Rildo Aparecido Volpini

Lineage‐Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage‐negative (Lin−) cells to test the hypothesis that Lin− cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham (laparotomy only, untreated); Nx (five‐sixth nephrectomy and untreated); NxLC1 (five‐sixth nephrectomy and receiving 2 × 106 Lin− cells on postnephrectomy day 15); and NxLC3 (five‐sixth nephrectomy and receiving 2 × 106 Lin− cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)‐1β, tumor necrosis factor‐α, and IL‐6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein‐1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin‐dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin− cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin− cell‐treated rats than in untreated rats. Lin− cell treatment significantly improved survival. These data suggest that Lin− cell treatment protects against chronic renal failure. STEM CELLS 2009;27:682–692

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.

N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation

Sepsis is a common cause of acute kidney injury (AKI) and acute lung injury. Oxidative stress plays as important role in such injury. The aim of this study was to evaluate the effects that the potent antioxidant N-acetylcysteine (NAC) has on renal and pulmonary function in rats with sepsis. Rats, treated or not with NAC (4.8 g/l in drinking water), underwent cecal ligation and puncture (CLP) 2 days after the initiation of NAC treatment, which was maintained throughout the study. At 24 h post-CLP, renal and pulmonary function were studied in four groups: control, control + NAC, CLP, and CLP + NAC. All animals were submitted to low-tidal-volume mechanical ventilation. We evaluated respiratory mechanics, the sodium cotransporters Na-K-2Cl (NKCC1) and the α-subunit of the epithelial sodium channel (α-ENaC), polymorphonuclear neutrophils, the edema index, oxidative stress (plasma thiobarbituric acid reactive substances and lung tissue 8-isoprostane), and glomerular filtration rate. The CLP rats developed AKI, which was ameliorated in the CLP + NAC rats. Sepsis-induced alterations in respiratory mechanics were also ameliorated by NAC. Edema indexes were lower in the CLP + NAC group, as was the wet-to-dry lung weight ratio. In CLP + NAC rats, α-ENaC expression was upregulated, whereas that of NKCC1 was downregulated, although the difference was not significant. In the CLP + NAC group, oxidative stress was significantly lower and survival rates were significantly higher than in the CLP group. The protective effects of NAC (against kidney and lung injury) are likely attributable to the decrease in oxidative stress, suggesting that NAC can be useful in the treatment of sepsis.

Effects of Continuous Erythropoietin Receptor Activator in Sepsis-Induced Acute Kidney Injury and Multi-Organ Dysfunction

Background Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI. Methods Rats were randomized into three groups: control; CLP; and CLP+CERA (5 µg/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting—to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-κB)—and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1β, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection. Results Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-κB was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats. Conclusion CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

Sildenafil reduces polyuria in rats with lithium-induced NDI.

Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; α-, β-, and γ-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, γ-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.

N-acetylcysteine protects against renal injury following bilateral ureteral obstruction.

Background. Obstructive nephropathy decreases renal blood flow (RBF) and glomerular filtration rate (GFR), causing tubular abnormalities, such as urinary concentrating defect, as well as increasing oxidative stress. This study aimed to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on the protein expression of aquaporin 2 (AQP2) and endothelial nitric oxide synthase (eNOS), after the relief of bilateral ureteral obstruction (BUO). Methods. Adult male Wistar rats were divided into four groups: sham (sham operated); sham operated + 440 mg/kg body weight (BW) of NAC daily in drinking water, started 2 days before and maintained until 48 h after the surgery; BUO (24-h BUO only); BUO + NAC-pre (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started 2 days before BUO); and BUO + NAC-post (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started on the day of BUO relief). Experiments were conducted 48 h after BUO relief. Results. Serum levels of thiobarbituric reactive substances, which are markers of lipid peroxidation, were significantly lower in NAC-treated rats than in the BUO group rats. The administration of NAC provided significant protection against post-BUO GFR drops and reductions in RBF. Renal cortices and BUO rats presented decreased eNOS protein expression of eNOS in the renal cortex of BUO group rats, whereas it was partially recovered in BUO + NAC-pre group rats. Urine osmolality was significantly lower in BUO rats than in sham group rats or NAC-treated rats, the last also presenting less interstitial fibrosis. Post-BUO downregulation of AQP2 protein expression was averted in the BUO + NAC-pre group rats. Conclusions. This study demonstrates that NAC administration ameliorates the renal function impairment observed 48 h after the relief of 24-h BUO. Oxidative stress is important for the suppression of GFR, RBF, tissue AQP2 and eNOS in the polyuric phase after the release of BUO.

Sodium bicarbonate treatment reduces renal injury, renal production of transforming growth Factor-β, and urinary transforming growth Factor-β excretion in rats with doxorubicin-induced nephropathy

The aim of this study is to investigate the effect of sodium bicarbonate on doxorubicin-injected rats. Thirty female Wistar rats were injected with doxorubicin (3.5 mg/kg of body weight, intravenously) and 30 rats with 0.15 mol/L of sodium chloride solution (group C). Fifteen days later, we replaced the drinking water with a 0.15-mol/L sodium bicarbonate solution for 10 of the animals injected with doxorubicin (group AD-B). Three months after the beginning of treatment, urine samples were collected to quantify albumin, creatinine, and transforming growth factor-beta (TGF-beta). The rats were killed, and the kidneys were removed for histological, morphometric, immunohistochemical, and RNA studies. All doxorubicin-injected animals showed structural renal changes. However, these alterations were less intense in rats treated with doxorubicin plus sodium bicarbonate (P < 0.05). The percentage of glomerulosclerosis was 0.11% +/- 0.08% in group C, 14.7% +/- 12.8% in group AD (rats treated with doxorubicin only), and 4.38% +/- 1.9% in group AD-B, and the percentage of tubulointerstitial damage was 0. 01% +/- 0.03% in group C, 54.6% +/- 20.3% in group AD, and 16.6% +/- 10.3% in group AD-B. The immunostaining for TGF-beta in the renal cortex and glomeruli was more intense in the animals injected with doxorubicin only. A greater renal cortical TGF-beta messenger RNA content was observed in the animals injected with only doxorubicin that did not receive sodium bicarbonate (P < 0.05). These animals also presented a greater rate of urinary TGF-beta excretion reported as picograms of TGF-beta per milligram of urinary creatinine (P < 0.05), which was 202 +/- 11 pg/mg in group C, 1, 103 +/- 580 pg/mg in group AD, and 299 +/- 128 pg/mg in group AD-B. However, albuminuria was more intense in the sodium bicarbonate-treated animals (P < 0.05). The animals from group AD also showed higher immunostaining scores for vimentin and albumin in tubule cells (P < 0.05). In conclusion, treatment with sodium bicarbonate reduces structural renal damage, albumin reabsorption, and renal TGF-beta production in rats with doxorubicin-induced nephropathy.

Vitamin D Deficiency Aggravates Chronic Kidney Disease Progression after Ischemic Acute Kidney Injury

Background Despite a significant improvement in the management of chronic kidney disease (CKD), its incidence and prevalence has been increasing over the years. Progressive renal fibrosis is present in CKD and involves the participation of several cytokines, including Transforming growth factor-β1 (TGF-β1). Besides cardiovascular diseases and infections, several studies show that Vitamin D status has been considered as a non-traditional risk factor for the progression of CKD. Given the importance of vitamin D in the maintenance of essential physiological functions, we studied the events involved in the chronic kidney disease progression in rats submitted to ischemia/reperfusion injury under vitamin D deficiency (VDD). Methods Rats were randomized into four groups: Control; VDD; ischemia/reperfusion injury (IRI); and VDD+IRI. At the 62 day after sham or IRI surgery, we measured inulin clearance, biochemical variables and hemodynamic parameters. In kidney tissue, we performed immunoblotting to quantify expression of Klotho, TGF-β, and vitamin D receptor (VDR); gene expression to evaluate renin, angiotensinogen, and angiotensin-converting enzyme; and immunohistochemical staining for ED1 (macrophages), type IV collagen, fibronectin, vimentin, and α-smooth mucle actin. Histomorphometric studies were performed to evaluate fractional interstitial area. Results IRI animals presented renal hypertrophy, increased levels of mean blood pressure and plasma PTH. Furthermore, expansion of the interstitial area, increased infiltration of ED1 cells, increased expression of collagen IV, fibronectin, vimentin and α-actin, and reduced expression of Klotho protein were observed. VDD deficiency contributed to increased levels of plasma PTH as well as for important chronic tubulointerstitial changes (fibrosis, inflammatory infiltration, tubular dilation and atrophy), increased expression of TGF-β1 and decreased expression of VDR and Klotho protein observed in VDD+IRI animals. Conclusion Through inflammatory pathways and involvement of TGF-β1 growth factor, VDD could be considered as an aggravating factor for tubulointerstitial damage and fibrosis progression following acute kidney injury induced by ischemia/reperfusion.

Inhibition of Nuclear Factor-κB Activation Attenuates Tubulointerstitial Nephritis Induced by Gentamicin

Background: Animals treated with gentamicin can show residual areas of interstitial fibrosis in the renal cortex. This study investigated the expression of nuclear factor-ĸB (NF-ĸB), mitogen-activated protein (MAP) kinases and macrophages in the renal cortex and structural and functional renal changes of rats treated with gentamicin or gentamicin + pyrrolidine dithiocarbamate (PDTC), an NF-ĸB inhibitor. Methods: 38 female Wistar rats were injected with gentamicin, 40 mg/kg, twice a day for 9 days, 38 with gentamicin + PDTC, and 28 with 0.15 M NaCl solution. The animals were killed 5 and 30 days after these injections and the kidneys were removed for histological and immunohistochemical studies. The results of the immunohistochemical studies were scored according to the extent of staining. The fractional interstitial area was determined by morphometry. Results: Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. Increased ED-1, MAP kinases and NF-ĸB staining were also observed in the renal cortex from all gentamicin-treated rats compared to control (p < 0.05). The animals killed on day 30 also presented fibrosis in the renal cortex despite the recovery of renal function. Treatment with PDTC reduced the functional and structural changes induced by gentamicin. Conclusions: These data show that inhibition of NF-ĸB activation attenuates tubulointerstitial nephritis induced by gentamicin.

Acute kidney injury in human leptospirosis: an immunohistochemical study with pathophysiological correlation

Tubulointerstitial nephritis is a common clinicopathological finding in leptospirosis. Clinically, nonoliguric acute kidney injury (AKI), hypokalemia, sodium, and magnesium wasting frequently occur in leptospirosis. The exact mechanisms of renal involvement remain largely unclear. Immunohistochemistry to detect expression of the endogenous sodium/hydrogen exchanger isoform 3 (NHE 3), aquaporin 1 and 2, α-Na+K+ATPase, and sodium–potassium–chloride cotransporter in its NKCC2 isoform was performed on kidneys removed during autopsy of human leptospirosis cases and kidneys removed during autopsy of human non-leptospirosis cases with and without evidence of acute tubular necrosis (ATN). A decrease in NHE 3, aquaporin 1, and α-Na+K+ATPase expression occurred in proximal convoluted tubule cells. Expression of aquaporin 1 was preserved along the descending thin limb of the loop of Henle in the outer medulla. α-Na+K+ATpase expression was essentially preserved in the distal tubules, i.e., the thick ascending limb of the loop of Henle, macula densa, and distal convoluted tubule. Aquaporin 2 expression in the collecting tubules was enhanced compared to those of non-leptospirotic kidneys. NKCC2 cotransport isoform was expressed in the thick ascending limb of the loop of Henle and was essentially preserved in leptospirotic kidneys. Primary injury of the proximal convoluted tubules is regarded as the hallmark of the kidney in leptospirosis. Sodium and water transport are particularly affected with increased distal potassium excretion, hypokalemia, and polyuria. Enhanced expression of aquaporin 2 in medullary collecting tubules is probably an attempt to retain water during the nonoliguric phase of renal failure.