Organ regeneration necessitates precise coordination of accelerators and brakes to restore organ function. However, the mechanisms underlying this intricate molecular crosstalk remain elusive. In this study, the level of proenkephalin-A (PENK-A), expressed by renal proximal tubular epithelial cells, decreases significantly with the loss of renal proximal tubules and increased at the termination phase of zebrafish kidney regeneration. Notably, this change contrasts with the role of hydrogen peroxide (H2O2), which acts as an accelerator in kidney regeneration. Through experiments with penka mutants and pharmaceutical treatments, we demonstrate that PENK-A inhibits H2O2 production in a dose-dependent manner, suggesting its involvement in regulating the rate and termination of regeneration. Furthermore, H2O2 influences the expression of tcf21, a vital factor in the formation of renal progenitor cell aggregates, by remodeling H3K4me3 in renal cells. Overall, our findings highlight the regulatory role of PENK-A as a brake in kidney regeneration.

Ischemic preconditioning (IPC) is effective in limiting subsequent ischemic acute kidney injury in experimental models. MicroRNAs are an important class of post-transcriptional regulator and show promise as biomarkers of kidney injury. We evaluated the time- and dose-dependence of benefit from IPC in a rat model of functional (bilateral) ischemia-reperfusion injury (IRI). We found optimal protection from subsequent injury following short, repetitive sequences of preconditioning insult. We subsequently used hybridization array and microRNA sequencing to characterize microRNA signatures of protective IPC and of IRI. These approaches identified a profile of microRNA changes consequent on IRI, that were limited by prior IPC. To localize these signals within the kidney, we used laser capture microdissection and RT-qPCR to measure microRNA abundance in nephron segments, pinpointing microRNA changes principally to glomeruli and proximal tubules. Our data describe a unique microRNA signature for IRI in the rat kidney. Pulsatile IPC reduces kidney damage following IRI and diminishes this microRNA signal. We have also identified candidate microRNAs that may act as biomarkers of injury and therapeutic targets in this context.

The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Acute kidney injury (AKI) carries high morbidity and mortality, and effective treatments are lacking. Preclinical models support involvement of micro-RNAs (miRs) in AKI pathogenesis, although effects on the kidney transcriptome are unclear. We previously showed that injection of cord blood endothelial colony forming cell-derived exosomes, enriched in miR-486-5p, prevented ischemic AKI in mice. To further define this, we studied direct effects of miR-486-5p in mice with kidney ischemia-reperfusion injury. RNA-Seq was used to compare the impact of miR-486-5p and exosomes on the transcriptome of proximal tubules and kidney endothelial cells 24 hours after ischemia-reperfusion. In mice with AKI, injection of miR-486-5p mimic increased its levels in proximal tubules and endothelial cells, and improved plasma creatinine, histological injury, neutrophil infiltration, and apoptosis. Additionally, miR-486-5p inhibited expression of its target phosphatase and tensin homolog, and activated protein kinase B. In proximal tubules, miR-486-5p or exosomes reduced expression of genes associated with ischemic injury and the tumor necrosis factor (TNF) pathway, and altered distinct apoptotic genes. In endothelial cells, genes associated with metabolic processes were altered by miR-486-5p or exosomes, although TNF pathway genes were not affected. Thus, our results suggest that miR-486-5p may have therapeutic potential in AKI.

Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.

Chronic kidney disease (CKD) is a commonly occurring complex renal syndrome that causes overall mortality in many diseases. The clinical manifestations of CKD include renal tubulointerstitial fibrosis and loss of renal function. Metallothionein-I/II (MT-I/II) is potentially expressed in the liver and kidney, and possesses antioxidant and metal detoxification properties. However, whether MT-I/II expression is associated with the prognosis of nephropathy remains unknown. In this study, we investigated the MT-I/II level in human CKD, using immunohistochemistry. MT-I/II is located on the proximal tubules and is notably reduced in patients with CKD. MT-I/II expression was significantly correlated with the functional and histological grades of CKD. In an aristolochic acid (AAI)-induced nephropathy mouse model, MT-I/II was abundantly increased after AAI injection for 7 days, but decreased subsequently compared to that induced in the acute phase when injected with AAI for 28 days. Furthermore, we found that ammonium pyrrolidinedithiocarbamate (PDTC) restored AAI-induced MT-I/II reduction in HK2 cells. The injection of PDTC ameliorated AAI-induced renal tubulointerstitial fibrosis and reduced the concentrations of blood urea nitrogen and creatinine in mouse sera. Taken together, our results indicate that MT-I/II reduction is associated with advanced CKD, and the retention of renal MT-I/II is a potential therapeutic strategy for CKD.

At 20 degrees when active transport of an organic acid - fluorescein in superficial proximal tubules of surviving rat kidney does not depend on the external Na, the capability of acetate (as well as succinate and D,L-leucine) of stimulating the fluorescein uptake in the tubules was studied. As it turned out, acetate and leucine stimulated the fluorescein uptake at 120 mM of Na in the bath medium but had no effect at 10 mM of Na. Succinate has a biphasic effect on the fluorescein transport (stimulation at low succinate concentration), which does not depend on Na concentration in the medium. The effect of acetate disappears with the presence of ouabain in the medium. The stimulation of fluorescein uptake by acetate is accompanied by somewhat increased degree of reduction of cellular pyridine nucleotides. Pyridine nucleotides are reduced more markedly in the presence of ouabain or in the absence of Na from the bath medium. The disappearance of acetate effect on the fluorescein transport is explained by the fact of transition of cell mitochondria in the inactive state 4, due to the decrease in the level of cellular ADP when Na, K-ATPase is inhibited by ouabain or by diminution of tissue Na+ concentration.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. In this report, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI. We found that Pax2 and Pax8 are upregulated after severe AKI and correlate with chronic injury. Surprisingly, we then discovered that proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to preconditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of S3 proximal tubule cells that display features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic preconditioning, and female sex. Taken together, our results identify a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both injury response and protection from ischemic AKI.

A microfluidic multi-organ chip emulates the tissue culture microenvironment, enables interconnection of organ equivalents and overcomes interspecies differences, making this technology a promising and powerful tool for preclinical drug screening. In this study, we established a microfluidic chip-based model that enabled non-contact cocultivation of liver spheroids and renal proximal tubule barriers in a connecting media circuit over 16 days. Meanwhile, a 14-day repeated-dose systemic administration of cyclosporine A (CsA) alone or in combination with rifampicin was performed. Toxicity profiles of the two different doses of CsA on different target organs could be discriminated and that concomitant treatment with rifampicin from day6 onwards decreased the CsA concentration and attenuated the toxicity compared with that after treatment with CsA for 14 consecutive days. The latter is manifested with the changes in cytotoxicity, cell viability and apoptosis, gene expression of metabolic enzymes and transporters, and noninvasive toxicity biomarkers. The on chip coculture of the liver and the proximal tubulus equivalents showed its potential as an effective and translational tool for repeated dose multi-drug toxicity screening in the preclinical stage of drug development.

A mechanism of inhibitory action of inorganic anions (SCN- and SO 2/4-) on the transport of an organic acid (fluorescein) into proximal tubules was investigated in slices of the rat's kidney outer cortex. The peritubular S14CN- is shown to be accumulated in the cortical tissue by means of simple diffusion, this accumulation not depending on the presence of fluorescein in the medium. The peritubular 35SO 2/4- is accumulated in the tissue against the gradient of its electrochemical potential. It is very likely that the peritubular sulphate penetrates via intercellular contacts into the tubular lumen to be then actively reabsorbed by the Na+-dependent transport system in the luminal membrane. The sulphate accumulation is not affected by fluorescein added to the medium. It is concluded that the inhibition of organic acid transport after replacement of Cl- in the medium by SCN- or SO 2/4- may not be considered as a competition for a common carrier between organic and inorganic anions.

Diabetic nephropathy is a major source of end-stage renal failure, affecting about one-third cases of diabetes mellitus. It has long been accepted that diabetic nephropathy is mainly characterized by glomerular defects, while clinical observations have implied that renal tubular damage is closely linked to kidney dysfunction at the early stages of diabetic nephropathy. In this study, we conducted pathohistological analyses focusing on renal tubular lesions in the early-stage diabetic kidney with the use of a streptozotocin (STZ)-induced diabetes mellitus mouse model. The results revealed that histological alterations in renal tubules, shown by a vacuolar nucleic structure, accumulations of PAS-positive substance, and accelerated restoration stress, occur initially without the presence of glomerular lesions in the early-stage diabetic kidney, and that these tubular defects are localized mainly in proximal renal tubules. Moreover, enhanced expression of RAGE, suggesting an aberrant activation of AGEs-RAGE signaling pathway, and accumulation of oxidative modified mitochondria through the impaired autophagy/lysosome system, were also seen in the damaged diabetic proximal renal tubules. Our findings indicate that proximal tubular defects are the initial pathological events increasingly linked to the progression of diabetic nephropathy, and that controlling renal tubular damage could be an effective therapeutic strategy for the clinical treatment of diabetic nephropathy.

Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.

Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases.

Many anatomical regions in the kidney, including proximal tubules, differ between males and females. While such differences in renal structures and functions under various physiological and pharmacological conditions have been identified, information relating to molecular mechanisms behind this gender disparity remain unknown. To understand gene expression differences in proximal tubules from human male and female kidneys, we reported on kidney cellular landscape using single-cell RNA sequencing. Differential gene expression profiles were observed in proximal tubules, between the sexes. Interestingly, the SLC22 family of anion transporters, including SLC22A6 and SLC22A8, had different expression profiles between male and female proximal tubule clusters but not sex-dependent abundance at the protein level. Moreover, in different species, we revealed a shared and species-specific differential gene expression between human and mouse kidney proximal tubules. Taken together, at single-cell resolution, this transcriptomic map represents a baseline description of gender biased genes in human kidney proximal tubules, which provide important insights for further studies of physiological differences in kidney.

Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70-4 with no apparent change in the aggresome-autophagosome markers. Angiotensin II induced aggregation of RPL27 specifically in proximal tubules, again without apparent change in antiaggregating proteins or the aggresome-autophagosome markers. Albumin endocytosis was unaffected by the hormone administration. Taken together, we find that the renal proximal tubules display aggresome formation and autophagy. Despite an increase in aggregation-prone protein load in these tubules during hormone treatment, renal proximal tubules seem to have sufficient capacity for removing protein aggregates from the cells.

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival.

The roles of angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of two-kidney, 1-clip (2K1C) Goldblatt hypertension have not been investigated previously. The present study tested the hypothesis that deletion of the AT1a receptor or NHE3 selectively in the proximal tubules of the kidney attenuates the development of 2K1C hypertension using novel mouse models with proximal tubule-specific deletion of AT1a receptors or NHE3. 2K1C Goldblatt hypertension was induced by placing a silver clip (0.12 mm) on the left renal artery for 4 weeks in adult male wild-type (WT), global Agtr1a−/−, proximal tubule (PT)-specific PT-Agtr1a−/− or PT-Nhe3−/− mice, respectively. As expected, telemetry blood pressure increased in a time-dependent manner in WT mice, reaching a maximal response by Week 3 (p < 0.01). 2K1C hypertension in WT mice was associated with increases in renin expression in the clipped kidney and decreases in the nonclipped kidney (p < 0.05). Plasma and kidney Ang II were significantly increased in WT mice with 2K1C hypertension (p < 0.05). Tubulointerstitial fibrotic responses were significantly increased in the clipped kidney (p < 0.01). Whole-body deletion of AT1a receptors completely blocked the development of 2K1C hypertension in Agtr1a−/− mice (p < 0.01 vs. WT). Likewise, proximal tubule-specific deletion of Agtr1a in PT-Agtr1a−/− mice or NHE3 in PT-Nhe3−/− mice also blocked the development of 2K1C hypertension (p < 0.01 vs. WT). Taken together, the present study provides new evidence for a critical role of proximal tubule Ang II/AT1 (AT1a)/NHE3 axis in the development of 2K1C Goldblatt hypertension.

Proteinuria is a marker of incipient kidney injury in many disorders, including obesity. Previously, we demonstrated that megalin, a receptor endocytotic protein in the proximal tubule, is downregulated in obese mice, which was prevented by inhibition of dipeptidyl protease 4 (DPP4). Obesity is thought to be associated with upregulation of intra-renal angiotensin II (Ang II) signaling via the Ang II Type 1 receptor (AT₁R) and Ang II suppresses megalin expression in proximal tubule cells in vitro. Therefore, we tested the hypothesis that Ang II will suppress megalin protein via activation of DPP4. We used Ang II (200 ng/kg/min) infusion in mice and Ang II (10(-8) M) treatment of T35OK-AT₁R proximal tubule cells to test our hypothesis. Ang II-infused mouse kidneys displayed increases in DPP4 activity and decreases in megalin. In proximal tubule cells, Ang II stimulated DPP4 activity concurrent with suppression of megalin. MK0626, a DPP4 inhibitor, partially restored megalin expression similar to U0126, a mitogen activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase (MEK) 1/2 inhibitor and AG1478, an epidermal growth factor receptor (EGFR) inhibitor. Similarly, Ang II-induced ERK phosphorylation was suppressed with MK0626 and Ang II-induced DPP4 activity was suppressed by U0126. Therefore, our study reveals a cross talk between AT₁R signaling and DPP4 activation in the regulation of megalin and underscores the significance of targeting DPP4 in the prevention of obesity related kidney injury progression.

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.