Renal Injury Responses Research Articles

Renal Injury Response in an Adult Pkd2 Mouse Model

Renal Injury Response in an Adult Pkd2 Mouse Model

Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis.

Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis. Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing. A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1-S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters ("new PTC clusters") were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages. These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.

C-Phycocyanin isolated from Microcystis aeruginosa Kützing mitigates renal injury induced by potassium dichromate via toll-like receptor-4 down regulation in rats

C-Phycocyanin is among the most promising microalgal derived molecules with an array of pharmacological activities. Toll-like receptors (TLRs) have a vital role in regulating inflammatory response in renal injury. The aim of this study was to evaluate the possible renal therapeutic effect of phycocyanin isolated from Microcystis aeruginosa Kutzing harvested from high rate algal pond on potassium dichromate (PD)-induced renal injury in rats. Rats were assigned randomly into: Group I: Normal control group. Group II: Rats injected with a single injection of PD (15 mg/kg; s.c) and served as renal injury group. Group III and IV: Rats received C-phycocyanin (25 & 50 mg/Kg; orally), after PD injection, daily for 7 days. Injection of PD induced a marked renal injury, evidenced by a significant increase in kidney functions and renal contents of malondialdehyde (MDA), tumor necrosis factor–alpha (TNF-α), toll-like receptor 4 (TLR4), heat shock protein 70 (HSP70) and insulin growth factor-1 (IGF-1) In addition PD reduced renal content of reduced glutathione (GSH) and exhibited tubular degeneration and necrosis. C-phycocyanin has renal therapeutic effect against renal injury induced by PD through improving kidney function, down regulation of oxidative stress and TLR4/TNF-α/HSP70 inflammatory pathway as well as modulation of IGF-1.

Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses.

Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin-positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC-/-) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC-/- mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.

Sodium bicarbonate loading limits tubular cast formation independent of glomerular injury and proteinuria in Dahl salt-sensitive rats.

Sodium bicarbonate (NaHCO3) slows the decline in kidney function in patients with chronic kidney disease (CKD), yet the mechanisms mediating this effect remain unclear. The Dahl salt-sensitive (SS) rat develops hypertension and progressive renal injury when fed a high salt diet; however, the effect of alkali loading on kidney injury has never been investigated in this model. We hypothesized that NaHCO3 protects from the development of renal injury in Dahl salt-sensitive rats via luminal alkalization which limits the formation of tubular casts, which are a prominent pathological feature in this model. To examine this hypothesis, we determined blood pressure and renal injury responses in Dahl SS rats drinking vehicle (0.1 M NaCl) or NaHCO3 (0.1 M) solutions as well as in Dahl SS rats lacking the voltage-gated proton channel (Hv1). We found that oral NaHCO3 reduced tubular NH4+ production, tubular cast formation, and interstitial fibrosis in rats fed a high salt diet for 2 weeks. This effect was independent of changes in blood pressure, glomerular injury, or proteinuria and did not associate with changes in renal inflammatory status. We found that null mutation of Hv1 also limited cast formation in Dahl SS rats independent of proteinuria or glomerular injury. As Hv1 is localized to the luminal membrane of TAL, our data suggest that alkalization of the luminal fluid within this segment limits cast formation in this model. Reduced cast formation, secondary to luminal alkalization within TAL segments may mediate some of the protective effects of alkali loading observed in CKD patients.

Abstract P247: Tumor Necrosis Factor-alpha Receptors (Type 1 & Type 2) are Differentially Expressed in Renal Tissues During Chronic Dietary Intake of High Salt and Angiotensin II Treatment

Tumor necrosis factor-alpha (TNF-α) exerts natriuresis that is mediated by its’ receptor type 1 (TNFR1) while its’ receptor type 2 (TNFR2) is involved in mediating inflammatory renal injury. To determine the differential roles of these receptors in angiotensin II (AngII) induced salt-sensitive hypertension and associated renal injury, protein expressions of TNFR1 and TNFR2 were examined in mice (n=6-7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic blood pressure (SBP) in these mice was measured by tail-cuff plethysmography and 24 hour urine collections were made using metabolic cages at the start and at the end of treatment period when the kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal slices was performed by measuring the staining area as well as the intensity of receptors’ immunoreactivities using NIS Elements Software (Nikon), which allowed the semi-quantitation of positive staining and the intensity of these proteins. The results were expressed in percent area of positive staining and the relative intensity. HS intake alone did not alter mean SBP (HS; 77±1 vs NS; 76±3 vs mmHg) but it caused an exaggeration of AngII induced increases in mean SBP (AngII+HS; 104±2 vs AngII+NS; 95±2 mmHg). The area of TNFR1 staining was higher in HS (6.0± 0.9 vs 3.2 ±0.7%; P<0.05) than NS group but it was not significantly different between AngII+HS (5.0± 0.7%) and AngII+NS groups (6.3±0.7%). Similar qualitative differences were also observed in relative intensity in protein expressions. TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII+NS group and even greater in AngII + HS group. These data suggest that the increases in TNFR1 activity due to HS alone facilitate salt excretion that results no change in SBP in response to HS intake. However, such HS induced increases in TNFR1 activity was compromised in elevated AngII condition causing more salt retention and thus, exaggerated hypertensive response. On the other hand, HS induced increases in TNFR2 activity in elevated AngII condition facilitates enhanced renal injury response.

Hypertensive and Renal Injury Responses to Nitric Oxide deficiency are Unaffected by Tyrosine Kinase Inhibition in Mice

Tyrosine kinases act as second messengers that play a prominent role in intracellular signaling induced by inflammatory responses. Nitric oxide (NO) deficiency leads to hypertension (HTN) and associated inflammatory renal injury (RI). To examine the role for TKs in NO deficient form of HTN and RI, the responses to chronic administration of a NO synthase inhibitor, nitro‐L‐arginine methyl ester (L‐NAME; 100 mg/kg/day) for 4 weeks were evaluated in mice (n=7 in each group) treated with or without a tyrosine kinase inhibitor, Imatinib (75 mg/kg/day). Both the drugs were given in the drinking water. Systemic blood pressure (SBP) was measured by tail‐cuff plethysmography and a 24 hr urine collection was made using metabolic cages at the start (0 day) as well as every 7th day of the experimental period. At the end of the 4 wk period, the mice were sacrificed and kidneys were isolated to determine the renal injury scores (glomerulosclerosis, GS by PAS staining and renal interstitial fibrosis, RIF by Trichrome staining). L‐NAME treatment for 4 wks increased mean SBP (107± 3 mmHg) compared to that in control mice (88±2 mmHg). Imatinib co‐treatment did not alter significantly this SBP response to L‐NAME (103±4 mmHg). No significant differences were observed between the changes in urinary sodium, potassium or protein excretion rates in the L‐NAME and the L‐NAME+Imatinib treated mice. Compared to control mice, L‐NAME treated mice have significantly higher scores for GS (7.5±1.5 vs 3.7±0.4%) and RIF (3.0±0.7 vs 0.8±0.1%) which is similar in mice co‐treated with Imatinib (GS, 6.2±0.5% and RIF, 2.2±0.4%). These data suggest that the mechanism involved in the HTN and RI responses to NO inhibition is mostly independent of tyrosine kinase activation.

IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia-reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1β. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1β and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation.

Changes in metabolic profiles during acute kidney injury and recovery following ischemia/reperfusion.

Changes of metabolism have been implicated in renal ischemia/reperfusion injury (IRI). However, a global analysis of the metabolic changes in renal IRI is lacking and the association of the changes with ischemic kidney injury and subsequent recovery are unclear. In this study, mice were subjected to 25 minutes of bilateral renal IRI followed by 2 hours to 7 days of reperfusion. Kidney injury and subsequent recovery was verified by serum creatinine and blood urea nitrogen measurements. The metabolome of plasma, kidney cortex, and medulla were profiled by the newly developed global metabolomics analysis. Renal IRI induced overall changes of the metabolome in plasma and kidney tissues. The changes started in renal cortex, followed by medulla and plasma. In addition, we identified specific metabolites that may contribute to early renal injury response, perturbed energy metabolism, impaired purine metabolism, impacted osmotic regulation and the induction of inflammation. Some metabolites, such as 3-indoxyl sulfate, were induced at the earliest time point of renal IRI, suggesting the potential of being used as diagnostic biomarkers. There was a notable switch of energy source from glucose to lipids, implicating the importance of appropriate nutrition supply during treatment. In addition, we detected the depressed polyols for osmotic regulation which may contribute to the loss of kidney function. Several pathways involved in inflammation regulation were also induced. Finally, there was a late induction of prostaglandins, suggesting their possible involvement in kidney recovery. In conclusion, this study demonstrates significant changes of metabolome kidney tissues and plasma in renal IRI. The changes in specific metabolites are associated with and may contribute to early injury, shift of energy source, inflammation, and late phase kidney recovery.

IL-37 Inhibits IL-18-Induced Tubular Epithelial Cell Expression of Pro-Inflammatory Cytokines and Renal Ischemia-Reperfusion Injury.

Cytokines and chemokines produced by tubular epithelial cells as well as infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37 is a newly described IL-1 family member, which inhibits IL-18 dependent pro-inflammatory cytokine production through its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia injury is unknown. In this study renal tubular epithelial cells were shown to basally express IL-18 receptors and IL-18 binding protein. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Exposure of tubular epithelial cells to exogenous IL-37 (300ng/mL) down-regulated IL-18 induced expression of TNFα, IL-6 and IL-1β. Moreover, pro-inflammatory cytokine expression was augmented in IL-37mRNA silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL37. In a mouse ischemia injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IFNγ, IL-6 and IL-1β, and improved histological infiltration, injury and kidney function. This report demonstrates human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 and suggests IL-37 production by tubular epithelial cells is an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses, and promote kidney function after renal ischemia injury and transplantation.

Abstract 383: Hypertensive and Renal Injury Responses to Angiotensin II and High Salt Intake in Mice Lacking the Gene for Interleukin-10

Endogenous interleukin-10 (IL-10) exerts immune down-regulating action on the generation of tumor necrosis factor-alpha (TNF-α). The present study examined the hypothesis that IL-10 plays a protective role in hypertension and renal injury induced by angiotensin II (AngII) and high salt (HS) diet by minimizing TNF-α production. Systemic blood pressure (BP; monitored by implanted radio-telemetry), TNF-α level in plasma and in the kidney (by ELISA) as well as renal injury (glomerulosclerosis, GS by PAS staining and renal interstitial fibrosis, RIF by Trichrome staining) responses to chronic infusion of AngII (400 ng/min; osmotic minipump) for 2 wks were evaluated in wild-type (WT; n=11) and IL-10 gene knockout mice (KO; n=11) which were fed either normal (NS; 0.03% NaCl, n=5) or HS (4% NaCl; n=6) diets. On the last day of the experiment, a 24 hr urine collection was made using metabolic cages prior to sacrificing the mice for the collection of plasma and renal tissue samples. The mean baseline BP in KO was lower (104±3 vs 116±4 mmHg) than that in WT. Increase in BP in AngII+HS treated KO was lower (Δ 20±5 vs Δ 39±2 mmHg) than that in WT but similar in AngII+NS treated KO and WT (Δ 40±3 vs Δ 47±7 mmHg). In AngII+HS treated WT, TNF-α was higher in plasma (69±6 vs 34±4 pg/mL) and in renal tissue (208±15 vs 95±11 pg/mg protein) compared to values in WT treated with AngII+NS. In AngII+HS treated KO, TNF-α was lower in plasma (20±3 vs 180±44 pg/mL) and in renal tissue (205±23 vs 277±23 vs pg/mg protein) compared to values in KO treated with AngII+NS. The urinary nitrate/nitrite excretion rate was higher in AngII+NS (0.56±0.25 vs 0.08±0.01 mM/24 hr) and AngII+HS (1.23±0.12 vs 0.18±0.02 mM/24 hr) treated KO compared to the correspondingly treated WT. The eNOS protein expression was higher in KO treated with AngII+NS (~2 folds) or AngII+HS (~3 folds) compared to those in treated WT. GS (24.6±1.3 vs 13.8±2.1 %) and RIF (10.6±1.1 vs 7.8±0.5 %) changes were greater in AngII+NS treated KO than those in treated WT. However, the changes were minimal in HS treated groups. In conclusion, these data demonstrate that there exists an interaction of IL-10 and eNOS activity in the regulation of TNF-α in the kidney that provides a protective role by minimizing hypertension and renal injury induced by Ang II and HS intake.

Abstract 384: Tumor Necrosis Factor-alpha Antagonism Attenuates Hypertension and Renal Injury Induced by Chronic Blockade of Nitric Oxide and High Salt Intake in Mice

Earlier, we demonstrated that systemic blockade of nitric oxide (NO) generation by acute infusion of nitro-L-arginine methyl ester (L-NAME) in mice increases plasma and renal levels of tumor necrosis factor-alpha (TNF-α). In the present study, we examined the role of TNF-α in mediating hypertension and renal injury responses to chronic NO blockade by evaluating the responses to chronic administration of L-NAME (0.05 μg/g/min; by osmotic mini-pump) for 3 wks in mice (C57BL6) treated with or without TNF-α receptor antagonist, etanercept (ETN; 5 mg, ip, every alternate day). These mice were given either normal (NS; 0.03% NaCl) or high salt (HS; 4% NaCl) containing diets. Systemic blood pressure (BP) was measured by tail-cuff plethysmography. At the end of the experimental period, mice were sacrificed to collect plasma and renal tissue samples to measure TNF-α level by ELISA and to determine the extent of tissue injury such as glomerulosclerosis (GS; PAS staining) and renal interstitial fibrosis (RIF; Trichrome staining). L-NAME treatment caused increase in BP which was greater in HS (104±3 to 139±3 mmHg; n=6) than in NS group (96±6 to 124±6 mmHg; n=6). ETN treatment significantly attenuated this increase in BP in both L-NAME+HS (119±11 mmHg; n=6) and in L-NAME+NS (115±8 mmHg, n=6) groups. Renal tissue level of TNF-α was higher in L-NAME+NS (664±100 vs 325±73 pg/mg protein) and in L-NAME+HS (391±38 vs 114±17 pg/mg protein) groups compared to the levels in groups given NS (n=5) or HS (n=5) diet alone. However, the changes in plasma level of TNF-α due to chronic L-NAME treatment were minimal. ETN treatment lowered renal TNF-α level in L-NAME+NS (356±60 pg/mg protein) and in L-NAME+HS (259±65 pg/mg protein) groups. L-NAME treatment significantly increased GS (7.1±1.0 vs 17.9±2.3 % sclerosis area) and RIF, (1.5±0.1 vs 5.8±0.6 % fibrotic area) in HS group. However, these changes were minimal in NS group (GS, 10.3±1.0 vs 13.2±1.5 % and RIF, 1.3±0.1 vs 2.4±0.3 %). ETN treatment markedly attenuated the GS (11.8±0.4% and 11.9±0.6 %) and RIF (2.6±0.3% and 1.9±0.3%) both in L-NAME+HS and in L-NAME+NS groups. These data demonstrate that an enhancement in TNF-α activity is involved in mediating hypertension and renal injury responses to chronic NO blockade and HS intake.

Protective role of the endothelial isoform of nitric oxide synthase in ANG II-induced inflammatory responses in the kidney

In the present study, we examine the hypothesis that the nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a protective role in the development of ANG II-induced hypertension and renal injury by minimizing oxidative stress and the inflammation induced by TNF-α. Systolic blood pressure (SBP) and renal injury responses to chronic infusions of ANG II (via implanted minipumps) were evaluated for 2 wk in wild-type (WT) and in eNOS knockout mice (KO) cotreated with or without a superoxide (O2(-)) scavenger, tempol (400 mg/l in the drinking water), or a TNF-α receptor blocker, etanercept (5 mg/kg/day ip). In study 1, when ANG II was given at a dose of 25 ng/min, it increased mean SBP in WT mice (Δ36 ± 3 mmHg; n = 7), and this effect was attenuated in mice pretreated with tempol (Δ24 ± 3 mmHg; n = 6). In KO mice (n = 9), this dose of ANG II resulted in severe renal injury associated with high mortality. To avoid this high mortality in KO, study 2 was conducted with a lower dose of ANG II (10 ng/min) that increased SBP slightly in WT (Δ17 ± 7 mmHg; n = 6) but exaggeratedly in KO (Δ48 ± 12 mmHg, n = 6) associated with severe renal injury. Cotreatment with either tempol (n = 6) or etanercept (n = 6) ameliorated the hypertensive, as well as the renal injury responses in KO compared with WT. These data demonstrate a protective role for eNOS activity in preventing renal inflammatory injury and hypertension induced by chronic increases in ANG II.

TNF-α type 2 receptor mediates renal inflammatory response to chronic angiotensin II administration with high salt intake in mice

Tumor necrosis factor-alpha (TNF-α) has been implicated in salt-sensitive hypertension and renal injury (RI) induced by angiotensin II (ANG II). To determine the receptor type of TNF-α involved in this mechanism, we evaluated the responses to chronic ANG II infusion (25 ng/min by implanted minipump) given with high-salt diet (HS; 4% NaCl) for 2 wk in gene knockout mice for TNF-α receptor type 1 (TNFR1KO; n = 6) and type 2 (TNFR2KO; n = 6) and compared the responses with those in wild-type (WT; C57BL/6; n = 6) mice. Blood pressure in these mice was measured by implanted radiotelemetry as well as by tail-cuff plethysmography. RI responses were assessed by measuring macrophage cell infiltration (CD68(+) immunohistochemistry), glomerulosclerosis (PAS staining), and interstitial fibrosis (Gomori's trichrome staining) in renal tissues at the end of the treatment period. The increase in mean arterial pressure induced by ANG II + HS treatment was not different in these three groups of mice (TNFR1KO, 114 ± 1 to 161 ± 7 mmHg; TNFR2KO, 113 ± 1 to 161 ± 3 mmHg; WT, 110 ± 3 to 154 ± 3 mmHg). ANG II + HS-induced RI changes were similar in TNFR1KO mice but significantly less in TNFR2KO mice (macrophage infiltration, 0.02 ± 0.01 vs. 1.65 ± 0.45 cells/mm(2); glomerulosclerosis, 26.3 ± 2.6 vs. 35.7 ± 2.2% area; and interstitial fibrosis, 5.2 ± 0.6 vs. 8.1 ± 1.1% area) compared with the RI changes in WT mice. The results suggest that a direct activation of TNF-α receptors may not be required in inducing hypertensive response to chronic ANG II administration with HS intake, but the induction of inflammatory responses leading to renal injury are mainly mediated by TNF-α receptor type 2.

Involvement of TNF‐α receptor type 2, but not the type 1, in angiotensin II induced renal tissue injury in mice

Although TNF‐α has been implicated in the hypertensive and renal injury (RI) responses to angiotensin II (AngII), the role for its receptors (type 1 and 2) in these responses are not yet clearly defined. In this study, their roles were assessed by evaluating the responses to chronic administration of AngII (25 ng·min−1 by implanted minipump) for 2 weeks in knockout mice for either type 1 (TNFR1KO; n=6) or type 2 (TNFR2KO; n=6) receptors and compared these results with those in wild type (WT; C57BL6; n=6) mice. RI responses were assessed by measuring glomerulosclerosis index (GSI; PAS staining), interstitial collagen deposition index (CDI; Gomori's trichrome staining) and CD68+ macrophage cell infiltration (MCI; immunohistochemistry). Hypertensive responses (monitored by radiotelemetry) to AngII were not different in these three groups of mice (TNFR1KO, 88 ± 5 to 155 ± 6 mmHg; TNFR2KO, 90 ± 5 to 141 ± 16 mmHg; WT, 89 ± 6 to 147 ± 14 mmHg). Compared to the values in WT, RI parameters were similar in TNFR1KO (GSI, 22.6 ± 4.2% vs 26.3 ± 2.6%; CDI, 8.1 ± 1.1% vs 7.1 ± 2.0% and MCI, 2.2 ± 0.6 vs 1.1 ± 0.2 cells/mm2tissue) but were significantly lower in TNFR2KO (GSI, 17.3 ± 3.1%; CDI 5.2 ± 0.6% and MCI, 0.02 ± 0.001 cells/mm2tissue). These data demonstrate that hypertensive response to AngII is independent of activation of TNFR1 or TNFR2 but inflammatory process leading to RI is modulated by TNFR2, but not by TNFR1 receptor. (NHLBI grant #66432)

Independent assessment of a wide‐focus, low‐pressure electromagnetic lithotripter: absence of renal bioeffects in the pig

To assess the renal injury response in a pig model treated with a clinical dose of shock waves (SWs) delivered at a slow rate (27 SW/min) using a novel wide focal zone (18 mm), low acoustic pressure (<20 MPa) electromagnetic lithotripter (Xi Xin-Eisenmenger, XX-ES; Xi Xin Medical Instruments Co. Ltd., Suzhou, PRC). The left kidneys of anaesthetized female pigs were treated with 1500 SWs from either an unmodified electrohydraulic lithotripter (HM3, Dornier MedTech America, Inc., Kennesaw, GA, USA; 18 kV, 30 SW/min) or the XX-ES (9.3 kV, 27 SW/min). Measures of renal function (glomerular filtration rate, GFR, and renal plasma flow) were collected before and after SW lithotripsy, and kidneys were harvested for histological quantification of vascular haemorrhage, expressed as a percentage of the functional renal volume (FRV). A fibre-optic probe hydrophone was used to characterize the acoustic field, and the breakage of gypsum model stones was used to compare the function of the two lithotripters. Kidneys treated with the XX-ES showed no significant change in renal haemodynamic function and no detectable tissue injury. Pigs treated with the HM3 had a modest decline from baseline ( approximately 20%) in both GFR (P > 0.05) and renal plasma flow (P = 0.064) in the treated kidney, but that was not significantly different from the control group. Although most HM3-treated pigs showed no evidence of renal tissue injury, two had focal injury measuring 0.1% FRV, localized to the renal papillae. The width of the focal zone for the XX-ES was approximately 18 mm and that of the HM3 approximately 8 mm. Peak positive pressures at settings used to treat pigs and break model stones were considerably lower for the XX-ES (17 MPa at 9.3 kV) than for the HM3 (37 MPa at 18 kV). The XX-ES required fewer SWs to break stones to completion than did the HM3, with a mean (sd) of 634 (42) and 831 (43) SWs, respectively (P < 0.01). However, conditions were different for these tests because of differences in physical configuration of the two machines. The absence of renal injury with the wide focal zone XX-ES lithotripter operated at low shock pressure and a slow SW rate suggests that this lithotripter would be safe when used at the settings recommended for patient treatment. That the injury was also minimal using the Dornier HM3 lithotripter at a slow SW rate implies that the reduced tissue injury seen with these two machines was because they were operated at a slow SW rate. As recent studies have shown stone breakage to be improved when the focal zone is wider than the stone, a wide focal zone lithotripter operated at low pressure and slow rate has the features necessary to provide better stone breakage with less tissue injury.

Recommendations for the management of special populations: renal disease in diabetes

During the past decade, the incidence of end-stage renal disease (ESRD) has risen dramatically, primarily due to an increase in the incidence of diabetes. In patients with diabetes, both hyperglycemia and hypertension are independent risk factors for renal disease. Hypertension is also a risk factor in nondiabetic renal disease and contributes to renal dysfunction by increasing glomerular pressure, glomerular capillary damage, and proteinuria. The resultant nephron damage increases glomerular pressure and damage within remnant functional nephrons, further contributing to deterioration of renal function. In addition to its role in systemic hypertension, angiotensin II has direct effects on the kidney through elevation of glomerular capillary pressure and upregulation of components of the renal injury response. These direct effects of angiotensin II on the kidney support the inclusion of agents that target the renin-angiotensin system (RAS) into treatment regimens for patients at risk for renal disease. Several clinical trials have established the benefits of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) in patients with diabetes. The ACE inhibitors have been shown to delay renal decline in patients with type 1 diabetes, whereas the renoprotective effect of these agents in patients with type 2 diabetes is less clear. The ARBs have been shown to provide significant benefits in patients with type 2 diabetes, both at early (microalbuminuria) and late (proteinuria) stages of renal decline. In the Irbesartan Diabetic Nephropathy Trial (IDNT) and the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study, ARB therapy significantly reduced the progression of overt nephropathy (composite of doubling of serum creatinine, ESRD, and death), a benefit that has not been shown for ACE inhibitors. Moreover, in RENAAL, losartan significantly reduced the incidence of the individual end point of ESRD. The benefits of ARB therapy in IDNT and RENAAL were associated with significant reductions in proteinuria and were independent of blood pressure reductions. In RENAAL, proteinuria was a strong predictor of both renal and cardiovascular events. These findings underscore the importance of RAS blockade as a strategy for improving clinical outcomes in patients with renal disease.

Smad7-dependent Regulation of Heme Oxygenase-1 by Transforming Growth Factor-β in Human Renal Epithelial Cells

Heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, is induced as a beneficial and adaptive response in cells/tissues exposed to oxidative stress. Transforming growth factor-beta1 (TGF-beta1) is a regulatory cytokine that has been implicated in a variety of renal diseases where it promotes extracellular matrix deposition and proinflammatory events. We hypothesize that the release of TGF-beta1 via autocrine and/or paracrine pathways may induce HO-1 and serve as a protective response in renal injury. To understand the molecular mechanism of HO-1 induction by TGF-beta1, we exposed confluent human renal proximal tubule cells to TGF-beta1 and observed a significant induction of HO-1 mRNA at 4 h with a maximal induction at 8 h. This induction was accompanied by increased expression of HO-1 protein. TGF-beta1 treatment in conjunction with actinomycin D or cycloheximide demonstrated that induction of HO-1 mRNA requires de novo transcription and, in part, protein synthesis. Exposure to TGF-beta1 resulted in marked induction of Smad7 mRNA with no effect on Smad6 expression. Overexpression of Smad7, but not Smad6, inhibited TGF-beta1-mediated induction of endogenous HO-1 gene expression. We speculate that the induction of HO-1 in the kidney is an adaptive response to the inflammatory effects of TGF-beta1 and manipulations of the Smad pathway to alter HO-1 expression may serve as a potential therapeutic target.