A model of hemodialysis after acute kidney injury in rats.

BACKGROUND: Acute kidney injury (AKI) is frequent among critically ill patients. Renal replacement therapy (RRT) is often required to deal with severe complications of AKI. This technique is however associated with side effects such as hemodynamic instability and delayed renal recovery. In this study, we aimed to describe a novel model of hemodialysis in rats with AKI and depict a dialysis membrane performance. METHODS: Eighteen Sprague-Dawley rats received 0.75% adenine-rich diet to induce AKI. After 2 weeks, nine underwent an arterio-venous extracorporeal circulation (ECC) (ECC group) for 2 h without a dialysis membrane on the circuit and nine received a hemodialysis session (HD group) for 2 h with an ECC circuit. All rats were hemodynamically monitored, and glomerular filtration rate (GFR) was measured by transcutaneous fluorescence after the injection of FITC-Sinistrin. Blood samples were collected at different time points to assess serum creatinine and serum urea concentrations and to determine the Kt/V. Sinistrin concentration was also quantified in both plasma and dialysis effluent. RESULTS: After 2 weeks of adenine-rich diet, rats exhibited a decrease in GFR. Both serum urea and serum creatinine concentrations increased in the ECC group but remained stable in the HD group. We found no significant difference in serum creatinine and serum urea concentrations between groups. At the end of experiments, mean serum urea was 36.7 mmol/l (95%CI 19.7-46.9 mmol/l) and 23.6 mmol/l (95%CI 15.2-33.5 mmol/l) in the ECC and HD groups, respectively (p = 0.15), and mean serum creatinine concentration was 158.0 µmol/l (95%CI 108.1-191.9 µmol/l) and 114.0 µmol/l (95%CI 90.2-140.9 µmol/l) in the ECC and HD groups, respectively (p = 0.11). The Kt/V of the model was estimated at 0.23. Sinistrin quantity in the ultrafiltrate raised steadily during the dialysis session. After 2 h, the median quantity was 149.2 µg (95% CI 99.7-250.3 µg). CONCLUSIONS: This hemodialysis model is an acceptable compromise between the requirement of hemodynamic tolerance which implies reducing extracorporeal blood volume (using a small dialyzer) and the demonstration that diffusion of molecules through the membrane is achieved.

Intrarenal urothelium proliferation: an unexpected early event following ischemic injury.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process.

[Renal hemodynamics and development of renal fibrotic lesions during hypertension]. / Dissociation entre l'hémodynamique rénale et les lésions histologiques rénales induites par l'hypertension artérielle.

Hypertension is frequently associated with the development of renal fibrosis leading to chronic renal failure. The objective of the present study was to evaluate the role of blood pressure and renal hemodynamics on the development of renal lesions during hypertension. To this end, rats were treated with a NO synthase inhibitor, L-NAME, for 4 weeks. At this time point, systolic blood pressure reached 170 mmHg, renal blood flow dropped to 3.3 +/- 0.7 ml/min and kidneys displayed glomerular and tubulo-interstitial lesions as evidenced by histological analysis. Thereafter, L-NAME treatment was combined with an AT1 receptor antagonist, losartan (30 mg/kg/d), for an additional period of 4 weeks. Treatment with losartan for 4 additional weeks did not significantly modify hypertension (168 mmHg) either the degree of tubulo-interstitial lesions; in contrast, a significant regression of ischemic and sclerotic glomerular lesions was observed. In parallel, renal blood flow was significantly improved by losartan (5.2 +/- 0.8 ml/min). In addition a negative correlation was observed between renal blood flow and index of glomerulosclerosis (r = -0.82), whereas tubulo-intarstitial damage was positively correlated to systemic pressure (r = 0.93). In conclusion, inhibition of the local effects of angiotensin II alleviates the fall of renal blood flow consecutive to NO deficiency and reduces the morphological and functional lesions of glomeruli, independently of the changes in blood pressure. In contrast, tubulo-interstitial lesions are not correlated with the levels of renal blood flow and do not regress with the blockade of AT1 receptors when rats remain hypertensive.

Regulation of ANP clearance receptors by EGF in mesangial cells from NOD mice.

Mesangial cells from nonobese diabetic (NOD) mice (D-NOD) that develop diabetes at 2-4 mo express an increased density of atrial natriuretic peptide (ANP) clearance receptors [natriuretic peptide C receptor (NPR-C)] and produce less GMP in response to ANP than their nondiabetic counterparts (ND-NOD). Our purpose was to investigate how both phenotypic characteristics were regulated. Epidermal growth factor (EGF) and heparin-binding (HB)-EGF, but not platelet-derived growth factor or insulin-like growth factor I, inhibited (125)I-ANP binding to ND-NOD and D-NOD mesangial cells, particularly in the latter. NPR-C density decreased with no change in the apparent dissociation constant, and there was also a decrease in NPR-C mRNA expression. The EGF effect depended on activation of its receptor tyrosine kinase but not on that of protein kinase C, mitogen-activated protein kinases, or phosphoinositide-3 kinase. Activation of activator protein-1 (AP-1) was necessary, as shown by the inhibitory effect of curcumin and the results of the gel-shift assay. The cGMP response to physiological concentrations of ANP was greater in EGF-treated D-NOD cells. These studies suggest that EGF potentiates the ANP glomerular effects in diabetes by inhibition of its degradation by mesangial NPR-C via a mechanism involving AP-1.

Angiotensin II activates collagen type I gene in the renal vasculature of transgenic mice during inhibition of nitric oxide synthesis: evidence for an endothelin-mediated mechanism.

BACKGROUND: Hypertension is frequently associated with renal vascular fibrosis. The purpose of this study was to investigate whether angiotensin II (Ang II) is involved in this fibrogenic process. METHODS AND RESULTS: Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha(2) chain promoter [procolalpha(2)(I)]. Hypertension was induced by chronic inhibition of NO synthesis (N(G)-nitro-L-arginine methyl ester, L-NAME). Procolalpha(2)(I) activity started to increase in the renal vasculature after 4 weeks of L-NAME treatment (P<0.01) and at 14 weeks reached 3- and 8-fold increases over control in afferent arterioles and glomeruli, respectively (P<0.001). Losartan, an AT(1) receptor antagonist, given simultaneously with L-NAME prevented the increase of procolalpha(2)(I) levels and attenuated the development of renal vascular fibrosis without normalizing systolic pressure increase. Because we found previously that endothelin mediated renal vascular fibrosis in the L-NAME model, the interaction between Ang II, endothelin, and procolalpha(2)(I) was investigated in ex vivo and short-term in vivo experiments. In both conditions, the Ang II-induced activation of procolalpha(2)(I) in renal cortex was blocked by an endothelin receptor antagonist. CONCLUSIONS: During chronic inhibition of NO, the collagen I gene becomes activated, leading to the development of renal vascular fibrosis. Ang II is a major player in this fibrogenic process, and its effect on collagen I gene is independent of systemic hemodynamics and is at least partly mediated by the profibrogenic action of endothelin.

Mesangial cells from diabetic NOD mice constitutively express increased density of atrial natriuretic peptide C receptors.

BACKGROUND: Experimental evidence shows that natriuretic peptides (NPs) play a pathophysiological role in the glomerular hemodynamic abnormalities that occur in diabetes mellitus. METHODS: In this study, the cGMP response to NPs and the different subtypes of NP receptors were examined in mesangial cells derived from a genetic model of diabetes, the nonobese diabetic (NOD) mouse. Multiple mesangial cell lines were derived from diabetic (D-NOD) and nondiabetic (ND-NOD) adult mice and were studied at different passages. RESULTS: cGMP accumulation after stimulation by atrial NP (ANP) or C-type NP (CNP) was markedly inhibited in D-NOD cells irrespective of the glucose concentration (6 or 20 mM) in the culture medium. In contrast, NP receptor density measured from [125I]-ANP saturation binding curves was 7.5 times greater in D-NOD than in ND-NOD cells. No change in KD (200 pM in both cell lines) was observed. Competitive inhibition studies showed that 4-23 C-ANP, which is specific of clearance receptors (NPR-C), displaced 90% of the maximum fraction bound, suggesting the predominance of NPR-C in both cell lines. Further identification was obtained from RNase protection assay and reverse transcription-polymerase chain reaction, which also demonstrated the higher expression of NPR-C mRNA in D-NOD cells. In contrast, NPR-A mRNA was not modified. Increased expression of NPR-C in D-NOD cells was associated with an increase of ANP internalization rate at 37 degrees C, indicating that these receptors were functional. CONCLUSIONS: These studies demonstrate that the constitutive overexpression of NPR-C in D-NOD mesangial cells is associated with a decreased response of cGMP to ANP or CNP treatment. This could be due to the lesser availability of the peptides for binding to NPR-A or NPR-B or to an inhibitory effect on NP-dependent guanylate cyclase activity via the activation of NPR-C.

Characterization of B2-bradykinin receptors in rabbit principal cells of the collecting duct.

Bradykinin (BK) plays a key role in collecting duct functions. Using an established line of principal cells of the rabbit collecting duct (R.C. SV3), we examined the characteristics of the BK receptors in these cells. [3H]-BK bound specifically to R.C. SV3. Saturation binding analyses allowed KD (968 +/- 232 pM) and Bmax values (356 +/- 43 fmol/mg protein) to be calculated. Competitive displacement of [3H]-BK was observed with Hoe-140, a specific type 2 BK receptor (BKR-2) antagonist, but not with des arg9-BK, a BKR-1 agonist. The presence of BKR-2 was confirmed by the reverse-transcription polymerase chain reaction technique. BK stimulated cytosolic calcium and inositol phosphate formation in a dose-dependent manner (from 1 nM to 1 microM). BK also inhibited the arginine vasopressin dependent increase of cyclic adenosine monophosphate. This effect could not be related to the production of prostaglandin E2. These results demonstrate the presence of high-affinity BKR-2 in the principal cells of the rabbit collecting duct that are linked to phospholipase C activity and are involved in arginine vasopressin related regulatory loops.

Prostaglandin E2 enhances type 2-bradykinin receptor expression in human glomerular podocytes.

We examined the effect of prostaglandin E2 (PGE2) on bradykinin (BK) binding, BK-dependent intracellular calcium and inositol phosphate (i.p.) concentrations and BK mRNA in human glomerular visceral epithelial cells (hGVEC). PGE2 (10 nM) produced a concentration-dependent increase in [3H]-BK specific binding after a lag time of 24 h with a threshold at 0.1 nM. This increase appeared to be mediated exclusively by an increase in BK receptor (BKR)-2 expression. Scatchard analysis of [3H]-BK saturation binding showed that PGE2 produced an increase in the receptor site density without a change in the apparent dissociation constant. PGE2 also markedly stimulated cAMP production. This effect was thought to mediate the increase in expression of BKR-2 as 8-bromo cAMP and various cAMP-stimulating agents acted similarly. PGE2 did not change the BK-dependent intracellular IP3 and cytosolic calcium increases. The overexpression of BKR-2 in the presence of PGE2 was associated with an increase in mRNA as shown by the nuclease protection assay without any change in mRNA half-life. Cycloheximide, an inhibitor of protein synthesis, enhanced BKR-2 mRNA expression. In conclusion, treatment with PGE2 stimulates the synthesis of BKR-2 in hGVEC, possibly by interfering with an inhibitory protein involved in BKR-2 transcription.

Receptors for natriuretic peptides in a human cortical collecting duct cell line.

The aim of the present study was to analyze the expression of natriuretic peptide receptors in human collecting duct, by using a newly established SV40 cell line (HCD). ANP and C-type natriuretic peptide (CNP) induced a concentration-dependent increase in cGMP suggesting the presence of type-A (NPR-A) and type-B (NPR-B) receptors, respectively. Threshold concentrations were 1 pM and 1 nM, respectively, and stimulated over basal cGMP ratios were 500 and 160 at 0.1 microM ANP and CNP. The urodilatin concentration-response curve was similar to that of ANP. [125I]-ANP bound specifically to HCD cells in a time-dependent fashion, reaching a plateau-phase between one and two hours at 4 degrees C. Equilibrium saturation binding curves suggested a single group of receptor sites (Kd = 421 +/- 55 pM, Bmax = 49.2 +/- 8.8 fmol/mg protein, Hill coefficient = 1.44 +/- 0.1, N = 6). Binding of [125I]-ANP was not displaced by CNP or by C-ANP (4-23), a specific ligand of clearance receptors (NPR-C), and thus occurred mainly via NPR-A. Neither Northern blot analysis nor RT-PCR could detect NPR-C mRNA, although the latter was clearly identified in control human glomerular visceral epithelial cells. In contrast, PCR products with the expected lengths were obtained for NPR-A and NPR-B. In conclusion, HCD cells express both NPR-A and NPR-B, as demonstrated by mRNA and cGMP production studies, but fail to produce NPR-C. This suggests that the human cortical collecting duct is a target for ANP, CNP and urodilatin.

Dexamethasone upregulates ANP C-receptor protein in human mesangial cells without affecting mRNA.

The objective of this study was to examine the role of dexamethasone on the expression of natriuretic peptide B-type and C-type receptors (ANPR-B and ANPR-C) in cultured human mesangial cells, which only possess these two subtypes. Dexamethasone caused concentration- and time-dependent increases in 125I-labeled ANP binding, which were prevented by glucocorticoid receptor inhibition with RU-38486. A lag time of 24 h and a concentration of dexamethasone of at least 1 nmol/l were necessary for this effect to occur. Dexamethasone-induced upregulation of 125I-ANP binding resulted from increased receptor density. No change in dissociation constant (Kd) was observed. Only ANPR-C were affected by dexamethasone. Indeed, dexamethasone did not modify C-type natriuretic peptide (i.e., CNP)-dependent cGMP production by mesangial cells. Moreover, dexamethasone upregulated ANPR-C protein expression as shown by Western blot analysis and by an increase in ANPR-C immunoreactivity at the cell surface. In contrast, dexamethasone did not modify ANPR-C mRNA expression. In conclusion, glucocorticoids increase ANPR-C density on mesangial cells through a mechanism implying, successively, interaction with the glucocorticoid receptor and increase of ANPR-C protein synthesis at a posttranscriptional stage. Thus dexamethasone may influence availability of natriuretic peptides at their glomerular target sites.

Characterization of C-type natriuretic peptide receptors in human mesangial cells.

Our aim was to examine whether the human glomerulus was a target for C-type natriuretic peptide (CNP) and how A, B and C receptors of natriuretic peptides (ANPR-A, ANPR-B, ANPR-C) were distributed in glomerular mesangial and epithelial cells. CNP stimulated cyclic GMP production in cultured human mesangial and epithelial cells with similar threshold concentrations (1 nM) and maximum effects (basal value x 30 at 1 microM). In contrast, atrial natriuretic peptide (ANP) was only stimulatory in epithelial cells. [125I] CNP bound specifically to mesangial cells with a Kd of 0.47 nM and Bmax of 42 fmol/mg. Equilibrium of binding was obtained after four to five hours at +4 degrees C and nonspecific binding represented 10 to 20% of total binding. HS142-1 (100 micrograms/ml), a specific inhibitor of ANPR-A and ANPR-B, suppressed 90% of CNP-dependent cyclic GMP production whereas it had little effect on [125I]-CNP binding, suggesting that C receptors were largely predominant in mesangial cells. No biological effect of CNP on mesangial cells, including change in basal or angiotensin II-induced contractility and inhibition of basal or serum-dependent proliferation, could be demonstrated. Similar results were obtained with 8-bromo-cyclic GMP and sodium nitroprusside. Intraglomerular localization of ANPR-A, ANPR-B and ANPR-C mRNA was studied using reverse transcriptase-polymerase chain reaction with amplification of their corresponding cDNA by different primers. Amplification products were identified on gel electrophoresis by their predicted sizes and sequencing. ANPR-A, ANPR-B and ANPR-C mRNA were present in epithelial cells whereas only ANPR-B and ANPR-C mRNA were detected in mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-gamma induces dipeptidylpeptidase IV expression in human glomerular epithelial cells.

Because dipeptidylpeptidase IV (DPP IV) is present in vivo on glomerular visceral epithelial cells and possesses immunogenic properties, as shown by the capability of anti-DPP IV antibody to induce the Heymann model of glomerulonephritis, we studied the expression and regulation of DPP IV in cultured human glomerular visceral epithelial cells. DPP IV is an ectoenzyme, as indicated by the rapid detection of the product of the reaction in the incubation medium of intact cells and the staining of paraformaldehyde-fixed cells in the presence of a specific anti-DPP IV antibody. DPP IV activity was inhibited by diisopropylfluorophosphate and phenylmethyl sulphonylfluoride. Its optimum pH was alkaline (7.7-8) and it exhibited a Km value of 0.94 mM. DPP IV expression was induced in cells treated by interferon-gamma (IFN-gamma). The effect was significant after a 3-day treatment with 100 U/ml. It increased with time, reaching a plateau after 11 days, and was dose-dependent with a maximum at a concentration of 1000 U/ml. Staining of the cells with anti-DPP IV antibody was also increased after a 6-day treatment with 100 U/ml IFN-gamma. It was shown by Northern analysis that, after 24 hr of exposure to 500 U/ml of IFN-gamma, DPP IV mRNA transcript was stimulated. Transcriptional activation by IFN-gamma did not require new protein synthesis. Interleukin-1 (IL-1) and cyclic AMP had a small stimulatory effect, whereas dexamethasone and phorbol esters were inefficient. These results suggest that DPP IV of glomerular epithelial cells may be up-regulated by IFN-gamma from activated T lymphocytes in glomerular diseases and during lymphocyte-mediated graft rejection.

Release of cyclic nucleotides from the apical and basolateral poles of cultured human glomerular epithelial cells.

We have examined the intracellular accumulation and the release of cGMP and cAMP from the apical and basolateral poles of SV40-transformed human glomerular visceral epithelial cells (HGVEC) cultured on filters. After treatment of the cells by atrial natriuretic peptide (ANP), cGMP did not accumulate in the cells and was rapidly released mainly into the apical medium (86% of its total secretion). Its apical secretion was inhibited by 83% in the presence of probenecid, an organic acid transport inhibitor, and by 90% in the presence of nocodazole, a microtubule disrupter. cAMP was released more slowly than cGMP after it had accumulated in the cells. Apical polarization of the secretion still existed but was less marked than for cGMP (70% of its total secretion). Similarly, probenecid and nocodazole inhibited to a lesser degree cAMP egression (66 and 33%, respectively, for the apical secretion). Polarization of HGVEC was confirmed by the vectorialized secretion of lactate dehydrogenase. These results indicate that cGMP release from HGVEC is essentially apical and depends on a probenecid-sensitive organic acid transport and also on an intact microtubular network. cAMP release appears to be less polarized and less sensitive to the two drugs studied. Vectorially secreted cGMP in the urinary space in response to ANP could act downstream in the nephron and thus permit ANP to stimulate cells which do not necessarily possess ANP receptors.