormonal milieu.     

Renal tubular protein absorption in the rat

Radioiodinated protein solutions, 20 nl in volume, were injected into surface proximal and distal convoluted tubules of the rat kidney. The unabsorbed fraction was collected in ureteral urine, and the percentage recovered was expressed as a function of the injection site. Recovery increased progressively from more distal sites of injection indicating absorption along the proximal tubule of human serum albumin, insulin, and ribonuclease. Fractional absorption of albumin along the proximal tubule varied from 0 to 20% of the injected load, and was similar when the injectate concentration was 20, 40, or 100 mg/100 ml. Fractional absorption along the proximal tubule of insulin and ribonuclease, smaller proteins, was 30 to 50% of the injected load, and was similar with insulin concentrations of 0.09 mg/100 ml and 40 mg/100 ml and ribonuclease concentration of 40 mg/100 ml. In addition to this constant fractional absorption of each protein in the proximal tubule, smaller amounts were absorbed when injections were made in distal convoluted tubules.     

Localization of NBC1 variants in rat kidney

Na+-HCO3- cotransporter (NBC1) plays a major role in bicarbonate reabsorption from proximal tubules. In a previous immunohistochemical study on human kidney, we showed that the kidney-type transporter (kNBC1) was abundantly expressed in the basolateral membranes of proximal tubules while the expression of pancreatic-type transporter (pNBC1) was undetectable. In the present study we tried to determine the localization of NBC1 variants in rat kidney using the antibodies against the unique N-terminal regions of kNBC1 and pNBC1. In Western blot analysis on the membrane-enriched fraction from rat kidney both anti-kNBC1 and anti-pNBC1 antibodies yielded a approximately 130 kDa band. In immunohistochemical analysis with confocal microscopy the anti-kNBC1 antibody produced a strong and exclusively basolateral labeling in proximal tubules. On the other hand, the occasional pNBC1 labeling was detected in the apical membranes of proximal tubules. The electron microscopic observation further supported the basolateral localization of kNBC1 as well as the localization of pNBC1 on the basis of the brush border. Acute metabolic acidosis did not change the protein expression levels as well as the intracellular distribution of both NBC1 variants in rat kidney. These results are consistent with a view that kNBC1 is the dominant variant that mediates bicarbonate reabsorption from rat renal proximal tubules. They also indicate that species difference may exist regarding the distribution of NBC1 variants in kidney.     

顺铂诱发大鼠肾损伤的组织病理学变化观察

目的 :探讨顺铂 (Cisplatin,CP)诱导大鼠肾损伤的组织病理学变化。方法 :Wistar成年大鼠腹腔注射 CP7mg/kg。于给药后不同时间取肾 ,常规石蜡切片、HE染色和 PAS反应。结果 :CP组鼠引起肾皮质损伤 ,可见肾小体肿大毛细血管基膜损伤 ,近端小管上皮浊肿、空泡形成乃至细胞脱落死亡。结论 :CP可引起严重肾小管损伤 ,损伤恢复缓慢。     

Expression of monocyte chemoattractant protein-1 in proximal tubular epithelial cells in a rat model of progressive kidney failure

Impairment of kidney function in various types of glomerular disease is associated with tubulointerstitial changes. Monocyte chemoattractant protein-1 (MCP-1) is up-regulated in the tubulointerstitium and in the glomeruli in many human and experimental kidney disorders. We investigated the localization of MCP-1 expression in a rat model of progressive kidney failure. Male Wistar rats were subjected to subtotal nephrectomy (n = 30) or sham surgery (n = 30). Immunohistochemistry with immunoelectron microscopy and in situ hybridization were used to examine the expression of MCP-1 protein and messenger ribonucleic acid (mRNA) in the kidney, respectively. MCP-1 protein and mRNA were hardly detected in both glomeruli and tubulointerstitium of control rats. However, in the rats subjected to nephrectomy, MCP-1 expression was increased in the tubular cells of the remnant kidney, accompanied by significant macrophage infiltration. MCP-1 was observed mainly in the proximal tubular cells and only weakly in distal tubular cells. No significant expression of MCP-1 protein or mRNA was noted in the glomeruli. Immunoelectron microscopy showed the presence of MCP-1 in the rough endoplasmic reticulum of proximal tubular cells, confirming that MCP-1 is produced in proximal tubular cells. MCP-1 was also observed in endocytic vesicles adjacent to the brush border of proximal tubular cells, suggesting incorporation of MCP-1 from the tubular lumen. Our findings indicate localized expression of MCP-1 in proximal tubular cells in the remnant kidney and suggest that MCP-1 in proximal tubular cells is involved in tubulointerstitial damage in chronic kidney failure associated with glomerular hypertension.     

Renin and renin mRNA in proximal tubules of the rat kidney.

The present study was undertaken to assess the presence of renin enzymatic activity and renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD), and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405 +/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST, although its abundance per mm tubule was about 1/500th that found in one glomerulus. Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was confirmed by restriction digestion. CEI administration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular signals in control of renin synthesis in the proximal tubule than in the vascular compartment.     

Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.

Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension.     

Can urine osteopontin levels,which may be correlated with nutrition intake and body composition,be used as a new biomarker in the diagnosis of nephrolithiasis?

Background and aimThe nephrolithiasis has a multifactorial etiology resulting from the interaction of metabolic, genetic and environmental factors. Parameters such as nutrition and urinary osteopontin (OPN) level may affect kidney stone formation. The purpose of this study is to evaluate the correlation between urinary OPN level and kidney stone formation and effect of nutrition on OPN level in nephrolithiasis.Materials and methodsThis study was conducted on 88 volunteers including 44 healthy individuals and 44 patients diagnosed with nephrolithiasis and aging between 20 and 65 years. Some serum parameters and urinary OPN levels of the individuals were analyzed. Several anthropometric measurements of the individuals were taken and calculated their body mass index. Additionally, 24-hour dietary recall and water intakes were recorded and the participants completed food-frequency questionnaire for the evaluation of their nutritional status.ResultsUrinary OPN (ng/mL) levels of patients were lower than that of control group (p<0.05). Dietary energy, carbohydrate, poly-unsaturated fatty acid (PUFA) and n-6 fatty acids intakes and urinary OPN levels of male patients were positively correlated (p<0.05). Additionally, there was a negative correlation between their urinary OPN (ng/mL) and serum creatinine (mg/dL) levels of female patients (p<0.05). Body weight, waist circumference, hip circumference and body muscle mass values of healthy males were positively correlated with their urinary OPN levels (p<0.05).ConclusionsResults of the study showed that low urinary OPN levels were correlated with increased kidney stone risk, and dietary habits can affect urinary OPN level.     

Expression of transforming growth factor-beta-inducible gene-h3 in normal and cyclosporine-treated rat kidney

Up-regulation of transforming growth factor-beta (TGF-beta) is known to play an important role in the tubulointerstitial injury of chronic cyclosporin A (CsA) nephropathy, but the expression of the TGF-beta-inducible gene-h3 (betaig-h3) is undetermined. In this study we examined betaig-h3 expression and its relationship to tubulointerstitial injury in a rat model of chronic CsA nephropathy. Sprague-Dawley rats kept on a low-salt diet (0.05% sodium) were treated daily for 4 weeks with subcutaneous injections of vehicle (olive oil, 1 mL/kg) or CsA (15 mg/kg). The expression of betaig-h3 messenger RNA (mRNA) and protein was evaluated with the use of in situ hybridization, immunohistochemical methods, and immunoblotting. We also compared renal function, histologic findings (tubulointerstitial fibrosis), and expression of TGF-beta1 among treatment groups. In vehicle-treated kidney, betaig-h3 mRNA and protein were constitutively expressed in the outer medulla and cortex, which was confined to the terminal portion of afferent arterioles, the S3 segment of the proximal tubules, and distal convoluted tubules. CsA treatment significantly up-regulated betaig-h3 expression in the interstitium, especially in expanded and fibrotic areas. Quantitative analysis revealed that CsA induced a significant (twofold) increase in betaig-h3 mRNA and protein, and this increase was correlated with up-regulation of TGF-beta1 expression (r =.943, P <.001) and the tubulointerstitial fibrosis score (r =.746, P =.05). Our observations indicate that an increase in betaig-h3 expression, along with TGF-beta1 up-regulation, is closely associated with tubulointerstitial fibrosis in a rat model of chronic CsA nephropathy.     

Detection of glucagon receptor mRNA in the rat proximal tubule: potential role for glucagon in the control of renal glucose transport

Glucagon is known to affect glomerular filtration rate and renal tubular solute and fluid transport, although it is only thought to act directly on the thick ascending limb (TAL) and collecting duct (CD). Indeed, previous studies have detected glucagon-sensitive adenylate cyclase exclusively in these nephron segments, suggesting the presence of glucagon receptors. In the present study, we have demonstrated for the first time that glucagon receptor mRNA is expressed in the rat proximal tubule, as well as in the TAL and CD. By autoradiography, we have also shown that specific binding of glucagon occurs in both the renal cortex and medulla. In addition, using proximal tubule brush-border membrane (BBM) vesicles for studies of glucose transport, we have established that glucagon stimulates glucose uptake via a facilitative GLUT-mediated transport process (by 58%; P < 0.005), whereas cAMP stimulates only the sodium glucose-linked transporter ('SGLT')-mediated glucose uptake (by 53%; P < 0.05). Taken together, these findings suggest that glucagon could have a role in controlling proximal tubular transport function, including glucose reabsorption, but unlike in the TAL and CD, the proximal tubule glucagon receptor might not be coupled primarily to adenylate cyclase.     

Autoradiographic study of tobramycin uptake by proximal and distal tubules of normal and pyelonephritic rats.

Multiple factors may modify the pharmacokinetics of aminoglycosides and affect their nephrotoxic potential. In the present study, the influence of Escherichia coli pyelonephritis on the renal handling of [3H]tobramycin was investigated. The accumulation of [3H]tobramycin in proximal and distal tubules in both normal and infected rats was compared. Following induction of pyelonephritis, disturbed intrarenal localization of the drug was noted. Grain counts were affected in both proximal and distal tubules. Decreased labeling was observed at all time intervals in the proximal tubules. Electron microscopy showed that radioactivity was associated mostly with lysosomes in both normal and infected rats 1 and 24 h following the injection of the drug. We could detect significantly higher amounts of drug in the distal tubules of the pyelonephritic kidney than the normal levels at 10 min and 24 h postinjection. The drug did not seem to be associated with any particular organelle and was evenly distributed within the distal tubular cells. The present study shows that the transport of tobramycin within the infected nephron is disturbed. These data might shed some light on the influence of infection on the intrarenal pharmacology of aminoglycosides.     

Expression of osteopontin in cisplatin-induced tubular injury

Osteopontin (OPN) is considered as a key protein in cell regeneration. OPN is thought to have many functions in cell-cell binding and cell-matrix binding via OPN receptors in various organs. But there is little information on the precise role of OPN. To clarify the functional role of OPN in tubular injury, we performed in situ hybridization and immunohistochemical analysis of the expression of OPN in a renal cortical necrosis model induced by cisplatin from the acute injury to late recovery phases. In the acute injury phase, both mRNA and protein of OPN were markedly induced in damaged tubular lumens with cell debris. In the late recovery phase, on the other hand, OPN protein and mRNA were observed in dilated and flattened tubular epithelial cells showing a regenerative appearance. Most of these cells were also immunostained with CD44, a receptor of OPN. PCNA staining was also co-localized with these expressions. In light of the CD44 function regulating cell proliferation, these findings suggest that OPN may contribute to regeneration of tubular epithelial cells during the acute to late recovery phases of cortical tubular damage induced by cisplatin.     

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

In previous studies we have shown that the gene encoding cholecystokinin (CCK) is expressed in spermatogenic cells of several mammalian species. In the present study we show that a gene homologous to the CCK-related hormone, gastrin, is expressed in the human testis. The mRNA hybridizing to a human gastrin cDNA probe in the human testis was of the same size (0.7 kb) as gastrin mRNA in the human antrum. By in situ hybridization the gastrinlike mRNA was localized to seminiferous tubules. Immunocytochemical staining of human testis revealed gastrinlike peptides in the seminiferous tubules primarily at a position corresponding to spermatids and spermatozoa. In ejaculated spermatozoa gastrinlike immunoreactivity was localized to the acrosome. Acrosomal localization could also be shown in spermatids with electron microscopy. Extracts of the human testis contained significant amounts of progastrin, but no bioactive amidated gastrins. In contrast, ejaculated sperm contained mature carboxyamidated gastrin 34 and gastrin 17. The concentration of gastrin in ejaculated human spermatozoa varied considerably between individuals. We suggest that amidated gastrin (in humans) and CCK (in other mammals) are released during the acrosome reaction and that they may be important for fertilization.