IU Indianapolis ScholarWorks :: Browsing by Author "Sandoval, Ruben M."

Browsing by Author "Sandoval, Ruben M."

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A nonerythropoietic derivative of erythropoietin inhibits tubulointerstitial fibrosis in remnant kidney
(Springer, 2012) Imamura, Ryoichi; Isaka, Yoshitaka; Sandoval, Ruben M.; Ichimaru, Naotsugu; Abe, Toyofumi; Okumi, Masayoshi; Yazawa, Koji; Kitamura, Harumi; Kaimori, Jyunya; Nonomura, Norio; Rakugi, Hiromi; Molitoris, Bruce A.; Takahara, Shiro; Medicine, School of Medicine
Background: The tissue-protective effects of erythropoietin (EPO) have been extensively investigated, and EPO administration can raise the hemoglobin (Hb) concentration. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia-reperfusion injury as well as EPO. Methods: To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a tubulointerstitial model rat. We randomized remnant kidney model rats to receive saline, EPO, or CEPO for 8 weeks. Results: CEPO- and EPO-treated rats had improved serum creatinine levels compared with saline-treated remnant kidney model rats, although the Hb level was significantly increased in EPO-treated rats. Two-photon microscopy revealed that EPO/CEPO significantly ameliorated tubular epithelial cell damage assessed by endocytosis. In addition, CEPO or EPO protected endothelial cells with a sustained blood flow rate. EPO or CEPO suppressed the number of TUNEL-positive apoptotic cells with weak αSMA staining. Furthermore, PCR analysis demonstrated that TGF-β and type I collagen expression was attenuated in EPO- or CEPO-treated rats, accompanied by a significant decrease in interstitial fibrosis. Conclusion: We established a long-term therapeutic approach to protect tubulointerstitial injury with CEPO, and thus, the therapeutic value of this approach warrants further attention and preclinical studies.
Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications
(American Physiological Society, 2022) Molitoris, Bruce A.; Sandoval, Ruben M.; Yadav, Shiv Pratap S.; Wagner, Mark C.; Medicine, School of Medicine
For nearly 50 years the proximal tubule (PT) has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes. Several genetic diseases, nonselective PT cell defects, chronic kidney disease (CKD), and acute PT injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PT injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism, and transcytosis are being reexamined with the use of techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multireceptor complex that mediates albumin binding and uptake and directs proteins for lysosomal degradation after endocytosis. Albumin transcytosis is mediated by a pH-dependent binding affinity to the neonatal Fc receptor (FcRn) in the endosomal compartments. This reclamation pathway rescues albumin from urinary losses and cellular catabolism, extending its serum half-life. Albumin that has been altered by oxidation, glycation, or carbamylation or because of other bound ligands that do not bind to FcRn traffics to the lysosome. This molecular sorting mechanism reclaims physiological albumin and eliminates potentially toxic albumin. The clinical importance of PT albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussion of genetic disorders and therapeutic considerations.
The archaeal Dps nanocage targets kidney proximal tubules via glomerular filtration
(American Society for Clinical Investigation, 2019-09-03) Uchida, Masaki; Maier, Bernhard; Waghwani, Hitesh Kumar; Selivanovitch, Ekaterina; Pay, S. Louise; Avera, John; Yun, EJun; Sandoval, Ruben M.; Molitoris, Bruce A.; Zollman, Amy; Douglas, Trevor; Hato, Takashi; Medicine, School of Medicine
Nature exploits cage-like proteins for a variety of biological purposes, from molecular packaging and cargo delivery to catalysis. These cage-like proteins are of immense importance in nanomedicine due to their propensity to self-assemble from simple identical building blocks to highly ordered architecture and the design flexibility afforded by protein engineering. However, delivery of protein nanocages to the renal tubules remains a major challenge because of the glomerular filtration barrier, which effectively excludes conventional size nanocages. Here, we show that DNA-binding protein from starved cells (Dps) — the extremely small archaeal antioxidant nanocage — is able to cross the glomerular filtration barrier and is endocytosed by the renal proximal tubules. Using a model of endotoxemia, we present an example of the way in which proximal tubule–selective Dps nanocages can limit the degree of endotoxin-induced kidney injury. This was accomplished by amplifying the endogenous antioxidant property of Dps with addition of a dinuclear manganese cluster. Dps is the first-in-class protein cage nanoparticle that can be targeted to renal proximal tubules through glomerular filtration. In addition to its therapeutic potential, chemical and genetic engineering of Dps will offer a nanoplatform to advance our understanding of the physiology and pathophysiology of glomerular filtration and tubular endocytosis.
Chronic endothelin-1 infusion elevates glomerular sieving coefficient and proximal tubular albumin reuptake in the rat
(Elsevier, 2012) Saleh, Mohamed A.; Sandoval, Ruben M.; Rhodes, George J.; Campos-Bilderback, Silvia B.; Molitoris, Bruce A.; Pollock, David M.; Medicine, School of Medicine
Aim: We have previously found that chronic endothelin-1 (ET-1) infusion in Sprague-Dawley rats increases glomerular permeability to albumin (P(alb)) as assessed in vitro independent of blood pressure with no observed albuminuria. In this study, we hypothesized that ET-1 increases glomerular albumin filtration with accompanied increase in albumin uptake via the proximal tubule, which masks the expected increase in urinary albumin excretion. Main methods: Nonfasting Munich-Wistar Fromter rats were surgically prepared for in vivo imaging (n=6). Rats were placed on the microscope stage with the exposed kidney placed in a cover slip-bottomed dish bathed in warm isotonic saline. Rats were then injected i.v. with rat serum albumin conjugated to Texas Red that was observed to enter capillary loops of superficial glomeruli, move into Bowman's space, bind to the proximal tubular cell brush border and reabsorbed across the apical membrane. Glomerular sieving coefficient (GSC) was calculated as the ratio of conjugated albumin within the glomerular capillary versus that in Bowman's space. Rats were again studied after 2 weeks of chronic ET-1 (2 pmol/kg/min; i.v. osmotic minipump). Key findings: Glomerular sieving coefficient was significantly increased in rats following chronic ET-1 infusion (0.025 ± 0.005 vs. 0.017 ± 0.003, p<0.05). Mean fluorescence intensity for conjugated albumin within proximal tubules was increased by ET-1 infusion: 118.40 ± 6.34 vs. 74.27 ± 4.45 pixel intensity (p<0.01). Significance: These data provide in vivo evidence that ET-1 directly increases glomerular permeability to albumin and that albuminuria is prevented by increased PT albumin uptake in the rat.
Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide
(American Chemical Society, 2023) Bryniarski, Mark A.; Sandoval, Ruben M.; Ruszaj, Donna M.; Fraser-McArthur, John; Yee, Benjamin M.; Yacoub, Rabi; Chaves, Lee D.; Campos-Bilderback, Silvia B.; Molitoris, Bruce A.; Morris, Marilyn E.; Medicine, School of Medicine
Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.
Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)
(American Physiological Society (APS), 2014-07-15) Carrisoza-Gaytan, Rolando; Liu, Yu; Flores, Daniel; Else, Cindy; Lee, Heon Goo; Rhodes, George; Sandoval, Ruben M.; Kleyman, Thomas R.; Lee, Francis Young-In; Molitoris, Bruce; Satlin, Lisa M.; Rohatgi, Rajeev; Department of Medicine, IU School of Medicine
An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.
Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
(Taylor & Francis Online, 2014-03-01) Sandoval, Ruben M.; Wang, Exing; Molitoris, Bruce A.; Department of Medicine, School of Medicine
Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.
Functional studies of the kidney of living animals using multicolor 2-photon microscopy
(2002-09) Dunn, Kenneth W.; Sandoval, Ruben M.; Kelly, Katherine J.; Dagher, Pierre C.; Tanner, George A.; Atkinson, Simon J.; Bacallao, Robert L.; Molitoris, Bruce A.
Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.
Hydrodynamic Isotonic Fluid Delivery Ameliorates Moderate-to-Severe Ischemia-Reperfusion Injury in Rat Kidneys
(American Society of Nephrology, 2017-07) Collett, Jason A.; Corridon, Peter R.; Mehrotra, Purvi; Kolb, Alexander L.; Rhodes, George J.; Miller, Caroline A.; Molitoris, Bruce A.; Pennington, Janice G.; Sandoval, Ruben M.; Atkinson, Simon J.; Campos-Bilderback, Silvia B.; Basile, David P.; Bacallao, Robert L.; Cellular and Integrative Physiology, School of Medicine
Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P<0.05). Intravital confocal microscopy performed immediately after HIFD showed improved microvascular perfusion. Notably, HIFD also resulted in immediate enhancement of parenchymal labeling with the fluorescent dye Hoechst 33342. HIFD also associated with a significant reduction in the accumulation of renal leukocytes, including proinflammatory T cells. Additionally, HIFD significantly reduced peritubular capillary erythrocyte congestion and improved histologic scores of tubular injury 4 days after IRI. Taken together, these results indicate that HIFD performed after establishment of AKI rapidly restores microvascular perfusion and small molecule accessibility, with improvement in overall renal function.
In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury
(Elsevier, 2013) Hall, Andrew M.; Rhodes, George J.; Sandoval, Ruben M.; Corridon, Peter R.; Molitoris, Bruce A.; Medicine, School of Medicine
Mitochondrial dysfunction has been implicated in the pathogenesis of acute kidney injury due to ischemia and toxic drugs. Methods for imaging mitochondrial function in cells using confocal microscopy are well established; more recently, it was shown that these techniques can be utilized in ex vivo kidney tissue using multiphoton microscopy. We extended this approach in vivo and found that kidney mitochondrial structure and function can be imaged in anesthetized rodents using multiphoton excitation of endogenous and exogenous fluorophores. Mitochondrial nicotinamide adenine dinucleotide increased markedly in rat kidneys in response to ischemia. Following intravenous injection, the mitochondrial membrane potential–dependent dye TMRM was taken up by proximal tubules; in response to ischemia, the membrane potential dissipated rapidly and mitochondria became shortened and fragmented in proximal tubules. In contrast, the mitochondrial membrane potential and structure were better maintained in distal tubules. Changes in mitochondrial structure, nicotinamide adenine dinucleotide, and membrane potential were found in the proximal, but not distal, tubules after gentamicin exposure. These changes were sporadic, highly variable among animals, and were preceded by changes in non-mitochondrial structures. Thus, real-time changes in mitochondrial structure and function can be imaged in rodent kidneys in vivo using multiphoton excitation of endogenous and exogenous fluorophores in response to ischemia–reperfusion injury or drug toxicity.

Browsing by Author "Sandoval, Ruben M."

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