Regulation of Mg2+ Reabsorption and Transient Receptor Potential Melastatin Type 6 Activity by cAMP Signaling

The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg²⁺ channels participate in transcellular Mg²⁺ transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg²⁺ reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na⁺/Cl⁻ cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg²⁺ reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal—and low—Mg²⁺ diet. Increased urinary Mg²⁺ wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg²⁺ concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg²⁺ diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels.     

Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc−/−) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc−/− mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc−/− mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.     

大黄灵脾颗粒对缺血再灌注急性肾损伤大鼠肾纤维化的干预作用

目的:探讨大黄灵脾颗粒对缺血再灌注急性肾损伤模型大鼠肾纤维化的影响。方法:30只雄性SD大鼠随机分为正常组、模型组、大黄灵脾颗粒(中药)组,每组10只,通过夹闭肾蒂45 min后恢复肾脏血流再灌注建立模型,建模成功后次日各组大鼠开始灌胃治疗,正常组和模型组灌服生理盐水,中药组给予大黄灵脾颗粒灌胃。术后12周检测各组大鼠Scr、BUN、24 h尿蛋白等生化指标;HE染色观察肾组织病理;Masson染色观察肾纤维化情况;免疫组化法检测肾组织TGF-β1和α-SMA蛋白的表达水平。结果:各组大鼠Scr、BUN差异无统计学意义(P>0.05);与模型组比较,HE染色显示中药组肾组织损伤轻于模型组,且中药组24 h尿蛋白显著低于模型组(P<0.05);Masson染色显示中药组纤维化程度较模型组明显减轻,中药组肾组织TGF-β1和α-SMA蛋白的表达水平也显著低于模型组(P<0.05)。结论:大黄灵脾颗粒可改善AKI大鼠肾脏病理,降低尿蛋白水平,抑制TGF-β1和α-SMA的表达,并减轻AKI后肾纤维化的发生。     

Potential Therapeutic Targets for Cisplatin-Induced Kidney Injury: Lessons from Other Models of AKI and Fibrosis

The effectiveness of cisplatin, a mainstay in the treatment of many solid organ cancers, is hindered by dose-limiting nephrotoxicity. Cisplatin causes AKI in 30% of patients. Patients who do not develop AKI by clinical standards during treatment are still at risk for long-term decline in kidney function and the development of CKD. The connection between AKI and CKD has become increasingly studied, with renal fibrosis a hallmark of CKD development. To prevent both the short- and long-term effects of cisplatin, researchers must use models that reflect both types of pathology. Although a lot is known about cisplatin-induced AKI, very little is known about the mechanisms by which repeated low levels of cisplatin lead to fibrosis development. In this review, strategies used in various rodent models to prevent kidney injury, its progression to fibrosis, or both, are examined to gain mechanistic insights and identify potential therapeutic targets for cisplatin-induced kidney pathologies. Reviewing the results from these models highlights the diverse and highly complex role of cell death, cell senescence, endoplasmic reticulum stress, autophagy, and immune cell activation in acute and chronic kidney injuries. The use of several models of kidney injury is needed for development of agents that will prevent all aspects of cisplatin-induced kidney injury.     

Sonic Hedgehog Is a Novel Tubule-Derived Growth Factor for Interstitial Fibroblasts after Kidney Injury

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER^T2 reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis.     

Electrophysiological Properties of a Novel Ca2+-Activated K+ Channel Expressed in Human Osteoblasts

Intracellular Ca²⁺ mobilization plays important roles in cell survival, proliferation, and differentiation of osteoblasts. In this study, we identified a novel type of Ca²⁺-activated K⁺ channel in human osteoblasts and investigated its physiological roles. Using RT-PCR methods and single-channel analysis in the patch-clamp technique, we found that BK and IK channels were genetically expressed in human osteoblasts and had electrophysiological properties similar to those reported previously for the channels in other organs (conductance, voltage dependence, and sensitivity to intracellular Ca²⁺). Taking advantage of the fact that ATP induces elevation of the intracellular Ca²⁺ concentration in human osteoblasts, we successfully demonstrated that ATP-induced hyperpolarization was effectively inhibited by the IK channel blockers charybdotoxin and clotrimazole and by a P2 purinergic receptor antagonist, suramin, but not by the BK channel blockers tetraethylammonium chloride and iberiotoxin under the current-clamp mode of whole-cell clamp. The present study is the first to demonstrate the electrophysiological properties and functional expression of IK channels in human osteoblasts, findings which suggest that IK channels are regulators of membrane potential that give rise to intracellular Ca²⁺ mobilization by physiological stimulation.     

Combined Deletion of Vhl and Kif3a Accelerates Renal Cyst Formation

A subset of familial and sporadic clear cell renal cell carcinomas (ccRCCs) is believed to develop from cystic precursor lesions. Loss of function of the von Hippel-Lindau tumor suppressor gene (VHL) predisposes renal epithelial cells to loss of the primary cilium in response to specific signals. Because the primary cilium suppresses renal cyst formation, loss of the cilium may be an initiating event in the formation of ccRCC. To test this hypothesis, we analyzed the consequences of inducible renal epithelium–specific deletion of Vhl together with ablation of the primary cilium via deletion of the kinesin family member 3A (Kif3a) gene. We developed a microcomputed tomography–based imaging approach to allow quantitative longitudinal monitoring of cystic burden, revealing that combined loss of Vhl and Kif3a shortened the latency of cyst initiation, increased the number of cysts per kidney, and increased the total cystic burden. In contrast with findings in other cystic models, cysts in Kif3a mutant mice did not display accumulation of hypoxia-inducible factor 1-α (HIF1α), and deletion of both Hif1a and Kif3a did not affect cyst development or progression. Vhl/Kif3a double mutation also increased the frequency of cysts that displayed multilayered epithelial growth, which correlated with an increased frequency of misoriented cystic epithelial cell divisions. These results argue against the involvement of HIF1α in promoting renal cyst growth and suggest that the formation of simple and atypical renal cysts that resemble ccRCC precursor lesions is greatly accelerated by the combined loss of Vhl and the primary cilium.     

Role of intracellular Ca2+ stores in smooth muscle of human penile erectile tissue

Objective: In human erectile tissue smooth muscle contraction and detumescence are highly dependent on an increase in cytosolic [Ca²⁺]. The Ca²⁺ influx can be derived from the extracellular space or from intracellular sarcoplasmic stores. The role of both pathways was evaluated in an organ bath study on human cavernosal strips. Patients and methods: The tissue was obtained from 12 patients with chronic erectile dysfunction. The effects of Ca²⁺-free solution, ryanodine, caffeine and of nifedipine on electrically and adrenergically induced contractions were evaluated. Results: Following an incubation period of 10 min in Ca²⁺-free solution the electrically induced contraction was reduced to 20%, whereas the contraction induced by phenylephrine (PE) was only reduced to 64 ± 6% (mean ± SEM). Ryanodine inhibited the PE-contraction to 30 ± 6% and the additional application of caffeine or nifedipine further reduced the contraction to 11% and 8%. Conclusion: The results give evidence for a role of intracellular Ca²⁺-stores in human cavernosal tissue. Whether the more marked effect of ryanodine in tissue from patients with erectile failure in comparison with similar experiments in rabbit cavernosal tissue might be a sign of an increased cavernosal contractility in these patients remains to be shown in future experiments with normal erectile tissue. Received: 4 March 1997 / Accepted: 10 November 1997     

Renal parenchymal histopathology predicts life‐threatening chronic kidney disease as a result of radical nephrectomy

The preoperative prediction of post‐radical nephrectomy renal insufficiency plays an important role in the decision‐making process regarding renal surgery options. Furthermore, the prediction of both postoperative renal insufficiency and postoperative cardiovascular disease occurrence, which is suggested to be an adverse consequence caused by renal insufficiency, contributes to the preoperative policy decision as well as the precise informed consent for a renal cell carcinoma patient. Preoperative nomograms for the prediction of post‐radical nephrectomy renal insufficiency, calculated using patient backgrounds, are advocated. The use of these nomograms together with other types of nomograms predicting oncological outcome is beneficial. Post‐radical nephrectomy attending physicians can predict renal insufficiency based on the normal renal parenchymal pathology in addition to preoperative patient characteristics. It is suggested that a high level of global glomerulosclerosis in nephrectomized normal renal parenchyma is closely associated with severe renal insufficiency. Some studies showed that post‐radical nephrectomy severe renal insufficiency might have an association with increased mortality as a result of cardiovascular disease. Therefore, such pathophysiology should be recognized as life‐threatening, surgically‐related chronic kidney disease. On the contrary, the investigation of the prediction of mild post‐radical nephrectomy renal insufficiency, which is not related to adverse consequences in the postoperative long‐term period, is also promising because the prediction of mild renal insufficiency might be the basis for the substitution of radical nephrectomy for nephron‐sparing surgery in technically difficult or compromised cases. The deterioration of quality of life caused by post‐radical nephrectomy renal insufficiency should be investigated in conjunction with life‐threatening matters.     

Scattered Deletion of PKD1 in Kidneys Causes a Cystic Snowball Effect and Recapitulates Polycystic Kidney Disease

In total, 1 in 1000 individuals carries a germline mutation in the PKD1 or PKD2 gene, which leads to autosomal dominant polycystic kidney disease (ADPKD). Cysts can form early in life and progressively increase in number and size during adulthood. Extensive research has led to the presumption that somatic inactivation of the remaining allele initiates the formation of cysts, and the progression is further accelerated by renal injury. However, this hypothesis is primarily on the basis of animal studies, in which the gene is inactivated simultaneously in large percentages of kidney cells. To mimic human ADPKD in mice more precisely, we reduced the percentage of Pkd1-deficient kidney cells to 8%. Notably, no pathologic changes occurred for 6 months after Pkd1 deletion, and additional renal injury increased the likelihood of cyst formation but never triggered rapid PKD. In mildly affected mice, cysts were not randomly distributed throughout the kidney but formed in clusters, which could be explained by increased PKD-related signaling in not only cystic epithelial cells but also, healthy-appearing tubules near cysts. In the majority of mice, these changes preceded a rapid and massive onset of severe PKD that was remarkably similar to human ADPKD. Our data suggest that initial cysts are the principal trigger for a snowball effect driving the formation of new cysts, leading to the progression of severe PKD. In addition, this approach is a suitable model for mimicking human ADPKD and can be used for preclinical testing.     

Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

BackgroundAKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear.MethodsWe used mice with myeloid or macrophage cell–specific deletion of Irf4 (MΦ Irf4 ^−/−) to evaluate Irf4’s role in renal macrophage polarization and development of fibrosis after severe AKI.ResultsSurprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow–derived monocytes (BMDMs) from MΦ Irf4 ^−/− mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 ^−/− BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 ^−/− mice or in wild-type mice with inhibition of AKT activity.ConclusionsDeletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.     

Renal functional outcomes for tumours in a solitary kidney managed by ablative or extirpative techniques

Study Type – Therapy (case series)
Level of Evidence 4

OBJECTIVE

To present the renal functional outcomes for patients treated with open partial nephrectomy (OPN) or radiofrequency ablation (RFA) for tumours in a solitary kidney, as renal masses in a solitary kidney present a challenging treatment dilemma.

PATIENTS AND METHODS

A retrospective review of institutional databases identified 89 patients with 98 renal tumours in a solitary kidney managed by RFA or OPN under cold ischaemia between January 1997 and September 2007. The choice of therapy was based on patient and surgeon preference, tumour characteristics and comorbidities. Renal function was calculated using the modified Modification of Diet in Renal Disease equation.

RESULTS

Outcomes from 47 patients treated by RFA and 42 by OPN were analysed at a median follow‐up of 18.1 and 30.0 months, respectively (P = 0.02). The median age (65.9 vs 59.6 years, P = 0.03) and American Society of Anesthesiology score (3.0 vs 2.0, P = 0.01) were both higher in patients treated with RFA. The median tumour size was greater for tumours managed by OPN (3.9 vs 2.8 cm, P = 0.001), while the median preoperative glomerular filtration rate (GFR) was lower in the RFA group (46.5 vs 55.9 mL/min/1.73 m² for OPN, P = 0.04). Compared to RFA, patients treated with OPN had a greater decline in GFR at all times evaluated, including soon after the procedure (15.8% vs 7.1%), 12 months after surgery (24.5% vs 10.4%) and at the last follow‐up (28.6% vs 11.4%, all P < 0.001). For patients with a pretreatment GFR of > 60 or > 30 mL/min/1.73 m², there was a new onset of decline in GFR of <60 and <30 mL/min/1.73 m² in none and 7% of patients after RFA, and in 35% and 17% after OPN.

CONCLUSION

Ablative techniques, which obviate ischaemic insults, might be a particularly attractive option for managing tumours in solitary renal units at risk of declining function. Renal functional outcomes compare favourably to extirpative surgery using cold ischaemia.     

血清胱抑素C评价慢性肾脏病患者早期肾损害的临床研究

目的:探讨血清胱抑素C(CystatinC,CystC)在评价慢性肾脏病患者早期肾功能损害的临床价值。方法:收集自2005年7月～2006年2月住院的慢性肾脏病患者88例,采用乳胶颗粒增强免疫透射比浊分析法(PETIA)测定血清CystC浓度,同时测定血清肌酐浓度(Scr,碱性苦味酸法)及内生肌酐清除率(Ccr,标准24h留尿计算法),以99mTc-二乙三胺五醋酸(99mTc-DTPA)法测得的肾小球滤过率(GFR)作为诊断评价的金标准,比较CystC、Scr、Ccr与GFR的相关性。采用受试者工作特征曲线下面积(AUCROC)和似然比(LR)评价CystC的可靠性。结果:患者血清CystC、Ccr、Scr与GFR均呈显著相关(P<0.01),以CystC与GFR的相关程度最密切;上述3个指标AUCROC分别为0.980、0.941、0.914,且CystC与Scr之间具有统计学意义(P<0.05),而与Ccr间无统计学差异(P>0.05),LR以血清CystC为最佳。结论:与Ccr和Scr相比,血清CystC是一个更为准确、可靠的反映肾小球滤过功能的指标,对早期诊断慢性肾脏病患者肾小球滤过功能的损害具有重要价值。     

Potential Benefits of Renal Diets on Cardiovascular Risk Factors in Chronic Kidney Disease Patients

Dietary manipulation, including protein, phosphorus, and sodium restriction, when coupled with the vegetarian nature of the renal diet and ketoacid supplementation can potentially exert a cardiovascular protective effect in chronic renal failure patients by acting on both traditional and nontraditional cardiovascular risk factors. Blood pressure control may be favored by the reduction of sodium intake and by the vegetarian nature of the diet, which is very important also for lowering serum cholesterol and improving plasma lipid profile. The low protein and phosphorus intake has a crucial role for reducing proteinuria and preventing and reversing hyperphosphatemia and secondary hyperparathyroidism, which are major causes of the vascular calcifications, cardiac damage, and mortality risk of uremic patients. The reduction of nitrogenous waste products and lowering of serum PTH levels may also help ameliorate insulin sensitivity and metabolic control in diabetic patients, as well as increase the responsiveness to erythropoietin therapy, thus allowing greater control of anemia. Protein-restricted diets may have also anti-inflammatory and anti-oxidant properties.

Thus, putting aside the still debatable effects on the progression of renal disease and the more admitted effects on uremic signs and symptoms, it is possible that a proper nutritional treatment early in the course of renal disease may be useful also to reduce the cardiovascular risk in the renal patient. However, conclusive data cannot yet be drawn because quality studies are lacking in this field; future studies should be planned to assess the effect of renal diets on hard outcomes, as cardiovascular events or mortality.     

腹腔镜下马蹄肾合并肾肿瘤根治性切除术（附2例报告）

目的：探讨并研究马蹄肾合并肾肿瘤的腹腔镜治疗方法、可行性分析及预后评价。方法：2008年10月～2010年12月对2例马蹄肾合并单侧肾脏肿瘤患者行腹腔镜下肾脏肿瘤根治性切除术。术前经B超、IVU和CT检查明确诊断,手术采用Karl Storz高清视频采集系统、超声刀、双极电凝,Hem—o-lok及其它腹腔镜常用器械共同完成。结果：2例手术均获得成功,无中转开放。手术时间分别为180min、100min（平均140min）,其中马蹄肾峡部处理时间为60min、36min（平均48min）;术中平均出血量约150ml。术后绝对卧床2～3天,术后5～7天拔除导尿管,2例患者均未出现继发出血、感染等并发症。随访无局部复发及远处转移。结论：对于符合根治手术条件的马蹄肾合并肾肿瘤患者,首选的治疗仍为根治性肾切除术。然而腹腔镜手术可充分发挥其创伤小、出血少、恢复快等微创优势。术前充分估计变异血管,细致的腹膜后腔解剖,以及妥善处理峡部,是手术治疗的关键所在。     

Upregulation of TNF Receptor Type 2 in Human and Experimental Renal Allograft Rejection

An important role of TNF interacting with TNFR2 has been shown in different models of ischemic, nephrotoxic and immune-mediated renal injury. To systematically evaluate the expression of TNFR2 in renal allograft rejection, we investigated human renal allograft biopsies and, in addition, established an experimental transplantation model in rats to verify the human data under standardized conditions.
The expression of TNFR2 was analyzed in 96 human renal allograft biopsies with different disease entities. In a 6-day and a 28-day experimental protocol, TNFR2 was examined in kidney specimens and in the urine of control, uni-nephrectomized and transplanted rats ± cyclosporine treatment (n = 114).
In human biopsies and in rat allografts on day 6 with acute allograft rejection, significantly elevated expression of TNFR2 was observed in tubular epithelial cells, podocytes, B cells and monocytes/macrophages. The expression level was associated with renal function. The TNFR2 expression level at day 28 was significantly lower compared to day 6.
TNFR2 is markedly upregulated both in human and experimental acute renal allograft rejection. Our data are robust and consistent between different species, suggesting a role for TNFR2 in the early course of rejection.