Unique Transcriptional Programs Identify Subtypes of AKI.

Two metrics, a rise in serum creatinine concentration and a decrease in urine output, are considered tantamount to the injury of the kidney tubule and the epithelial cells thereof (AKI). Yet neither criterion emphasizes the etiology or the pathogenetic heterogeneity of acute decreases in kidney excretory function. In fact, whether decreased excretory function due to contraction of the extracellular fluid volume (vAKI) or due to intrinsic kidney injury (iAKI) actually share pathogenesis and should be aggregated in the same diagnostic group remains an open question. To examine this possibility, we created mouse models of iAKI and vAKI that induced a similar increase in serum creatinine concentration. Using laser microdissection to isolate specific domains of the kidney, followed by RNA sequencing, we found that thousands of genes responded specifically to iAKI or to vAKI, but very few responded to both stimuli. In fact, the activated gene sets comprised different, functionally unrelated signal transduction pathways and were expressed in different regions of the kidney. Moreover, we identified distinctive gene expression patterns in human urine as potential biomarkers of either iAKI or vAKI, but not both. Hence, iAKI and vAKI are biologically unrelated, suggesting that molecular analysis should clarify our current definitions of acute changes in kidney excretory function.

SDF-1 activates papillary label-retaining cells during kidney repair from injury.

The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795-804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

Urinary neutrophil gelatinase-associated lipocalin predicts mortality and identifies acute kidney injury in cirrhosis.

BACKGROUND: Kidney failure predicts mortality in patients with cirrhosis. Identification of kidney failure etiology and recognition of those at the highest mortality risk remains a challenge. AIMS: We hypothesized that urinary neutrophil gelatinase-associated lipocalin (uNGAL) predicts mortality and identifies hepatorenal syndrome (HRS) in patients with cirrhosis. METHODS: Prospectively enrolled patients with cirrhosis were investigated by uNGAL immunoblot upon hospital admission. Kidney failure type was determined blinded to NGAL measurements. RESULTS: One hundred eighteen patients were enrolled. Fifty-two (44 %) patients had normal kidney function, 14 (12 %) stable chronic kidney disease, 17 (14 %) prerenal azotemia, 20 (17 %) HRS, and 15 (13 %) intrinsic acute kidney injury (iAKI). Patients with HRS had uNGAL levels intermediate between prerenal azotemia [median (IQR) 105 (27.5-387.5) vs. 20 (15-45) ng/mL, p = 0.004] and iAKI [325 (100-700), p < 0.001]. Fifteen (13 %) patients died. In unadjusted analysis, uNGAL predicted inpatient mortality (OR 2.00, 95 % CI 1.36-2.94) and mortality or liver transplantation (OR 2.01, 95 % CI 1.42-2.85). In multiple regression models, uNGAL > 110 ng/mL (OR 6.05, 95 % CI 1.35-27.2) and HRS (OR 6.71, 95 % CI 1.76-25.5) independently predicted mortality, adjusting for age and serum creatinine >1.5 mg/dL. CONCLUSIONS: uNGAL strongly predicts short-term inpatient mortality in both unadjusted and adjusted models. Patients with HRS may have uNGAL levels intermediate between those with prerenal azotemia and iAKI. Further studies are needed to determine if uNGAL can improve discrimination of HRS from other types of acute kidney injury and predict short- and long-term cirrhosis outcomes.

Afferent mechanisms of sodium retention in cirrhosis and hepatorenal syndrome.

Cirrhosis induces extra-cellular fluid volume expansion, which when the disease is advanced can be severe and poorly responsive to therapy. Prevention and/or effective therapy for cirrhotic edema requires understanding the stimulus that initiates and maintains sodium retention. Despite much study, this stimulus remains unknown. Work over the last several years has shown that signals originating in the liver can influence a variety of systemic functions, including extra-cellular fluid volume control. We review work on the afferent mechanisms triggering sodium retention in cirrhosis and suggest that the data are most consistent with the existence of a sensor in the hepatic circulation that contributes to normal extra-cellular fluid volume control (that is, a 'volume' sensor) and that in cirrhosis, the sensor is pathologically activated by the hepatic circulatory abnormalities caused by the disease. Detailed analysis of the hepatic circulation in normal conditions and cirrhosis is needed.

Increased complexity of vasopressin's vascular actions.

Vasopressin is becoming a widely used pressor in conditions with severe hypotension. Like several other hormones important in cardiovascular and extracellular fluid control, however, vasopressin can activate several receptors that when pharmacologically or pathologically stimulated may result in conflicting effects. In the present issue of Critical Care, Rehberg and colleagues examined the hypothesis that blockade of vasopressin V2 receptor during septic shock may be beneficial. Their tantalizing results indicate that future work must consider the precise vasopressin receptors that are stimulated and/or inhibited.

Proliferation and migration of label-retaining cells of the kidney papilla.

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with "chains" of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

Endogenous and exogenous vasopressin during hemodialysis.

Intradialytic hypotension likely results from hypovolemia as well as patient and dialysis-specific factors. An impaired vasoconstrictive response to volume loss during hemodialysis has been demonstrated and increasing evidence suggests that deficiency in the hormone arginine vasopressin may be a contributing factor. Although vasopressin is widely recognized for its role in the regulation of serum osmolality, vasopressin is also an important regulator of blood pressure in health and in various disease states. That vasopressin deficiency contributes to the pathogenesis of intradialytic hypotension is suggested by several observations. First, vasopressin levels typically fall during hemodialysis when a rise might be expected as a result of volume loss. Second, therapies that prevent a fall in osmolality during dialysis, including dialysis against a high sodium bath and isolated ultrafiltration, have been shown to improve intradialytic blood pressure stability. Finally, and perhaps most importantly, the administration of low-dose exogenous vasopressin during dialysis has been shown to support blood pressure and improve volume removal. Further research is needed to determine the effect of chronic vasopressin (or selective V1a agonist) administration during dialysis on volume removal, inter- and intradialytic blood pressure control, and, ultimately, clinical outcomes in end-stage renal disease patients on dialysis.

The renal papilla is a niche for adult kidney stem cells.

Many adult organs contain stem cells, which are pluripotent and are involved in organ maintenance and repair after injury. In situ, these cells often have a low cycling rate and locate in specialized regions (niches). To detect such cells in the kidney, we administered a pulse of the nucleotide bromodeoxyuridine (BrdU) to rat and mouse pups and, after a long (more than 2-month) chase, examined whether the kidney contained a population of low-cycling cells. We found that in the adult kidney, BrdU-retaining cells were very sparse except in the renal papilla, where they were numerous. During the repair phase of transient renal ischemia, these cells entered the cell cycle and the BrdU signal quickly disappeared from the papilla, despite the absence of apoptosis in this part of the kidney. In vitro isolation of renal papillary cells showed them to have a plastic phenotype that could be modulated by oxygen tension and that when injected into the renal cortex, they incorporated into the renal parenchyma. In addition, like other stem cells, papillary cells spontaneously formed spheres. Single-cell clones of these cells coexpressed mesenchymal and epithelial proteins and gave rise to myofibroblasts, cells expressing neuronal markers, and cells of uncharacterized phenotype. These data indicate that the renal papilla is a niche for adult kidney stem cells.

The kidney papilla is a stem cells niche.

Stem cells are characterized by low cycle time, which has allowed us to identify such cells in the mature kidney. These putative stem cells are located mostly outside the renal tubule and are concentrated in the papilla of the kidney potentially under the urinary epithelium of the papilla. Clonal analysis of these cells shows that they can differentiate into epithelial, neuronal, and other uncharacterized cells. Induction of ischemic renal failure resulted in increased proliferation of these papillary cells. Injection of these cells under the renal capsule led to their incorporation into various tubule segments. It is likely that these stem cells sense a "damage" signal from the cortex resulting in proliferation followed by migration to the site of injury.

A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.

To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.

Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease with mitochondrial DNA (mtDNA) alterations and is caused by mutations in the nuclear gene encoding thymidine phosphorylase (TP). The cardinal clinical manifestations are ptosis, ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Skeletal muscle shows mitochondrial abnormalities, including ragged-red fibers and cytochrome c oxidase deficiency, together with mtDNA depletion, multiple deletions or both. In MNGIE patients, TP mutations cause a loss-of-function of the cytosolic enzyme, TP. As a direct consequence of the TP defect, thymidine metabolism is altered. High blood levels of this nucleoside are likely to lead to mtDNA defects even in cells that do not express TP, such as skeletal muscle. We hypothesize that high concentrations of thymidine affect dNTP (deoxyribonucleoside triphosphate) metabolism in mitochondria more than in cytosol or nuclei, because mitochondrial dNTPs depend mainly on the thymidine salvage pathway, whereas nuclear dNTPs depend mostly on de novo pathway. The imbalance in the mitochondrial dNTP homeostasis affects mtDNA replication, leading to mitochondrial dysfunction.

The regulation of growth and metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney.

Native extracellular matrix (ECM) that is secreted and maintained by resident cells is of great interest for cell culture and cell delivery. We hypothesized that specialized bioengineered niches for stem cells can be established using ECM-derived scaffolding materials. Kidney was selected as a model system because of the high regional diversification of renal tissue matrix. By preparing the ECM from three specialized regions of the kidney (cortex, medulla, and papilla; whole kidney, heart, and bladder as controls) in three forms: (i) intact sheets of decellularized ECM, (ii) ECM hydrogels, and (iii) solubilized ECM, we investigated how the structure and composition of ECM affect the function of kidney stem cells (with mesenchymal stem cells, MSCs, as controls). All three forms of the ECM regulated KSC function, with differential structural and compositional effects. KSCs cultured on papilla ECM consistently displayed lower proliferation, higher metabolic activity, and differences in cell morphology, alignment, and structure formation as compared to KSCs on cortex and medulla ECM, effects not observed in corresponding MSC cultures. These data suggest that tissue- and region-specific ECM can provide an effective substrate for in vitro studies of therapeutic stem cells.

Endogenous and exogenous vasopressin in shock.

PURPOSE OF REVIEW: Vasopressin is critical for blood pressure regulation when cardiovascular homeostasis is threatened and some patients with shock have inappropriately low levels of hormone in plasma. The present review focuses on recent work that addresses the role of endogenous vasopressin in the pathogenesis of shock and the potential therapeutic indications and secondary effects of exogenous hormone in patients with shock. RECENT FINDINGS: Examples of types of shock resistant to catecholamine pressors in which exogenous vasopressin was effective in restoring arterial pressure continued to accumulate. Widespread determinations of plasma vasopressin in patients with shock suggest that endogenous vasopressin deficiency may be more frequent than previously thought. The generation of mice with deletion of vasopressin's V1a receptor highlighted the important role of the hormone on cardiovascular homeostasis. SUMMARY: Vasopressin administration is very effective in restoring arterial pressure in many forms of shock and this appears to be due, at least in part, to deficiency of endogenous hormone. Generation of mice lacking vasopressin V1a receptor open new and exciting avenues of inquiry to clarify the role of the hormone in cardiovascular homeostasis.

Adult renal stem cells and renal repair.

PURPOSE OF REVIEW: Recent studies that might help in the search for stem cells in adult kidney and clarify the origin of proliferating cells during kidney repair are reviewed. RECENT FINDINGS: Some of the most notable recent findings are as follows: (1) the 'stemness' profile may be determined by approximately 250 genes; (2) organ-specific stem-cell growth and differentiation are stimulated during the reparative phase following transient injury; (3) two bone marrow stem-cell types show a remarkable degree of differentiation potential; (4) some organs contain resident marrow-derived stem cells, and their differentiation potential may only be expressed during repair; (5) the metanephric mesenchyme contains pluripotent and self-renewing stem cells; (6) marrow-derived cells invade the kidney and differentiate into mesangial and tubular epithelial cells, and these processes are increased following renal injury; and (7) epithelial-to-mesenchymal transition generates renal fibroblasts. SUMMARY: While it remains unknown whether there is a stem cell in the adult kidney, characterization of the cell populations involved in renal repair and misrepair is allowing a new understanding of the mechanisms that are responsible for renal homeostasis. The most surprising results suggest a very prominent role for cells exogenous to the kidney. Two recently published transcription profiles of 'stemness' and the phenotype of pluripotent metanephric mesenchymal cells may help in the search for adult renal stem cells.

Stem cells in the kidney.

The kidney is derived from the ureteric bud and the metanephrogenic mesenchyme, and these two progenitor cells differentiate into more than 26 different cell types in the adult kidney. The ureteric bud contains the precursor of the epithelial cells of the collecting duct and the renal mesenchyme contains precursors of all the epithelia of the rest of the nephron, endothelial cell precursors and stroma cells, but the relatedness among these cells is unclear. A single metanephric mesenchymal cell can generate all the epithelial cells of the nephron (except the collecting duct), indicating that the kidney contains epithelial stem cells. It is currently unknown whether these stem cells also are present in the adult kidney but experience in other organs makes this likely. It also is unclear whether embryonic renal epithelial stem cells can generate other cell types, but preliminary studies in our laboratory suggest that they can differentiate into myofibroblasts, smooth muscle, and perhaps endothelial cells, indicating that they are pluripotent renal stem cells. The important problem to be solved now is the identification and location of adult renal stem cells. This article discusses work done in other organs and in renal development that we believe may be useful for the resolution of this problem.

Metanephric mesenchyme contains embryonic renal stem cells.

Renal epithelial cells derive from either cells of the metanephric mesenchyme or ureteric bud cells, but the origin of other renal cells is unclear. To test whether metanephric mesenchymal cells generate cells other than epithelial, we examined the developmental potential of a metanephric mesenchymal cell line (7.1.1 cells) and of primary cultures of metanephric mesenchymal cells. 7.1.1 Cells express both mesenchymal and epithelial markers and, on confluence, form well-defined monolayers expressing epithelial junctional proteins. However, 7.1.1 cells as well as primary cultures of metanephric mesenchymal cells also generate spindle-shaped cells that are positive for alpha-smooth muscle actin, indicating that they are myofibroblasts and/or smooth muscle; this differentiation pathway is inhibited by collagen IV and enhanced by fetal calf serum or transforming growth factor-beta(1). Transforming growth factor-beta(1) also induces expression of smooth muscle proteins, indicating that the cells differentiate into smooth muscle. 7.1.1 Cells as well as primary cultures of metanephric mesenchymal cells also express vascular endothelial growth factor receptor 2 and Tie-2, suggesting that the metanephric mesenchymal cells that generate epithelia may also differentiate into endothelial cells. The pluripotency of the 7.1.1 cells is self-renewing. The data suggest that the metanephric mesenchyme contains embryonic renal stem cells.

Altered thymidine metabolism due to defects of thymidine phosphorylase.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene. TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. We present evidence that thymidine metabolism is altered in MNGIE. TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls. All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers. In two patients, the renal clearance of thymidine was approximately 20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney. In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine. In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine. In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic. We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both. Therapies to reduce thymidine levels may be beneficial to MNGIE patients.