Calcium Oxalate Deposition in Renal Allografts: Morphologic Spectrum and Clinical Implications

Many aspects of calcium oxalate (CaOx) deposition in renal transplant biopsies are not known. Review of all renal transplant biopsies performed in a 7-year period showed that CaOx deposition could be classified into three groups. Group I: Seven biopsies within a month post-transplant displayed rare CaOx foci against a background of acute tubular necrosis or acute cell-mediated rejection. At follow-up, five grafts functioned well and two failed due to chronic allograft nephropathy. CaOx in this context was an incidental finding secondary to a sudden excretion of an end-stage renal disease-induced increased body burden of CaOx. Group II: Two biopsies performed 2 and 10 months post-transplant showed rare CaOx foci against a background of chronic allograft nephropathy, leading to graft loss. CaOx in this context reflected nonspecific parenchymal deposition due to chronic renal failure regardless of causes. Group III: One biopsy with recurrent PH1 characterized by marked CaOx deposition associated with severe tubulointerstitial injury and graft loss 6 months post-transplant. There were two previously reported cases in which CaOx deposition in the renal allografts was due the antihypertensive drug naftidrofuryl oxalate or increased intestinal absorption of CaOx. CaOx deposition in renal allografts can be classified in different categories with distinctive morphologic features and clinical implications.     

Klotho Prevents Renal Calcium Loss

Disturbed calcium (Ca²⁺) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca²⁺ absorption is primary, which causes increased urinary Ca²⁺ excretion, leading to elevated 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and its sequelae. Here, we assessed intestinal Ca²⁺ absorption, bone densitometry, renal Ca²⁺ excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho^−/− mice. We observed elevated serum Ca²⁺ and fractional excretion of Ca²⁺ (FE_Ca) in klotho^−/− mice. Klotho^−/− mice also showed intestinal Ca²⁺ hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D_9K increased. In the kidney, klotho^−/− mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D_28K, implying a failure to absorb Ca²⁺ through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-α-hydroxylase (1αOHase), and calbindin-D_9K excluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca²⁺ wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca²⁺ handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca²⁺ loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis.Characterization of a mouse that showed a phenotype comparable to human aging led to the identification of the hormone klotho.¹ Klotho^−/− mice have atherosclerosis, osteopenia, soft tissue calcifications, pulmonary emphysema, and altered glucose metabolism.¹ It has been suggested that the etiology of many of these findings is a primary defect in phosphorous [P(i)] and calcium (Ca²⁺) homeostasis.^2,3 Klotho^−/− mice have elevated serum levels of Ca²⁺.^1,4,5 The mechanism mediating hypercalcemia is poorly understood. A possible explanation invokes the role of klotho in vitamin D homeostasis. Klotho has been proposed to participate in a negative feedback circuit to inhibit 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] synthesis.^6,7 Specifically, klotho is necessary to transduce the signal of fibroblast growth factor 23 (FGF23) through the FGF receptor, thereby suppressing CYP1b expression, the enzyme that mediates the conversion of 25-hydroxyvitamin D into 1,25(OH)₂D₃. Thus, the absence of klotho results in increased serum levels of 1,25(OH)₂D₃ and reduced serum concentrations of the calciotropic hormone parathyroid hormone.^4,7,8 This would drive increased resorption of Ca²⁺ from bone, hyperabsorption from the intestine, increased serum levels of Ca²⁺, and consequently increase renal Ca²⁺ excretion. Definitive proof of this is lacking because the molecular control of Ca²⁺ homeostasis in klotho^−/− mice has yet to be delineated.Consistent with the above hypothesis is the observation that klotho^−/− mice display hypercalciuria^4,5,9 and that normalization of serum 1,25(OH)₂D₃ levels reverts many, but not all, of their abnormalities.⁶ The published literature supports an alternative, complementary hypothesis.^9–11 A primary defect in tubular Ca²⁺ handling might cause hypervitaminosis D and renal Ca²⁺ wasting observed in klotho^−/− mice. Consistent with this idea, in vitro, klotho mediates an increase in cell surface expression of transient receptor potential vanilloid 5 (TRPV5)^10,11 the distal convoluted tubule/connecting tubule (DCT/CNT) channel responsible for the transcellular absorption of Ca²⁺.¹² This process is itself implicit to Ca²⁺ homeostasis as TRPV5 is the predominant regulator of urinary Ca²⁺ excretion.¹³ Therefore, we set out to test the hypothesis that klotho^−/− mice have a primary renal Ca²⁺ leak that contributes to a secondary increase in 1,25(OH)₂D₃ synthesis and its consequences.     

MicroRNA-24 Antagonism Prevents Renal Ischemia Reperfusion Injury

Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI.     

Hydrogen Sulfide-Induced Hypometabolism Prevents Renal Ischemia/Reperfusion Injury

Hydrogen sulfide (H₂S) can induce a hypometabolic, hibernation-like state in mammals when given in subtoxic concentrations. Pharmacologically reducing the demand for oxygen is a promising strategy to minimize unavoidable hypoxia-induced injury such as ischemia/reperfusion injury during renal transplantation. Here we show that H₂S reduces metabolism in vivo, ex vivo, and in vitro. Furthermore, we demonstrate the beneficial effects of H₂S-induced hypometabolism in a model of bilateral renal ischemia/reperfusion injury using three different treatment strategies. The results demonstrate striking protective effects on survival, renal function, apoptosis, and inflammation. A hypometabolic state induced by H₂S might have therapeutic potential to protect kidneys that suffer from hypoxia.The toxicity of hydrogen sulfide (H₂S) has long been studied because of its involvement in deadly industrial and agricultural accidents.¹ Recently, an unknown property of the gas was revealed, namely the ability to induce hypometabolism in naturally nonhibernating mammals.^2,3 Mice exposed to subtoxic concentrations of gaseous H₂S rapidly and reversibly enter a hibernation-like state. During H₂S treatment, metabolic parameters rapidly decrease: an approximately 60% reduction in carbon dioxide (CO₂) production and oxygen (O₂)-consumption within minutes of exposure (Figure 1A), which can decline even further to more than 90%.^2,4 In addition, the core body temperature decreases to near-ambient temperature and heart rate and breathing frequency are significantly lower.² The demand for O₂ is reduced to such an extent that H₂S-treated mice can survive in 5% O₂ for over 6 h, whereas untreated controls die within 15 min.⁴ In vitro, H₂S can reversibly reduce mitochondrial O₂ consumption^5,6 and mitochondrial membrane potential (Figure 1B). Ex vivo, H₂S can reduce O₂ consumption and total ATP content of the isolated perfused kidney (Figure 1, C and D). H₂S also has antioxidant capacity, either by direct scavenging of reactive O₂ or nitrogen species or indirectly by increasing cellular glutathione levels.³ We hypothesized that a state of extremely low metabolism induced by exposure to gaseous H₂S would provide protection during periods of ischemia and reperfusion by reducing the demand for O₂.Open in a separate windowFigure 1.Metabolic suppression by H₂S and experimental design. (A) Exposure to 100 ppm H₂S causes a rapid reduction in CO₂ production of a single mouse. (B) NRK-52E proximal tubular cells loaded with the mitochondrial membrane potential indicating fluorescent dye JC-1 were exposed to different concentrations of sodium hydrosulfide (a donor of H₂S in solution) for 20 min (**P < 0.01) (C and D) Rat kidneys in an isolated perfused kidney setup were exposed to 1 mM sodium hydrosulfide (n = 4) for 30 min, and O₂ consumption and ATP were compared with controls (n = 3) (*P < 0.05). (E) Schematic of experimental design showing different H₂S treatment regimens. (F) Relative CO₂ production of animals during the period of ischemia, corrected for body weight (n = 7) (**P < 0.01). (G) Average breathing frequency of animals 5 min before, during, and 30 min after ischemia (n = 5). Open circles indicate periods in which animals received 100 ppm H₂S.To investigate the protective potential of H₂S-induced hypometabolism, we used a model of bilateral renal ischemia/reperfusion in the mouse. We evaluated four different treatment regimens (Figure 1E), comparing pretreatment, post-treatment, and pre- and post-treatment with 100 ppm H₂S (n = 6 to 7 per group). In both pretreated groups, C57BL/6 mice were first treated with H₂S for 30 min to induce hypometabolism. Our initial experiments showed that the induction of hypometabolism typically takes place within the first 10 min of exposure (Figure 1A). After the pretreatment period, renal blood flow was interrupted for 30 min by placing nontraumatic vascular clamps over both renal pedicles. To study the effects of H₂S on reperfusion damage alone, post-treatment with H₂S started 5 min before removal of the clamps and lasted for 35 min. The pre- and post-treatment group received H₂S starting 30 min before ischemia until 30 min after reperfusion. To separate the effects of H₂S from the already well known protective effects of hypothermia, core body temperature of all animals was maintained at 37°C during and after the procedure. Induction of hypometabolism in H₂S-treated animals was confirmed by lowered breathing frequency and CO₂ production, measured using closed-system respirometry (Figure 1, F and G).Bilateral ischemia caused excessive renal damage in the control group, leading to an impaired 3-d survival caused by renal failure (Figure 2A). Both groups in which mice were pretreated with H₂S had 100% survival after 3 d (P < 0.001), whereas mice that only received H₂S during reperfusion showed similar survival to the control group (P = NS). Serum creatinine and urea measurements were performed to quantify the renal function loss associated with bilateral renal ischemia/reperfusion. Control and post-treatment animals showed highly elevated levels of creatinine and urea (Figure 2B, Supplementary Figure 1), whereas animals pretreated with H₂S had only slightly higher levels than sham-operated animals (P = NS). These measurements indicate massive renal failure in the control and post-treatment groups, which is the most likely cause of the diminished survival in these groups.Open in a separate windowFigure 2.H₂S-induced hypometabolism prevents mortality and renal damage after renal ischemia. (A) Three-day survival of animals after reperfusion. (B) Renal function as measured by serum creatinine after 1 d of reperfusion. *P < 0.05 versus control, ^†P < 0.001 versus sham. (C) Apoptosis after 1 d of reperfusion was scored in sections stained for active Caspase 3 using immunohistochemistry. Apoptotic tubular cells were counted at 400× magnification in ten nonoverlapping fields (***P < 0.001). (D) Structural damage as assessed in periodic acid–Schiff-stained sections after 1 d of reperfusion. *P < 0.05 versus control, ***P < 0.001 versus control, ^†P < 0.001 versus sham. (E) Influx of leukocytes and granulocytes into the renal interstitium was scored in sections stained for Mac-1 (solid bars) or Ly-6G (dashed bars) using immunohistochemistry. *P < 0.05 versus control. (F through I) Representative photomicrographs of Ly-6G stained sections. (J through N) Representative periodic acid–Schiff-stained renal sections with necrotic area artificially colored red, indicating the extent of necrotic damage found in each group. [For B through E: sham (n = 5), control (n = 7), and H₂S-treated groups (n = 6)].We assessed structural renal damage in periodic acid–Schiff-stained sections and found a similar pattern to the renal function measurements, as expected. Massive acute tubular necrosis was detected in control animals at day 1, whereas mice in both pretreated groups had no or minimal renal damage (Figure 2, D, J through N, Supplementary Figure 3). Post-treatment with H₂S showed a significant reduction in tubular damage compared with controls, although it was not as extensive as in pretreated animals. After 3 d, a similar pattern was seen (Supplementary Figure 2). Post-treatment did not have significant protective effects at this time point, although these results are confounded to some extent, because animals with large amounts of renal damage had already deceased at this point.Active Caspase3 staining using immunohistochemistry indicated that ischemia/reperfusion injury (IRI)-induced apoptosis is also prevented by H₂S pretreatment. (Figure 2C, Supplementary Figure 4). A less pronounced but statistically significant effect was seen in the post-treatment group. Real-time PCR measurements showed that mRNA expression of proapoptotic Bax was 2.5 times higher in control kidneys compared with sham-operated animals (Supplementary Figure 5A). Expression was not significantly increased in animals pretreated with H₂S. The expression of anti-apoptotic BCL-2 did not differ between groups (Supplementary Figure 5B), indicating that the anti-apoptotic effects of H₂S are not mediated through induction of BCL-2 mRNA expression. Whether H₂S directly or indirectly inhibits increased expression of Bax is not clear. Transmission electron microscopy of a few samples implies that H₂S treatment protected against loss of mitochondrial integrity and mitochondrial swelling (Supplementary Figure 6). In literature, proapoptotic as well as anti-apoptotic effects of H₂S are described,^5,7–9 and it is not known whether H₂S can directly modulate apoptotic pathways, or that increased mitochondrial integrity and reduced mitochondrial stress caused by reduced mitochondrial activity caused the reduction in Caspase 3 activity in the post-treatment group.We studied the inflammatory component of IRI by immunohistochemical staining for Mac-1 (CD11b, which is present on macrophages, monocytes, granulocytes, and natural killer cells¹⁰) and Ly-6G (which is expressed on mature granulocytes). (Figure 2, E through I, Supplementary Figure 7). The influx of Mac-1 and Ly-6G-positive cells was greatly reduced by H₂S pretreatment (P < 0.05) but was not significantly affected by post-treatment.These results indicate that the reduction in metabolism before ischemia is highly protective in reducing ischemia-induced injury with predictable onset, such as during transplantation or surgical intervention. The mechanism of H₂S-induced hypometabolism is unknown as of yet but is most likely mediated through reversible inhibition of complex IV (cytochrome oxidase),¹¹ the terminal enzyme of the mitochondrial electron transport chain. Inhibition of this complex might be the mechanism of the reduction in mitochondrial membrane potential caused by H₂S treatment. It seems unlikely that H₂S directly and effectively inhibits necrotic, apoptotic, and inflammatory pathways after an ischemic insult. The observation that protection is greatest when H₂S is given before and during, but much less when given directly after the hypoxic period, supports the notion that the reduction in O₂ demand during hypoxia prevents the activation of these detrimental pathways. The moderate effects of H₂S in the post-treatment group could be caused by the inhibition of reactive O₂ species production by decreasing mitochondrial activity. Protection could also be mediated through direct antioxidative action, or increased glutathione levels caused by H₂S.³Recent literature shows beneficial effects of gaseous H₂S on survival in models of hypoxia⁴ and hemorrhagic shock.¹² Other groups have studied the protective effects of soluble forms of H₂S (such as sodium hydrosulfide or sodium sulfide) in models of ischemia. These studies show beneficial effects of H₂S on renal,¹³ cardiac,⁵ hepatic,¹⁴ and pulmonary ischemia.¹⁵ One paper suggests an association between H₂S treatment and reduced activation of multiple signal transduction molecules, such as p38, ERK, and JNK; however, a direct relationship between H₂S and kinase activation was not proven. We found that phosphorylation of ERK1/2 was stimulated by ischemia in our model, but no modulation was seen in H₂S-treated animals (Supplementary Figure 8). Our study shows a novel relation between H₂S treatment and hypometabolism, which has not been previously investigated. The protective effects of H₂S treatment posthypoxia are less pronounced in our experiments. However, a recent paper indicated that injection of sodium sulfide just before reperfusion in a model of myocardial infarction caused a great reduction in infarct size and protected mitochondrial integrity and function.⁵ This indicates that post-treatment with H₂S might still be a promising intervention in cutting back on the detrimental effects of hypoxia after the event. We conclude that hypometabolism induced by gaseous H₂S is a novel treatment regimen with high therapeutic potential in reducing renal damage associated with ischemic insults.     

草酸钙结晶肾损伤模型小鼠肾小管上皮细胞转分化的实验研究

目的:研究乙醛酸盐诱导所致草酸钙结晶肾损伤模型小鼠中肾小管上皮细胞转分化的发生情况。方法:选择C57BL/6J小鼠连续腹腔注射乙醛酸盐建立草酸钙结晶肾损伤模型,应用HE染色及冯库萨染色分别观察肾组织结构变化及钙盐沉积情况,并应用免疫荧光双染、Western-blot观察肾小管上皮细胞间充质转分化(epithelial mesenchymal transtion,EMT)的情况。结果:随着连续腹腔注射乙醛酸盐时间的延长,小鼠肾组织HE染色结果显示,近端肾小管管腔逐渐扩张,且肾小管上皮细胞逐渐出现肿胀及变形,基底膜逐渐裸露;冯库萨染色结果显示近端小管腔内黑色钙盐沉积逐渐增加;免疫荧光双染、Western blot结果均显示肾小管上皮标志E-cadherin及Pan-ck逐渐丢失,而间质标志α-SMA及Vimentin的表达则逐渐增加,Western blot检测结果显示随着乙醛酸盐干预的增强,Rho相关卷曲螺旋形成蛋白激酶(rho associated coiled coil forming protein kinase,ROCKI)表达也逐渐增加,且在干预第3天即达高峰。结论:乙醛酸盐诱导所致草酸钙结晶肾损伤模型早期即出现EMT,启动了肾间质纤维化的进程。     

N,N-dimethyltryptamine Prevents Renal Ischemia-Reperfusion Injury in a Rat Model

BackgroundIschemia reperfusion (I/R) injury remains one of the most challenging fields of organ transplantation. It is highly associated with the use of expanded criteria donors that might conclude to delayed graft function or early or late graft failure.ObjectiveTo investigate the metabolic, microcirculatory parameters, and histologic changes under the effect of N,N-dimethyltryptamine (DMT) in a renal I/R model in rats.MethodIn 26 anesthetized rats both kidneys were exposed. In the control group (n = 6) no other intervention happened. In 20 other animals, the right renal vessels were ligated, and after 60 minutes the right kidney was removed. The left renal vessels were clamped for 60 minutes then released, followed by 120 minutes of reperfusion. In the I/R group (n = 10), there was no additive treatment, while in I/R + DMT group (n = 10) DMT was administered 15 minutes before ischemia. Blood samples were taken, laser Doppler measurement was performed, and both kidneys were evaluated histologically.ResultsMicrocirculation (blood flux units [BFU]) diminished in all groups, but remarkably so in the I/R + DMT group. This group compensated better after the 30th minute of reperfusion. The control and I/R + DMT groups had similar BFUs after 120 minutes of reperfusion, but in the I/R group BFU was higher. Tubular necrosis developed in the I/R and I/R + DMT groups too; it was moderated under DMT effect, and severe without. Histologic injuries were less in I/R + DMT Group compared to non-treated animals.ConclusionHistologic changes characteristic to I/R injuries were reversible and microcirculation recovered at the end of 120 minutes reperfusion under the administration of DMT. DMT can be used for renoprotection in kidney transplantation.     

Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate

The presence of macromolecular substances is among the multiple factors that may influence the complex process of urinary stone formation. The aim of this study was to evaluate the inhibitory potency of crystal matrix protein (CMP). Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis and high–performance liquid chromatography (HPLC). The inhibitory potency of crystal aggregation was examined by the seed crystal method, the undiluted urine method, and the use of scanning electron microscopy (SEM). CMP showed the protein band of 31 kDa in SDS–PAGE. Anti–CMP polyclonal antibody and antihuman prothrombin antibody cross–reacted well with human prothrombin and CMP in Western blotting. CMP and human prothrombin had high inhibitory potency by the seed crystal method and undiluted urine method. Using SEM, we were able to observe the high inhibitory potency of human prothrombin and undiluted CMP on the aggregation of calcium oxalate crystals.     

Parstatin Prevents Renal Injury following Ischemia/Reperfusion and Radiocontrast Administration

Background/Aims: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the protease-activated receptor 1. Parstatin was recently found to be cardioprotective against myocardial ischemia/reperfusion (IR) injury. In the present study, it was hypothesized that parstatin would protect the kidneys in acute renal failure. Methods: We investigated the effects of parstatin on the renal dysfunction and injury caused either by renal IR injury or contrast-induced nephropathy (CIN) in two animal models. Renal IR injury was induced in rats by bilateral occlusion of renal arteries and veins for 45 min followed by 4 h of reperfusion, while CIN was induced in rabbits by intravenous injection of the radiocontrast medium Iopromide. Results: Treatment with parstatin 15 min before or immediately after renal ischemia attenuated the resulting renal dysfunction as demonstrated by the improved biochemical indicators (serum creatinine and fractional excretion of Na(+)) and scintigraphic analysis. The effect was dose depended and provided evidence for a more prominent protection of tubular than glomerulal function. Histopathological examination of the kidneys revealed severe renal damage, which was significantly suppressed by the parstatin. Similarly, administration of a single dose of parstatin before the induction of CIN significantly protected against the resulting renal dysfunction and histologically evidenced renal tubular injury. Conclusion: These results suggest that parstatin is able to act as nephroprotective agent and may be useful in enhancing the tolerance of the kidney against renal injury associated with clinical conditions of acute renal failure. Further investigation on the mechanism underlying the nephroprotective properties of parstatin is deemed necessary.     

Ultrasound Prevents Renal Ischemia-Reperfusion Injury by Stimulating the Splenic Cholinergic Anti-Inflammatory Pathway

AKI affects both quality of life and health care costs and is an independent risk factor for mortality. At present, there are few effective treatment options for AKI. Here, we describe a nonpharmacologic, noninvasive, ultrasound-based method to prevent renal ischemia-reperfusion injury in mice, which is a model for human AKI. We exposed anesthetized mice to an ultrasound protocol 24 hours before renal ischemia. After 24 hours of reperfusion, ultrasound-treated mice exhibited preserved kidney morphology and function compared with sham-treated mice. Ultrasound exposure before renal ischemia reduced the accumulation of CD11b⁺Ly6G^high neutrophils and CD11b⁺F4/80^high myeloid cells in kidney tissue. Furthermore, splenectomy and adoptive transfer studies revealed that the spleen and CD4⁺ T cells mediated the protective effects of ultrasound. Last, blockade or genetic deficiency of the α7 nicotinic acetylcholine receptor abrogated the protective effect of ultrasound, suggesting the involvement of the cholinergic anti-inflammatory pathway. Taken together, these results suggest that an ultrasound-based treatment could have therapeutic potential for the prevention of AKI, possibly by stimulating a splenic anti-inflammatory pathway.The immune response after ischemia-reperfusion injury (IRI) contributes to tissue damage and reduced GFR. CD45⁺ leukocyte infiltration begins as early as 30 minutes after reperfusion, with the appearance of CD4⁺ and CD8⁺ T cells, B220⁺ B cells, and the myeloid/monocyte populations (including Ly6G⁺ neutrophils, Ly6C⁺CCR2⁺ monocytes, and F4/80⁺ macrophages).^1,2 Attenuating this ensuing inflammatory response markedly reduces the development of IRI^3–9 by preventing tubular epithelial cell apoptosis, rarefaction, and scarring.¹⁰ The severity of tissue injury depends on the duration of ischemia and results in acute loss of kidney function, progressive kidney fibrosis,¹¹ and, in some cases, CKD or ESRD.^11,12Given the role of the innate immune system in the development of AKI,^1,13,14 treatments targeting inflammation could be valuable therapeutic tools. However, current immunosuppressive agents elicit adverse effects and increase the onset of various comorbid conditions.^15,16 An inherent splenic anti-inflammatory pathway has recently been described, and this pathway can be stimulated pharmacologically with nicotinic agonists or by electrical stimulation of the vagus nerve. Referred to as the cholinergic anti-inflammatory pathway, this cascade depends on the spleen, CD4⁺ T cells, and the α-7 nicotinic acetylcholine receptor (α7nAChR).¹⁷ This pathway modulates inflammation and benefits animals in models of myocardial ischemia,¹⁸ hepatic injury,¹⁹ sepsis and endotoxemia,^17,20,21 IRI,^22–24 and the response of humans injected with lipopolysaccharide.²⁵ Because of its efficacy in humans and the preclinical data from human tissues, the cholinergic anti-inflammatory pathway is a promising therapeutic target. However, improved methods to stimulate this anti-inflammatory pathway are needed.Using a modification of contrast-enhanced ultrasound (CEU), our original intent was to develop a method to precondition the renal vasculature before IRI.²⁶ This concept stems from observations that a modified CEU protocol improves blood flow in ischemic skeletal muscle.^27–29 Serendipitously, results from our initial studies revealed that prior ultrasound (US) exposure alone, in the absence of a contrast agent, prevented kidney IRI. Further studies indicated that the cholinergic anti-inflammatory pathway may be involved because of the dependence of the US treatment on an intact spleen and the α7nAChR. These studies provide evidence for a simple, portable, noninvasive, and nonpharmacologic approach to prevent AKI.     

The Dependence on Membrane Fluidity of Calcium Oxalate Crystal Attachment to IMCD Membranes

The development of urolithiasis is a multifaceted process, starting with urine supersaturation and ending with the formation of mature renal calculi. The retention of microcrystals by kidney tubule epithelium cell membranes has been proposed as a critical event in the process. To date, attachment of kidney stone constituent crystals to urothelial cells has been demonstrated both in vitro and in vivo yet the mechanism of crystal attachment remains unknown. We hypothesize that for effective stone crystal attachment to the epithelium there must be cell membrane rearrangement that would allow for long-range bonding between the stone crystal and the cell membrane. This rearrangement may be influenced by the physical state of the membrane. The current study examines calcium oxalate monohydrate (COM) crystal attachment to inner medullary collecting duct (IMCD) cells following changes in cell membrane fluidity. Radioactively labeled COM crystals were used to quantitate crystal attachment. Membrane fluidity was altered by changing temperature, cell membrane cholesterol content, or extended length of cell culture. Crystal attachment to IMCD cells was directly correlated to changes in membrane fluidity. This finding was consistently observed regardless of the method used to alter membrane fluidity. The results are consistent with the theory that the ability to form a crystal attachment region on the cell surface may be related to the ease of rearrangement of membrane components at the cell surface. Variations in the urothelial cell environment during certain pathological conditions in the kidney could induce these physical perturbations and prime kidney epithelial cells at or near the papillary tip to bind COM crystals. Received: 21 June 1996 / Accepted: 25 October 1996     

Membranes and Their Constituents as Promoters of Calcium Oxalate Crystal Formation in Human Urine

We have proposed that membranes of cellular degradation products are a suitable substrate for the nucleation of calcium oxalate (CaOx) crystals in human urine. Human urine is generally metastable with respect to CaOx. To demonstrate that cellular membranes present in the urine promote nucleation of CaOx we removed these substrates by filtration or centrifugation and induced crystallization by adding sodium oxalate, before and after filtration or centrifugation. In a separate experiment, membrane vesicles isolated from rat renal tubular brush border were added into the filtered or centrifuged urine before crystal induction. Crystals were counted using a particle counter. Urine, the pellet, and retentate were analyzed for the presence of membranes, lipids, and proteins. Lipids were further separated into different classes, identified, and quantified. Both filtration and centrifugation removed lipids, proteins, and membrane vesicles, causing a reduction in lipid and protein contents of the urine. More crystals formed in whole than in filtered or centrifuged urine. The number of crystals significantly increased when filtered urine was supplemented with various urinary components such as the retentate and phospholipids, which are removed during filtration. We also determined the urinary metastable limit with respect to CaOx. Filtration and centrifugation were associated with increased metastable limit which was reduced by the addition of membrane vesicles. These results support our hypothesis that urine normally contains promoters of CaOx crystal formation and that membranes and their constituents are the most likely substrate for crystal nucleation in the urine. Received: 11 August 1998 / Accepted: 26 July 1999     

Experimental Studies of Adhesion and Endocytosis of Calcium Oxalate Crystals in Renal Tubular Cells

The present investigation was designed to study the interactions between Madin–Darby canine kidney (MDCK) cells and calcium oxalate monohydrate (COM) crystals, the most abundant constituent of urinary crystals, and to clarify the significance of these crystal–cell interactions in stone pathogenesis. COM crystals adhered to the intact surface of MDCK cells by some biological mechanisms (biological adhesion) and, were then internalized into the cell (endocytosis). The microvilli of the cell appeared to play an important role in this process.
In the kidneys of rats with experimentally induced stones, most COM crystals adhered to the tubular cells and some crystals were engulfed, via endocytosis. Thus, these crystal–cell interactions might be one of the earliest processes in the formation of kidney stones. Further elucidation of the mechanism and the regulatory factors of this process may provide new insight into stone pathogenesis.     

Protein Electrostatic Surface Distribution Can Determine Whether Calcium Oxalate Crystal Growth is Promoted or Inhibited

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite and calcium oxalate. Despite their importance in such fundamental physiological processes as bone and tooth formation, however, there is remarkably little known of the protein structure-function relationships that govern crystal recognition. We have taken a model system approach to elucidate some of the relationships between protein surface chemistry and secondary crystal growth of biological minerals. We show here that the distribution of electrostatic surface charge on our model protein, Protein G, determined whether the secondary growth of calcium oxalate, the principal mineral phase of kidney stones, was promoted or inhibited when the proteins were preadsorbed at low and equivalent surface coverages of <10%. The native Protein G, which contains 10 surface carboxylates, increased the rate of calcium oxalate growth from aqueous solution under constant composition conditions up to 97%, whereas a site-directed mutant with six of the surface charges removed inhibited the growth rate by 60%. The adsorption isotherms of both proteins were determined and suggested that the differences in electrostatic surface properties also lead to differences in protein orientation on the crystal surface. These results demonstrate that differences in electrostatic surface potential of proteins can directly determine whether secondary calcium oxalate growth is promoted or inhibited, and a model is proposed that suggests the distribution of carboxylate residues determines the interrelated binding orientation and exposed surface chemistry of the adsorbed Protein G. Received: 4 May 1998 / Accepted: 1 November 1998     

Early Presence of Calcium Oxalate Deposition in Kidney Graft Biopsies is Associated with Poor Long-Term Graft Survival

Accumulated oxalate will be excreted after renal transplantation, creating an increased risk of tubular precipitation, especially in the presence of allograft dysfunction. We evaluated calcium oxalate (CaOx) deposition in renal allograft biopsies with early dysfunction, its association with acute tubular necrosis (ATN) and graft survival. We studied 97 renal transplant patients, submitted to a graft biopsy within 3 months post-transplant, and reanalyzed them after 10 years. We analyzed renal tissue under polarized light and quantified CaOx deposits. CaOx deposits were detected in 52.6% of the patients; 26.8% were of mild and 25.8% of moderate intensity. The deposits were more frequent in biopsies performed within 3 weeks post-transplant (82.4 vs. 63.0%, p < 0.05) and in allografts with more severe renal dysfunction (creatinine 5.6 mg/dL vs. 3.4 mg/dL, p < 0.001). ATN incidence was also higher in patients with CaOx deposits (47% vs. 24%, p < 0.001). Twelve-year graft survival was strikingly worse in patients with CaOx deposits compared to those free of deposits (49.7 vs. 74.1%, p = 0.013). Our study shows a high incidence of CaOx deposits in kidney allografts with early dysfunction, implying an additional risk for acute tubular injury, with a negative impact on graft survival.